US20210102213A1

US20210102213A1 - CCCTC-Binding Factor Variants

Info

Publication number: US20210102213A1
Application number: US17/118,378
Authority: US
Inventors: Rebecca Tayler COTTMAN; J. Keith Joung
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 2018-05-17
Filing date: 2020-12-10
Publication date: 2021-04-08
Also published as: CA3100726A1; AU2019269692A1; WO2019222670A1; EP3793584A4; US20190382767A1; US11041155B2; EP3793584A1; JP2021523719A

Abstract

Described herein are engineered CCCTC-binding factor (CTCF) variants that can bind to mutant CTCF binding sequences and method of using the same.

Description

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No. 16/415,989, filed May 17, 2019, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/672,682, filed on May 17, 2018 and U.S. Provisional Patent Application Ser. No. 62/828,277, filed on Apr. 2, 2019. The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. GM118158 awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 10, 2020, is named Sequence Listing.txt and is 1,104,397 bytes in size.

TECHNICAL FIELD

The invention relates, at least in part, to engineered CCCTC-binding factor variants with altered DNA-binding specificities.

BACKGROUND

CCCTC-binding factor (CTCF) is a multi-domain protein that acts as an essential genome organizer by maintaining higher-order chromatin structure while also having a role in cell differentiation and the promotion or repression of gene expression (Ong and Corces, Nature Reviews Genetics (2014); Phillips and Corces, Cell (2009)). CTCF maintains topologically associated domains (TADs) spanning MBs of the genome as well as smaller scale Sub-TADs leading to fine-tuned gene insulation or gene activation within gene clusters (Ali et al., Current Opinion in Genetics & Development (2016); Nora et al., Nature (2012); Rao et al., Cell (2014)). In addition, CTCF has been found to regulate mRNA splicing by influencing the rate of transcription and more recently been implicated in promoting homologous recombination repair at double-strand breaks (Shukla et al., Nature (2011); Hilmi et al., Science Advances (2017); Han et al., Scientific Reports (2016)). CTCF binds throughout the genome via an 11 finger zinc finger (ZF) array that recognizes CTCF binding sites (CBSs). The CBS is typically 40 bp in length with a highly conserved 15 bp core sequence.

SUMMARY

The present invention is based, at least in part, on the development of engineered CTCF variants that can bind to mutant CBSs with higher affinity than a wild-type CTCF.
The present invention relates to an engineered CCCTC-binding factor (CTCF) variant including at least one amino acid residue in at least one zinc finger that differs in sequence from the amino acid sequence of a wild-type CTCF, where the engineered CTCF variant binds to a mutant CTCF binding sequence (CBS) with a higher affinity than wild-type CTCF, the mutant CBS including at least one nucleotide base that differs in sequence from the nucleotide sequence of a consensus CBS, where the at least one amino acid residue that differs in sequence from the amino acid sequence of a wild-type CTCF is selected from the group consisting of the amino acid residues at the position(s) −1, +1, +2, +3, +5, and +6 of any of ZF7, ZF6, ZF5, ZF4, and ZF3 of the engineered CTCF variant.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CTCF binding sequence (CBS) that has a Thymine (T), Adenine (A), or Guanine (G) residue at position 2 of the consensus CBS motif, the engineered CTCF including an amino acid residue threonine, asparagine, or histidine at ZF7 position +3.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a G residue at position 2 of the consensus CBS motif, the engineered CTCF including the amino acid sequence DHLQT (SEQ ID NO: 8), EHLNV (SEQ ID NO: 9), AHLQV (SEQ ID NO: 10), EHLRE (SEQ ID NO: 11), DHLQV (SEQ ID NO: 12), EHLKV (SEQ ID NO: 13), EHLVV (SEQ ID NO: 15), DHLRT (SEQ ID NO: 16), or DHLAT (SEQ ID NO: 17) at ZF7 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, G, or C residue at position 3 of the consensus CBS motif, the engineered CTCF at ZF7 positions −1 to +3 including: the amino acid sequence RKHD (SEQ ID NO: 173) or RRSD (SEQ ID NO: 174), where the mutant CBS has a T residue at position 3 of the consensus CBS motif; the amino acid sequence RKAD (SEQ ID NO: 175), IPRI (SEQ ID NO: 176), RKHD (SEQ ID NO: 173), or RKDD (SEQ ID NO: 177), where the mutant CBS has a G residue at position 3 of the consensus CBS motif; or the amino acid sequence GIVN (SEQ ID NO: 178), ELLN (SEQ ID NO: 179), QALL (SEQ ID NO: 180) or PHRM (SEQ ID NO: 181), where the mutant CBS has a C residue at position 3 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, G, or A residue at position 5 of the consensus CBS motif, the engineered CTCF at ZF6 positions +2 to +6 including: the amino acid sequence NAMKR (SEQ ID NO: 30), GNMAR (SEQ ID NO: 182), EGMTR (SEQ ID NO: 183), SNMVR (SEQ ID NO: 184), or NAMRG (SEQ ID NO: 185), where the mutant CBS has a T residue at position 5 of the consensus CBS motif; or the amino acid sequence EHMGR (SEQ ID NO: 31), DHIVINR (SEQ ID NO: 32), THMKR (SEQ ID NO: 33), EHMRR (SEQ ID NO: 34), or THIVINR (SEQ ID NO: 35), where the mutant CBS has a G residue at position 5 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, G, or C residue at position 6 of the consensus CBS motif, the engineered CTCF at ZF6 positions −1 to +3 including: the amino acid sequence MNES (SEQ ID NO: 36) or HRES (SEQ ID NO: 37), where the mutant CBS has a T residue at position 6 of the consensus CBS motif; or the amino acid sequence RPDT (SEQ ID NO: 38), RTDI (SEQ ID NO: 39), or RHDT (SEQ ID NO: 40), where the mutant CBS has a G residue at position 6 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a C, A, or T residue at position 7 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 including: the amino acid sequence HGLKV (SEQ ID NO: 41), HRLKE (SEQ ID NO: 42), HALKV (SEQ ID NO: 43), SRLKE (SEQ ID NO: 44), or DGLRV (SEQ ID NO: 45), where the mutant CBS has a T residue at position 7 of the consensus CBS motif; the amino acid sequence HTLKV (SEQ ID NO: 46), or HGLKV (SEQ ID NO: 41), where the mutant CBS has an A residue at position 7 of the consensus CBS motif; or the amino acid sequence SRLKE (SEQ ID NO: 44), HRLKE (SEQ ID NO: 42) or NRLKE (SEQ ID NO: 47), where the mutant CBS has a C residue at position 7 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a C, A, or T residue at position 8 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 including: the amino acid sequence ATLKR (SEQ ID NO: 48), QALRR (SEQ ID NO: 49), GGLVR (SEQ ID NO: 50), or HGLIR (SEQ ID NO: 51), where the mutant CBS has a T residue at position 8 of the consensus CBS motif; the amino acid sequence ANLSR (SEQ ID NO: 52), TGLTR (SEQ ID NO: 53), HGLVR (SEQ ID NO: 54), or GGLTR (SEQ ID NO: 55), where the mutant CBS has an A residue at position 8 of the consensus CBS motif; the amino acid sequence HTLRR (SEQ ID NO: 56), TVLKR (SEQ ID NO: 57), ADLKR (SEQ ID NO: 58), or HGLRR (SEQ ID NO: 59), where the mutant CBS has a C residue at position 8 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, A, or C residue at position 10 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 including: the amino acid sequence AHLRK (SEQ ID NO: 60), wherein the mutant CBS has a T residue at position 10 of the consensus CBS motif; the amino acid sequence AKLRV (SEQ ID NO: 61), EKLRI (SEQ ID NO: 186), or AKLRI (SEQ ID NO: 63), where the mutant CBS has an A residue at position 10 of the consensus CBS motif; or the amino acid sequence TKLKV (SEQ ID NO: 64), wherein the mutant CBS has a C residue at position 10 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, A, or C residue at position 11 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 including: the amino acid sequence ATLRR (SEQ ID NO: 66) or RRLDR (SEQ ID NO: 67), where the mutant CBS has a T residue at position 11 of the consensus CBS motif; the amino acid sequence TNLRR (SEQ ID NO: 68), ANLRR (SEQ ID NO: 69), or GNLTR (SEQ ID NO: 70), where the mutant CBS has an A residue at position 11 of the consensus CBS motif; or the amino acid sequence AMLKR (SEQ ID NO: 71), HMLTR (SEQ ID NO: 72), AMLRR (SEQ ID NO: 73), or TMLRR (SEQ ID NO: 74), where the mutant CBS has a C residue at position 11 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a T, A, or C residue at position 13 of the consensus CBS motif, the engineered CTCF at ZF3 positions +2 to +6 including: the amino acid sequence QQLIV (SEQ ID NO: 75), SQLIV (SEQ ID NO: 76), QQLLV (SEQ ID NO: 77), GELVV (SEQ ID NO: 78), or QQLLI (SEQ ID NO: 79), where the mutant CBS has a T residue at position 13 of the consensus CBS motif; the amino acid sequence GQLIV (SEQ ID NO: 80), GQLTV (SEQ ID NO: 81), GKLVT (SEQ ID NO: 187), TELII (SEQ ID NO: 82) or QGLLV (SEQ ID NO: 83), where the mutant CBS has an A residue at position 13 of the consensus CBS motif; or the amino acid sequence QQLLT (SEQ ID NO: 84), GQLLT (SEQ ID NO: 85), GELLT (SEQ ID NO: 86), or QQLLI (SEQ ID NO: 79), where the mutant CBS has a C residue at position 13 of the consensus CBS motif.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has A, G, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF including: (i) the amino acid sequence AKLKK (SEQ ID NO: 88), AKLRK (SEQ ID NO: 89), AHLRV (SEQ ID NO: 90), AKLRV (SEQ ID NO: 61), or SKLRL (SEQ ID NO: 92) at ZF4 positions +2 to +6 of the engineered CTCF; (ii) the amino acid sequence ERLRV (SEQ ID NO: 93), NRLKV (SEQ ID NO: 94), SRLKE (SEQ ID NO: 44), or NRLKV (SEQ ID NO: 94) at ZF5 positions +2 to +6 of the engineered CTCF; (iii) the amino acid sequence RPDT (SEQ ID NO: 38), RTET (SEQ ID NO: 98), or RADV (SEQ ID NO: 99) at ZF6 positions −1 to +3 of the engineered CTCF; and (iv) the amino acid sequence DNLLA (SEQ ID NO: 100), SNLLV (SEQ ID NO: 101), DNLMA (SEQ ID NO: 102), or DNLRV (SEQ ID NO: 103) at ZF7 positions +2 to +6 of the engineered CTCF.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has G, G, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF including: (i) the amino acid sequence GHLKK (SEQ ID NO: 158), AHLRK (SEQ ID NO: 60), or GKLRI (SEQ ID NO: 106) at ZF4 positions +2 to +6 of the engineered CTCF; (ii) the amino acid sequence SRLKE (SEQ ID NO: 44), DALRR (SEQ ID NO: 108), DGLKR (SEQ ID NO: 109), or TRLRE (SEQ ID NO: 110) at ZF5 positions +2 to +6 of the engineered CTCF; (iii) the amino acid sequence at RPDTMKR (SEQ ID NO: 188) or RTENMKM (SEQ ID NO: 189) at ZF6 positions −1 to +6 of the engineered CTCF; and (iv) the amino acid sequence EHLKV (SEQ ID NO: 13), DHLLA (SEQ ID NO: 114), or HHLDV (SEQ ID NO: 115) at ZF7 positions +2 to +6 of the engineered CTCF.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has A, G, and A residues at positions 2, 5, and 11 of the consensus CBS motif, respectively, the engineered CTCF including: (i) the amino acid sequence SNLRR (SEQ ID NO: 116), GNLVR (SEQ ID NO: 117), GNLRR (SEQ ID NO: 118), GNLKR (SEQ ID NO: 119), ANLRR (SEQ ID NO: 69), NNLRR (SEQ ID NO: 121), or TNLRR (SEQ ID NO: 68) at ZF4 positions +2 to +6 of the engineered CTCF; (ii) the amino acid sequence EHMKR (SEQ ID NO: 123), EHMAR (SEQ ID NO: 34), THMKR (SEQ ID NO: 33), EHMNR (SEQ ID NO: 126), or EHMAR (SEQ ID NO: 127) at ZF6 positions +2 to +6 of the engineered CTCF; and (iii) the amino acid sequence DNLLT (SEQ ID NO: 128), DNLLV (SEQ ID NO: 129), DNLQT (SEQ ID NO: 130), DNLLA (SEQ ID NO: 100), DNLAT (SEQ ID NO: 132), DNLQA (SEQ ID NO: 133), DNLMA (SEQ ID NO: 102), or DNLMT (SEQ ID NO: 135) at ZF7 positions +2 to +6 of the engineered CTCF.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has G, G, and A residues at positions 2, 5, and 11 of the consensus CBS motif, respectively, the engineered CTCF including: (i) the amino acid sequence GNLVR (SEQ ID NO: 117), GNLRR (SEQ ID NO: 118), GNLAR (SEQ ID NO: 138), GNLMR (SEQ ID NO: 139), ANLRR (SEQ ID NO: 69), SNLRR (SEQ ID NO: 116), or NNLRR (SEQ ID NO: 121) at ZF4 positions +2 to +6 of the engineered CTCF; (ii) the amino acid sequence EHMNR (SEQ ID NO: 126), EHMKR (SEQ ID NO: 123), EHMRR (SEQ ID NO: 34), SHMNR (SEQ ID NO: 146), SHMRR (SEQ ID NO: 147), THMKR (SEQ ID NO: 33), or DHIVINR (SEQ ID NO: 32) at ZF6 positions +2 to +6 of the engineered CTCF; and (iii) the amino acid sequence EHLKV (SEQ ID NO: 13), EHLAE (SEQ ID NO: 151), STLNE (SEQ ID NO: 152), DHLQV (SEQ ID NO: 12), EHLNV (SEQ ID NO: 9), DHLNT (SEQ ID NO: 155), EHLQA (SEQ ID NO: 156), or HHLMH (SEQ ID NO: 157) at ZF7 positions +2 to +6 of the engineered CTCF.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has G, T, and T residues at positions 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF including: (i) the amino acid sequence GHLKK (SEQ ID NO: 158), AHLKK (SEQ ID NO: 159), TKLRL (SEQ ID NO: 160), TKLKL (SEQ ID NO: 161), GHLRK (SEQ ID NO: 162), THLKK (SEQ ID NO: 163), or AHLRK (SEQ ID NO: 60) at ZF4 positions +2 to +6 of the engineered CTCF; (ii) the amino acid sequence TRLKE (SEQ ID NO: 165) or SRLKE (SEQ ID NO: 44) at ZF5 positions +2 to +6 of the engineered CTCF; and (iii) the amino acid sequence RADN (SEQ ID NO: 167), RHDT (SEQ ID NO: 40), RRDT (SEQ ID NO: 169), RPDT (SEQ ID NO: 38), RTSS (SEQ ID NO: 171), or RNDT (SEQ ID NO: 172) at ZF6 positions −1 to +3 of the engineered CTCF.
In some embodiments, the engineered CTCF variant includes at least one amino acid residue in at least one zinc finger that differs in sequence from the amino acid sequence of a wild-type CTCF, where the engineered CTCF variant binds to a mutant CTCF binding sequence (CBS) with a higher affinity than wild-type CTCF, the mutant CBS including at least one nucleotide base that differs in sequence from the nucleotide sequence of a consensus CBS, where the at least one amino acid residue that differs in sequence from the amino acid sequence of a wild-type CTCF is selected from the group consisting of the amino acid residues at the position(s) −1, +1, +2, +3, +5, and +6 of any of ZF7, ZF6, ZF5, ZF4, and ZF3 of the engineered CTCF variant.
In some embodiments, the engineered CCCTC-binding factor (CTCF) variant that binds with a higher affinity than a wild-type CTCF to a mutant CTCF binding sequence (CBS) that differs from a consensus CBS at position 2 of the consensus CBS motif, the engineered CTCF including an amino acid residue threonine, asparagine, or histidine at ZF7 +3 position.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that has a C-to-G mutation at position 2 of the consensus CBS motif, the engineered CTCF including the amino acid sequence DHLQT (SEQ ID NO: 8), EHLNV (SEQ ID NO: 9), AHLQV (SEQ ID NO: 10), EHLRE (SEQ ID NO: 11), DHLQV (SEQ ID NO: 12), EHLKV (SEQ ID NO: 13), DHLQV (SEQ ID NO: 12), EHLVV (SEQ ID NO: 15), DHLRT (SEQ ID NO: 16), DHLAT (SEQ ID NO: 17), or DHLQT (SEQ ID NO: 8) at ZF7 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 3 of the consensus CBS motif, the engineered CTCF including the amino acid sequence RKHD (SEQ ID NO: 173), RRSD (SEQ ID NO: 174), GIVN (SEQ ID NO: 178), ELLN (SEQ ID NO: 179), or PHRM (SEQ ID NO: 181) at ZF7 positions −1 to +3.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 5 of the consensus CBS motif, the engineered CTCF including the amino acid sequence NAMKR (SEQ ID NO: 30), EHMGR (SEQ ID NO: 31), DHMNR (SEQ ID NO: 32), THMKR (SEQ ID NO: 33), EHMRR (SEQ ID NO: 34), or THMNR (SEQ ID NO: 35) at ZF6 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 6 of the consensus CBS motif, the engineered CTCF including the amino acid sequence MNES (SEQ ID NO: 36), HRES (SEQ ID NO: 37), RPDT (SEQ ID NO: 38), RTDI (SEQ ID NO: 39), or RHDT (SEQ ID NO: 40) at ZF6 positions −1 to +3.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 7 of the consensus CBS motif, the engineered CTCF including the amino acid sequence HGLKV (SEQ ID NO: 41), HRLKE (SEQ ID NO: 42), HALKV (SEQ ID NO: 43), SRLKE (SEQ ID NO: 44), DGLRV (SEQ ID NO: 45), HTLKV (SEQ ID NO: 46), or NRLKE (SEQ ID NO: 47) at ZF5 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 8 of the consensus CBS motif, the engineered CTCF including the amino acid sequence ATLKR (SEQ ID NO: 48), QALRR (SEQ ID NO: 49), GGLVR (SEQ ID NO: 50), HGLIR (SEQ ID NO: 51), ANLSR (SEQ ID NO: 52), TGLTR (SEQ ID NO: 53), HGLVR (SEQ ID NO: 54), GGLTR (SEQ ID NO: 55), HTLRR (SEQ ID NO: 56), TVLKR (SEQ ID NO: 57), ADLKR (SEQ ID NO: 58), or HGLRR (SEQ ID NO: 59) at ZF5 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 10 of the consensus CBS motif, the engineered CTCF including the amino acid sequence AHLRK (SEQ ID NO: 60), AKLRV (SEQ ID NO: 61), GGLGL (SEQ ID NO: 62), AKLRI (SEQ ID NO: 63), TKLKV (SEQ ID NO: 64), or SKLRV (SEQ ID NO: 65) at ZF4 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 11 of the consensus CBS motif, the engineered CTCF including the amino acid sequence ATLRR (SEQ ID NO: 66), RRLDR (SEQ ID NO: 67), TNLRR (SEQ ID NO: 68), ANLRR (SEQ ID NO: 69), GNLTR (SEQ ID NO: 70), AMLKR (SEQ ID NO: 71), HMLTR (SEQ ID NO: 72), AMLRR (SEQ ID NO: 73), or TMLRR (SEQ ID NO: 74) at ZF4 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at position 13 of the consensus CBS motif, the engineered CTCF including the amino acid sequence QQLIV (SEQ ID NO: 75), SQLIV (SEQ ID NO: 76), QQLLV (SEQ ID NO: 77), GELVV (SEQ ID NO: 78), QQLLI (SEQ ID NO: 79), GQLIV (SEQ ID NO: 80), GQLTV (SEQ ID NO: 81), TELII (SEQ ID NO: 82), QGLLV (SEQ ID NO: 83), QQLLT (SEQ ID NO: 84), GQLLT (SEQ ID NO: 85), GELLT (SEQ ID NO: 86), or QQLLI (SEQ ID NO: 79) at ZF3 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at positions 2, 6, 7, and 10 of the consensus CBS motif, the engineered CTCF including:
(i) the amino acid sequence AKLKK (SEQ ID NO: 88), AKLRK (SEQ ID NO: 89), AHLRV (SEQ ID NO: 90), AKLRV (SEQ ID NO: 61), or SKLRL (SEQ ID NO: 92) at ZF4 positions +2 to +6;
(ii) the amino acid sequence ERLRV (SEQ ID NO: 93), NRLKV (SEQ ID NO: 94), SRLKE (SEQ ID NO: 44), or NRLKV (SEQ ID NO: 94) at ZF5 positions +2 to +6;
(iii) the amino acid sequence RPDT (SEQ ID NO: 38), RTET (SEQ ID NO: 98), or RADV (SEQ ID NO: 99) at ZF6 positions −1 to +3; and
(iv) the amino acid sequence DNLLA (SEQ ID NO: 100), SNLLV (SEQ ID NO: 101), DNLMA (SEQ ID NO: 102), or DNLRV (SEQ ID NO: 103) at ZF7 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at positions 2, 6, 7, and 10 of the consensus CBS motif, the engineered CTCF including:
(i) the amino acid sequence GHLKK (SEQ ID NO: 158), AHLRK (SEQ ID NO: 60), or GKLRI (SEQ ID NO: 106) at ZF4 positions +2 to +6;
(ii) the amino acid sequence SRLKE (SEQ ID NO: 44), DALRR (SEQ ID NO: 108), DGLKR (SEQ ID NO: 109), or TRLRE (SEQ ID NO: 110) at ZF5 positions +2 to +6;
(iii) the amino acid sequence at RPDTMKR (SEQ ID NO: 188) or RTENMKM (SEQ ID NO: 189) at ZF6 positions −1 to +36; and
(iv) the amino acid sequence EHLKV (SEQ ID NO: 13), DHLLA (SEQ ID NO: 114), or HHLDV (SEQ ID NO: 115) at ZF7 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at positions 2, 5, and 11 of the consensus CBS motif, the engineered CTCF including:
(i) the amino acid sequence SNLRR (SEQ ID NO: 116), GNLVR (SEQ ID NO: 117), GNLRR (SEQ ID NO: 118), GNLKR (SEQ ID NO: 119), ANLRR (SEQ ID NO: 69), NNLRR (SEQ ID NO: 121), or TNLRR (SEQ ID NO: 68) at ZF4 positions +2 to +6;
(ii) the amino acid sequence EHMKR (SEQ ID NO: 123), EHMRR (SEQ ID NO: 34), THMKR (SEQ ID NO: 33), EHMNR (SEQ ID NO: 126), or EHMAR (SEQ ID NO: 127) at ZF6 positions +2 to +6; and
(iii) the amino acid sequence DNLLT (SEQ ID NO: 128), DNLLV (SEQ ID NO: 129), DNLQT (SEQ ID NO: 130), DNLLA (SEQ ID NO: 100), DNLAT (SEQ ID NO: 132), DNLQA (SEQ ID NO: 133), DNLMA (SEQ ID NO: 102), or DNLMT (SEQ ID NO: 135) at ZF7 positions +2 to +6.
In some embodiments, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at positions 2, 5, and 11 of the consensus CBS motif, the engineered CTCF including:
(i) the amino acid sequence GNLVR (SEQ ID NO: 117), GNLRR (SEQ ID NO: 118), GNLAR (SEQ ID NO: 138), GNLMR (SEQ ID NO: 139), ANLRR (SEQ ID NO: 69), SNLRR (SEQ ID NO: 116), or NNLRR (SEQ ID NO: 121) at ZF4 positions +2 to +6;
(ii) the amino acid sequence EHMNR (SEQ ID NO: 126), EHMKR (SEQ ID NO: 123), EHMRR (SEQ ID NO: 34), SHMNR (SEQ ID NO: 146), SHMRR (SEQ ID NO: 147), THMKR (SEQ ID NO: 33), or DHMNR (SEQ ID NO: 32) at ZF6 positions +2 to +6; and
(iii) the amino acid sequence EHLKV (SEQ ID NO: 13), EHLAE (SEQ ID NO: 151), STLNE (SEQ ID NO: 152), DHLQV (SEQ ID NO: 12), EHLNV (SEQ ID NO: 9), DHLNT (SEQ ID NO: 155), EHLQA (SEQ ID NO: 156), or HHLMH (SEQ ID NO: 157) at ZF7 positions +2 to +6.
In one embodiment, the engineered CTCF variant binds with a higher affinity than a wild-type CTCF to a mutant CBS that differs from a consensus CBS at positions 6, 7, and 10 of the consensus CBS motif, the engineered CTCF including:
(i) the amino acid sequence GHLKK (SEQ ID NO: 158), AHLKK (SEQ ID NO: 159), TKLRL (SEQ ID NO: 160), TKLKL (SEQ ID NO: 161), GHLRK (SEQ ID NO: 162), THLKK (SEQ ID NO: 163), or AHLRK (SEQ ID NO: 60) at ZF4 positions +2 to +6;
(ii) the amino acid sequence TRLKE (SEQ ID NO: 165) or SRLKE (SEQ ID NO: 44) at ZF5 positions +2 to +6; and
(iii) the amino acid sequence RADN (SEQ ID NO: 167), RHDT (SEQ ID NO: 40), RRDT (SEQ ID NO: 169), RPDT (SEQ ID NO: 38), RTSS (SEQ ID NO: 171), or RNDT (SEQ ID NO: 172) at ZF6 positions −1 to +3.
In some embodiments, the engineered CTCF variant interacts with cohesion to mediate the formation of an enhancer-promoter loop to modulate gene expression.
In another aspect, the invention features a method of treating a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an engineered CTCF variant described herein.
In some embodiments, the subject can have cancer.
In another aspect, the invention features a method of activating or repressing expression of a gene which is under the control of a CBS bearing one or more mutations, the method including contacting the engineered CTCF according to any one of claims 1-15 with a sequence of interest in the gene, such that the expression of the gene is regulated.
In another aspect, the invention features a pharmaceutical composition including an engineered CTCF variant described herein.
In another aspect, the invention features a gene expression system for regulation of a gene, the system including a nucleic acid encoding an engineered CTCF variant according described herein.
In another aspect, the invention features a method of altering the structure of chromatin including contacting an engineered CTCF variant according to any one of claims 1-15 with a sequence of interest to form a binding complex, such that the structure of the chromatin is altered.
In another aspect, the invention features a method of activating or repressing expression of a gene which is under the control of a CBS bearing one or more mutations, the method including contacting the CBS bearing one or more mutations with an engineered CTCF variant described herein.
In another aspect, the invention features a kit including an engineered CTCF variant described herein.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiment described, may be understood in conjunction with the accompanying figures, incorporated herein by reference.

FIG. 1: Diagram of an exemplary 11-finger CTCF zinc finger array protein-DNA interactions at the CTCF binding site. Each zinc finger of the 11-finger array contained a recognition alpha-helix where protein-DNA base contacts were made by amino acids in position −1, 2, 3 and 6 of each alpha-helix. Here, position −1, 3, and 6 were only depicted as positon 2 makes a cross strand contact with the opposite strand of the binding site that is not shown here. The sequence for the binding site was derived from ChIP-seq data (Nakahashi et al., 2013). The binding site was partitioned into three segments: 5′ flanking (gray-line), core (black-line), and 3′ flanking (light gray line). The position of each nucleotide within each segment are numbered. Dashes indicate known DNA-protein contacts (black) and theoretical DNA-protein contacts (gray) between the zinc finger array and the binding site. Zinc fingers 3-7 of the array (white) make protein-DNA contacts with the core sequence (bold, black lined). There was a possible 5-6 base pair gap (represented by horizontal dashed lines) between zinc finger 8 and zinc fingers 9-11 as suggested by ChIP-exo and DNAse I footprinting of CTCF bound DNA fragments (Hashimoto, H. et al., 2017). Note CTCF binds to its target site in the 3′-5′ direction with the N-terminal side of the protein binding to the 3′ end of the binding site. FIG. 1 discloses SEQ ID NO: 5544.

FIG. 2: Diagram of B2H Beta-galactosidase reporter assay. The B2H reporter assay used Gal11P-mediated recruitment of Gal4 to indicate binding. E. coli is transformed with two plasmids: one plasmid encoded for both a zinc finger-Gal11P fusion and an alpha N-terminal domain of RNA polymerase (α-NTD)-Gal4 fusion; the second plasmid contained a modifiable binding sequence upstream of a weak promoter that drives the expression of the lacZ gene, which encodes for β-galactosidase. A zinc finger-Gal11P fusion that was able to bind to the target sequence recruited the α-NTD-Gal4 fusion to the promoter, thereby inducing the expression of lacZ. This increase in β-galactosidase levels was detected by a simple colorimetric ONPG-based assay. The CTCF zinc finger array-gal11P fusion was bound to a CTCF binding site in this diagram, recruiting the α-NTD-Gal4 fusion to the promoter region upstream of lacZ, leading to expression.

FIG. 3: Fold activation in the B2H B-gal assay was greatest when CTCF zinc fingers 1-11 of 11 finger array interacts with full length target site. Five target sites (sequence indicated in the legend) were tested along with the full CTCF zinc finger array and four different subsets (indicated on the x-axis). The core sequence (black and bolded) which is the most highly conserved sequence of CTCF binding sites was tested independently and with different quantities of flanking sequence as derived from Hashimoto, H. et al. Mol. Cell. 2017 (black and light gray); Persikov, A and Singh, M. NAR. 2014 (medium gray); and Nakahashi, H. et al., Cell Rep. 2013 (very light gray and dark gray). Positive control reflects binding activity of a known 3-finger zinc finger that binds strongly in bacterial and human contexts to a known sequence. The negative control reflects baseline beta-galactosidase levels when the alpha N-terminal domain of RNA polymerase (α-NTD)-Gal4 fusion is not directly recruited to the promoter of lacZ. This baseline was used to calculate fold activation when the CTCF zinc finger array is fused to gal11P. FIG. 3 discloses SEQ ID NOS 5545-5548 and 5544, respectively, in order of appearance.

FIG. 4: CTCF zinc finger array is sensitive to sequence changes at certain positions of the core region within the CTCF binding site. Each of the four possible nucleotides at each position of the 40 bp reference CBS were tested for ability to bind the CTCF zinc finger array in the B2H y. Fold activation reflects binding activity above background β-galactosidase levels (Background β-gal levels are obtained from the levels of β-gal from samples with each binding site in the presence of the gal4-RNA polymerase fusion with no zinc finger array fused to gal11P). The reference sequence above is partitioned into three segments: 5′ flanking (dark gray lined), core (black lined), and 3′ flanking (gray lined). The position of each nucleotide within each segment are numbered. Dashes indicate known DNA-protein contacts (black) and theoretical DNA-protein contacts (gray) between the zinc finger array and the binding site. Core sequence 1-15 of the binding site (black, bold) interacts with zinc finger 3-7 of the array (white, black outline) and appear to be most sensitive to changes in the binding sequence. Alterations to the 5′ flanking sequence as well as the 3′ flanking sequence did not negatively impact binding. FIG. 4 discloses SEQ ID NO: 5544.

FIG. 5: Maximizing binding potential of the CTCF binding site. Modifications were made to the reference binding site (bottom) to combine nucleotide changes that, individually, showed increased binding activity of the CTCF zinc finger array. The core sequence motif is bold while changes made are underlined. Binding activity of the 11-finger CTCF zinc finger array was quantified in the B2H Beta-galactosidase reporter assay in triplicate. Fold activation reflects binding activity above background levels when no DNA binding protein is present. FIG. 5 discloses SEQ ID NOS 5549-5550 and 5544, respectively, in order of appearance.

FIG. 6: Diagram of B2H Beta-lactamase inhibitor selection. The selection system contained the same components as the reporter system except successful binding of the zinc finger array to the CBS drove BlaC expression, an inhibitor of the beta-lactamase class of antibiotics, instead of lacZ. Expression of BlaC allowed for growth on Carbenicillin plates. The selection was driven by the addition of Clavulanic acid, an inhibitor of beta lactamase inhibitors. Low level expression of BlaC can result in growth on Carbenicillin plates, but the addition of clavulanic acid inhibits BlaC activity and results in the depletion of false positives and further enrichment of strong binders to any modification made to the binding site. Libraries of mutations in the zinc finger array fused to gal11P were selected for binders to an altered binding sequence through low stringency conditions followed by selection on a gradient of clavulanic acid. Growth on the highest stringency end of the gradient indicated variants in the zinc finger array that are strong binders to the new binding sequence.

FIGS. 7A-7C: Binding activity of variants on altered CTCF binding sites. Variants picked from the high stringency gradient of the selective plates were tested for binding activity on sequences representing all four possible nucleotides at position 2 of the core sequence (gray star). Amino acid sequence of variants pulled out of the selection were listed above the heat map and the nucleotide present at position 2 of the core sequence was indicated on the y-axis. FIG. 7A: The nucleotide at position 2 is T. FIG. 7B: The nucleotide at position 2 is A. FIG. 7C: The nucleotide at Binding was quantified by the beta-galactosidase reporter system and colorimetric ONPG assay. Binding activity of wild-type CTCF zinc finger array on the wild-type binding site sequence was indicated by the white dot. A diagram of the ZF7 alpha recognition helix for each nucleotide change is on the left. It included the amino acid residues interacting with the triplet in the binding sequence. The amino acid at position 3 of the alpha helix was varied in the library and is indicated by an ‘X’. FIGS. 7A-C disclose “RKSXLGV” as SEQ ID NO: 5551.

FIG. 8: Increasing the variation within the recognition helix produced stronger binders. Four amino acids were targeted for variance in the library to allow for more flexibility in the selection and generate stronger binders to the modified binding site of choice. ZF7 targeting a C:G change at position 2 (gray star) of the core sequence was selected for variants using the expanded approach. Each amino acid codon was replaced with ‘VNS’ codons at the indicated sites (‘X’). Twelve colonies were picked from the high-stringency end of the selection and tested for their ability to bind to the CTCF binding site when the indicated nucleotide is at positon 2 of core sequence. Amino acid sequence of the variants selected are listed on the x-axis and the nucleotide at position two of the core sequence is on the y-axis. Wild-type zinc finger array binding activity on wild-type binding sequence is indicated by the white dot. FIG. 8 discloses “RKSXLGV” as SEQ ID NO: 5551, “AHLQV” as SEQ ID NO: 10, “DHLRT” as SEQ ID NO: 16, “DHLAT” as SEQ ID NO: 17, “DHLQT” as SEQ ID NO: 8, “DHLQV” as SEQ ID NO: 12, “SDLGV” as SEQ ID NO: 5552, “EHLKV” as SEQ ID NO: 13, “EHLVV” as SEQ ID NO: 15, “EHLNV” as SEQ ID NO: 9 and “EHLRE” as SEQ ID NO: 11.

FIGS. 9A-9C: Selected variants binding altered binding sites sequence at position 3 of core motif in CBS. Selections performed on library of variants centered around alterations in position −1 to 3 of recognition helix in ZF7 of the 11 finger CTCF zinc finger array. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 3 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by dashed lines. (A) Selections performed on A:T change in the binding site, (B) A:G change, (C) A:C change. Most variants pulled out had relaxed binding specificity instead of altered specificity. FIGS. 9A-C disclose “RKSD” as SEQ ID NO: 711, “RKHD” as SEQ ID NO: 173, “RRSD” as SEQ ID NO: 174, “RKAD” as SEQ ID NO: 175, “IPRI” as SEQ ID NO: 176, “RKDD” as SEQ ID NO: 177, “QALL” as SEQ ID NO: 180, “PHRM” as SEQ ID NO: 181, “ELLN” as SEQ ID NO: 179 and “GIVN” as SEQ ID NO: 178.

FIGS. 10A-10B: Selections performed targeting sequence changes at position 5 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 2 to 6 of the ZF6 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 5 of the core motif in the core motif of the CBS (gray star). Direct protein-DNA contacts were indicated by dashed lines. (A) Selections performed on C:T change in the binding site, (B) C:G change. No variants grew beyond the low stringency end of the gradient on selection plates for C:A change and were considered weak/insufficient binders. Most variants pulled out had relaxed binding specificity instead of altered specificity with the exception of THMKR′ (SEQ ID NO: 33) targeting C:G change in the binding sequence. FIGS. 10A-B disclose “GNMAR” as SEQ ID NO: 182, “NAMKR” as SEQ ID NO: 30, “EGMTR” as SEQ ID NO: 183, “NAMRG” as SEQ ID NO: 185, “GTMKM” as SEQ ID NO: 1255, “SNMVR” as SEQ ID NO: 184, “DHMNR” as SEQ ID NO: 32, “EHMRR” as SEQ ID NO: 34, “EHMGR” as SEQ ID NO: 31, “THMNR” as SEQ ID NO: 35 and “THMKR” as SEQ ID NO: 33.

FIGS. 11A-11C: Selections performed targeting sequence changes at position 6 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position −1 to 3 of ZF6 recognition helix. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 6 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by dashed lines. (A) Selections performed on A:T change in the binding site, (B) A:G change, (C) A:C change. Variants analyzed from the A:T selection had relaxed binding profile while variants from A:G selection showed strong binding for only the changed nucleotide. No good binders were identified in the A:C selection. FIGS. 11A-C disclose “MMES” as SEQ ID NO: 36, “QSGT” as SEQ ID NO: 1582, “HRES” as SEQ ID NO: 37, “RHDT” as SEQ ID NO: 40, “RPDT” as SEQ ID NO: 38, “RTDI” as SEQ ID NO: 39, “RADN” as SEQ ID NO: 167 and “ERKS” as SEQ ID NO: 1479.

FIGS. 12A-12C: Selections performed targeting sequence changes at position 7 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 4 to 6 of ZF5 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 7 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by a line. (A) Selections performed on G:T change in the binding site, (B) G:A change, (C) G:C change. FIGS. 12A-C disclose “DGLRV” as SEQ ID NO: 45, “HGLKV” as SEQ ID NO: 41, “HRLKE” as SEQ ID NO: 42, “HALKV” as SEQ ID NO: 43, “YKLKR” as SEQ ID NO: 5553, “SRLKE” as SEQ ID NO: 44, “HTLKV” as SEQ ID NO: 46 and “NRLKE” as SEQ ID NO: 47.

FIGS. 13A-13C: Selections performed targeting sequence changes at position 8 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 2 to 6 of ZF5 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 8 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by a line. (A) Selections performed on G:T change in the binding site, (B) G:A change, (C) G:C change. Note the different variants that appear with the same library being used to bind to the same changes in the sequence, but in a different position on the binding site. FIGS. 13A-13C disclose “GGLVR” as SEQ ID NO: 50, “QALRR” as SEQ ID NO: 49, “HGLIR” as SEQ ID NO: 51, “YKLKR” as SEQ ID NO: 5553, “ATLKR” as SEQ ID NO: 48, “GGLTR” as SEQ ID NO: 55, “HGLVR” as SEQ ID NO: 54, “ANLSR” as SEQ ID NO: 52, “TGLTR” as SEQ ID NO: 53, “HGLRR” as SEQ ID NO: 59, “ADLKR” as SEQ ID NO: 58, “HTLRR” as SEQ ID NO: 56 and “TVLKR” as SEQ ID NO: 57.

FIGS. 14A-14C: Selections performed targeting sequence changes at position 10 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 2 to 6 of ZF4 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 10 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by a line. (A) Selections performed on G:T change in the binding site, (B) G:A change, (C) G:C change. G:C selection did not produce any growth at the high stringency end of the gradient selective plates. Binding data reflects colonies picked from mid-tier region, which is why they did not perform well as binders. White dot indicates wild-type CTCF zinc finger array binding activity on wild-type binding sequence. FIGS. 14A-C disclose “GHLRK” as SEQ ID NO: 162, “AKLRL” as SEQ ID NO: 3311, “AHLRK” as SEQ ID NO: 60, “SKLKR” as SEQ ID NO: 3470, “GGLGL” as SEQ ID NO: 62, “AKLRI” as SEQ ID NO: 63, “AKLRV” as SEQ ID NO: 61, “EKLRI” as SEQ ID NO: 186, “SKLRV” as SEQ ID NO: 65 and “TKLKV” as SEQ ID NO: 64.

FIGS. 15A-15C: Selections performed targeting sequence changes at position 11 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 2 to 6 of ZF4 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 11 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by a line. (A) Selections performed on G:T change in the binding site, (B) G:A change, (C) G:C change. FIGS. 15A-C disclose “RRLDR” as SEQ ID NO: 67, “SKLKR” as SEQ ID NO: 3470, “ATLRR” as SEQ ID NO: 66, “GNLTR” as SEQ ID NO: 70, “ANLRR” as SEQ ID NO: 69, “TNLRR” as SEQ ID NO: 68, “AMLRR” as SEQ ID NO: 73, “AMLKR” as SEQ ID NO: 71, “HMLTR” as SEQ ID NO: 72 and “TMLRR” as SEQ ID NO: 74.

FIGS. 16A-16C: Selections performed targeting sequence changes at position 13 of the core motif in the CBS. Selections performed on library of variants centered around alterations in position 2 to 6 of ZF3 recognition helix, leaving the 4th position unchanged. ‘VNS’ codons were introduced at positions indicated by ‘X’ and selected against three different nucleotide changes at position 13 of the core motif in the CBS (gray star). Direct protein-DNA contacts are indicated by a line. (A) Selections performed on G:T change in the binding site, (B) G:A change, (C) G:C change. FIGS. 16A-C disclose “QQLLI” as SEQ ID NO: 79, “QQLLV” as SEQ ID NO: 77, “QQLIV” as SEQ ID NO: 75, “GELVV” as SEQ ID NO: 78, “GELVR” as SEQ ID NO: 5554, “SQLIV” as SEQ ID NO: 76, “QGLLV” as SEQ ID NO: 83, “GQLTV” as SEQ ID NO: 81, “GQLIV” as SEQ ID NO: 80, “GKLVT” as SEQ ID NO: 187, “TELII” as SEQ ID NO: 82, “GQLLT” as SEQ ID NO: 85, “QQLLT” as SEQ ID NO: 84, “GELLT” as SEQ ID NO: 86 and “ATLAD” as SEQ ID NO: 5555.

FIG. 17: Binding activity of multi-finger variants on multiple sequence changes to the CBS. Diagram of the recognition helices of zinc finger 4-7 out of the 11 finger array, binding to their respective triplets in the core motif of the CBS. Altered amino acids are indicated by ‘X’ and nucleotide changes to the wild-type CBS are indicated by a gray star in the diagram and by bolded letters. ZF1-3 and ZF8-11 were unmodified in this library Protein-DNA contacts are indicated by lines between the ZF recognition helices and the CBS sequence. Wild-type CTCF 11-finger zinc finger array binding strength to wild-type CBS is indicated by a white dot. The amino acid sequence of each variant recognition helix in ZF4-7 are listed on the y-axis and binding activity on the modified CBS (changes in red) or the wild-type CBS are reflected by B2H β-gal reporter assay. FIG. 17 discloses “CGTGGTGCGAAC” as SEQ ID NO: 5556, “CAAGCGTGGTGCGCT” as SEQ ID NO: 5557, “CCAGCAGGGGGCGCT” as SEQ ID NO: 5558, “ERLRV” as SEQ ID NO: 93, “RPDT” as SEQ ID NO: 38, “DNLLA” as SEQ ID NO: 100, “AKLKK” as SEQ ID NO: 88, “AKLRK” as SEQ ID NO: 89, “NRLKV” as SEQ ID NO: 94, “RTET” as SEQ ID NO: 98, “SNLLV” as SEQ ID NO: 101, “AHLRV” as SEQ ID NO: 90, “SRLKE” as SEQ ID NO: 44, “DNLMA” as SEQ ID NO: 102, “AKLRV” as SEQ ID NO: 61, “SKLRL” as SEQ ID NO: 92, “RADV” as SEQ ID NO: 99 and “DNLRV” as SEQ ID NO: 103.

FIG. 18: Binding activity of multi-finger variants on multiple sequence changes to the CBS. The same selection as before except now there is a C:G change at position 2 of the CBS, where previously there was a C:A change. Variants pulled out of this selection had binding activity on the modified CBS without binding to the wild-type CBS. Wild-type 11-finger ZF array only showed binding activity on wild-type CBS (white dot) and no ability to bind to the modified CBS. Interestingly, the dominant variant selected for in the library contained a mutation that occurs at position 9 of the recognition helix that was either introduced during oligo synthesis (0.05% chance of the wrong nucleotide at each position) or through PCR while constructing these libraries. FIG. 18 discloses “CGTGGTGCGAGC” as SEQ ID NO: 5559, “CGAGCGTGGTGCGCT” as SEQ ID NO: 5560, “CCAGCAGGGGGCGCT” as SEQ ID NO: 5558, “GHLKK” as SEQ ID NO: 158, “SRLKE” as SEQ ID NO: 44, “EHLKV” as SEQ ID NO: 13, “RPDT(MK)R” as SEQ ID NO: 5561, “AHLRK” as SEQ ID NO: 60, “DALRR” as SEQ ID NO: 108, “RTEN” as SEQ ID NO: 112, “DHLLA” as SEQ ID NO: 114, “DGLKR” as SEQ ID NO: 109, “RPDT” as SEQ ID NO: 38, “HHLDV” as SEQ ID NO: 115, “GKLRI” as SEQ ID NO: 106 and “TRLRE” as SEQ ID NO: 110.

FIG. 19: Binding activity of multi-finger variants on multiple sequence changes to the CBS. Variants from individual pooled high stringency selections were stitched together and selected against three changes to the wild-type CBS (indicated by gray stars or bolded). Variants were assayed for binding on the modified CBS and the wild-type CBS alongside wild-type CTCF zinc finger array. The variants picked out of the selection were able to bind to the modified CBS, but not the wild-type sequence. Inversely, the wild-type zinc finger array was able to bind to the wild-type CBS, but not the modified one. FIG. 19 discloses “DTYKLKR” as SEQ ID NO: 3, “CAGGGGAGGAAC” as SEQ ID NO: 5562, “CAAGGAGGGGACGCT” as SEQ ID NO: 5563, “CCAGCAGGGGGCGCT” as SEQ ID NO: 5558, “SNLRR” as SEQ ID NO: 116, “EHMKR” as SEQ ID NO: 123, “DNLLT” as SEQ ID NO: 128, “GNLVR” as SEQ ID NO: 117, “EHMRR” as SEQ ID NO: 34, “DNLLV” as SEQ ID NO: 129, “GNLRR” as SEQ ID NO: 118, “THMKR” as SEQ ID NO: 33, “DNLQT” as SEQ ID NO: 130, “GNLKR” as SEQ ID NO: 119, “EHMNR” as SEQ ID NO: 126, “DNLLA” as SEQ ID NO: 100, “ANLRR” as SEQ ID NO: 69, “DNLAT” as SEQ ID NO: 132, “DNLQA” as SEQ ID NO: 133, “NNLRR” as SEQ ID NO: 121, “DNLMA” as SEQ ID NO: 102, “TNLRR” as SEQ ID NO: 68, “EHMAR” as SEQ ID NO: 127 and “DNLMT” as SEQ ID NO: 135.

FIG. 20: Binding activity of multi-finger variants on multiple sequence changes to the CBS. Variants from individual pooled high stringency selections were stitched together and selected against three changes to the wild-type CBS (indicated by gray stars or bolded). Variants were assayed for binding on the modified CBS and the wild-type CBS alongside wild-type CTCF zinc finger array. The variants picked out of the selection were able to bind to the modified CBS, but not the wild-type sequence. Inversely, the wild-type zinc finger array was able to bind to the wild-type CBS, but not the modified one. FIG. 20 discloses “DTYKLKR” as SEQ ID NO: 3, “CAGGGGAGGAGC” as SEQ ID NO: 5564, “CGAGGAGGGGACGCT” as SEQ ID NO: 5565, “CCAGCAGGGGGCGCT” as SEQ ID NO: 5558, “GNLVR” as SEQ ID NO: 117, “EHMNR” as SEQ ID NO: 126, “EHLKV” as SEQ ID NO: 13, “GNLRR” as SEQ ID NO: 118, “EHMKR” as SEQ ID NO: 123, “EHLAE” as SEQ ID NO: 151, “GNLAR” as SEQ ID NO: 138, “EHMRR” as SEQ ID NO: 34, “STLNE” as SEQ ID NO: 152, “GNLMR” as SEQ ID NO: 139, “SHMNR” as SEQ ID NO: 146, “DHLQV” as SEQ ID NO: 12, “ANLRR” as SEQ ID NO: 69, “SHMRR” as SEQ ID NO: 147, “EHLNV” as SEQ ID NO: 9, “SNLRR” as SEQ ID NO: 116, “DHLNT” as SEQ ID NO: 155, “EHLQA” as SEQ ID NO: 156, “NNLRR” as SEQ ID NO: 121, “THMKR” as SEQ ID NO: 33, “DHMNR” as SEQ ID NO: 32 and “HHLMH” as SEQ ID NO: 157.

FIG. 21: Binding activity of multi-finger variants on multiple sequence changes to the CBS. Variants from individual pooled high stringency selections were stitched together and selected against three changes to the wild-type CBS (indicated by gray stars or bolded). Variants were assayed for binding on the modified CBS and the wild-type CBS alongside wild-type CTCF zinc finger array. The variants picked out of the selection were able to bind to the modified CBS, but not the wild-type sequence. Inversely, the wild-type zinc finger array was able to bind to the wild-type CBS (white dot), but not the modified one. FIG. 21 discloses “CGTGGTGCGACC” as SEQ ID NO: 5566, “RKSDLGV” as SEQ ID NO: 5, “CCAGCGTGGTGCGCT” as SEQ ID NO: 5567, “CCAGCAGGGGGCGCT” as SEQ ID NO: 5558, “GHLKK” as SEQ ID NO: 158, “TRLKE” as SEQ ID NO: 165, “RADN” as SEQ ID NO: 167, “AHLKK” as SEQ ID NO: 159, “RHDT” as SEQ ID NO: 40, “TKLRL” as SEQ ID NO: 160, “SRLKE” as SEQ ID NO: 44, “RRDT” as SEQ ID NO: 169, “TKLKL” as SEQ ID NO: 161, “RPDT” as SEQ ID NO: 38, “GHLRK” as SEQ ID NO: 162, “RTSS” as SEQ ID NO: 171, “RNDT” as SEQ ID NO: 172, “THLKK” as SEQ ID NO: 163 and “AHLRK” as SEQ ID NO: 60.

FIG. 22: Wild-type CTCF has binding activity to wild-type CTCF target site and no binding activity to two variant target sites. To confirm endogenous CTCF binds to the wild-type CBSs and not the variant binding sites, as seen in the B2H assay, in a human cell context, we harvested K562 cells, a human erythroleukemia cell line, and examined CTCF binding through ChIP-qPCR. CTCF was assayed for binding to a known CTCF target site and to two endogenous variant binding site sequences using a CTCF specific antibody to enrich for genomic DNA crosslinked to CTCF. Two sets of qPCR primers were designed for each binding site (indicated by 1.1, 1.2, etc). Binding was determined by enrichment of target site above 1% input of crosslinked and sonicated sample not treated with antibody, which is to represent the levels of the site of interest as a fold increase over the frequency of the site of interest in a sample unenriched with antibody. Antibody based enrichment of each sample is quantified by fold enrichment above untreated, and therefore unenriched, input. The negative control reflects background qPCR amplification levels of a target site that CTCF does not bind to. Anything above this negative level is considered enriched indicating CTCF binding while anything below is considered to not be unenriched, and therefore no binding by CTCF. Wild-type CTCF binds to the wild-type target site with no detectable binding to the variant binding sites as predicted by the bacterial B2H reporter assay

FIGS. 23A-23B: Exogenous wild-type and variant CTCF binding activity in human cells. Two endogenous variant binding site sequences, matching one of the five variant binding sites that CTCF variants were selected on, were identified in the human genome (Variant site 1 and Variant site 2). Both wild-type CTCF with a 3×HA tag and one of the 3×HA tagged engineered CTCF variants, selected to bind to the variant binding site sequence of Variant site 1 and Variant site 2, were assayed for binding in human cells through ChIP-qPCR. FIG. 23A: 3×HA tagged wild-type CTCF binds to wild-type CTCF binding site and does not bind to either variant binding site. Human K562 cells were transfected with plasmid expressing 3×HA tagged CTCF and processed with HA antibody to enrich specifically for the exogenous CTCF (3×HA tagged) and not endogenous CTCF (no tag) binding. A negative control is provided to show ChIP-qPCR levels with no enrichment for a region that is not occupied by CTCF. These results demonstrate exogenous wild-type CTCF has the same binding activity as endogenous CTCF. FIG. 23B: 3×HA tagged variant CTCF binds to variant binding sites and does not bind to wild-type CTCF binding site. K562 cells expressing variant CTCF tagged with 3×HA were analyzed by ChIP-qPCR and treated with HA specific antibody. The same sites as in FIGS. 22 and 23A were investigated for variant CTCF binding. The variant CTCF could bind to the variant sites as indicated by enrichment with variant specific HA antibody and no detectable binding was seen at the wild-type binding site as indicated by lack of HA antibody-based enrichment.

FIGS. 24A-24B: Changes in gene expression relative to wild-type control of genes located around variant binding sites. A variant CTCF selected to the G3 binding site sequence and variant CTCF selected to the Other binding site sequence were expressed in wild-type K562s. The variant CTCFs were fused to GFP and RNA was isolated from GFP+ cells 72 hours post nucleofection. cDNA was generated from the RNA and quantified by RT-qPCR. Gene expression levels across samples were normalized to a house keeping gene (HPRT). Changes in gene expression are relative to gene expression levels in wild-type K562s expressing wild-type CTCF tagged with GFP. FIG. 24A. Changes in gene expression of genes around G3 variant binding site in the presence of variant CTCF relative to the wild-type CTCF control. FIG. 24B. Changes in gene expression of genes around Other variant binding site relative to the wild-type control.

FIG. 25: Introduction of variant binding sites upstream of MYC leads to reduction of Endogenous MYC expression. The CTCF binding site ˜2 kb upstream of the MYC TSS was replaced with one of six different sequences used for CTCF variant selections (listed in table). The introduction of these sequences with 4-6 nucleotide changes from the wild-type CTCF binding site sequence result in a reduction of endogenous MYC expression to the same levels as when the CTCF binding site is deleted and loop formation is disrupted. WT_6 sequence has 4 point mutations from the native CTCF binding site, but these changes should have no impact on wild-type CTCF binding as indicated by results from the B2H reporter assay. This appears to be the case as MYC expression levels in the WT_6 cell line are comparable to wild-type K562 MYC expression levels. Because K562 vitality is linked to MYC expression, all variant cell lines were generated in a K562 cell line with exogenous MYC expressed off of a separate PGK promoter (exoMYC.K562). FIG. 25 discloses SEQ ID NOS 5568-5573, respectively, in order of appearance.

FIGS. 26A-26B: Variant CTCFs are able to bind the engineered G3 variant binding site and recover MYC expression. CTCF variants selected to bind to the G3 variant binding site sequence were expressed in the G3_3.K562 cell line. Cells were analyzed for MYC expression and CTCF occupancy on the DNA 72 hours post nucleofection. Residues of ZF helix of the variant and wild-type (indicated by (wt) are listed in the legend. G3 binding site sequence and interacting recognition helix of the CTCF zinc finger array is also diagramed. FIG. 26A. Endogenous MYC levels are recovered to wild-type levels in the G3_3 cell line when CTCF variants are expressed. Endogenous MYC expression levels were quantified by RT-qPCR and are relative to reduced endogenous MYC levels of G3_3 cell line. Endogenous MYC levels from the exoMYC.K562 cell line without any alterations to the CTCF binding site is shown as a positive control (separated by dashed lines). FIG. 26B. CTCF variants are able to bind to the introduced variant binding site in G3_3 cell line while the wild-type CTCF does not. CTCF Ab specific enrichment captures both wild-type and variant CTCF while HA Ab will only detect HA-tagged CTCF (transiently expressed). exoMYC.K562 is included as a control for ChIP-qPCR and is separated by dashed line. exoMYC.K52 has the native sequence at the CTCF binding site upstream of MYC and should demonstrate wild-type CTCF binding. The exogenously expressed CTCFs (variant and wild-type) are HA tagged and expressed in the G3_3 cell line. ChIP-qPCR was performed to investigate CTCF binding to the variant CTCF site replacing the wild-type site ˜2 kb upstream of MYC (MYC site). An endogenous G3 site elsewhere in the genome and a region with no known CTCF binding served as a positive and negative control respectively. The variant CTCFs are able to bind to the variant site as indicated by enrichment with both CTCF and HA antibody, while the wild-type CTCF does not. FIGS. 26A-B disclose “CAGGGGAGGAGC” as SEQ ID NO: 5564, “DTYKLKR” as SEQ ID NO: 3, “SNLRR” as SEQ ID NO: 116, “GNLRR” as SEQ ID NO: 118, “GNLVR” as SEQ ID NO: 117, “ANLRR” as SEQ ID NO: 69, “GNLMR” as SEQ ID NO: 139, “NNLRR” as SEQ ID NO: 121, “GNLAR” as SEQ ID NO: 138, “SKLKR” as SEQ ID NO: 3470, “EHMKR” as SEQ ID NO: 123, “EHMRR” as SEQ ID NO: 34, “EHMNR” as SEQ ID NO: 126, “SHMNR” as SEQ ID NO: 147, “SHMNR” as SEQ ID NO: 146, “THMKR” as SEQ ID NO: 33, “DHMNR” as SEQ ID NO: 32, “GTMKM” as SEQ ID NO: 1255, “DHLNT” as SEQ ID NO: 155, “EHLAE” as SEQ ID NO: 151, “DHLQV” as SEQ ID NO: 12, “EHLKV” as SEQ ID NO: 13, “STLQE” as SEQ ID NO: 225, “EHLNV” as SEQ ID NO: 9, “STLNE” as SEQ ID NO: 152, “EHLQA” as SEQ ID NO: 156, “HHLMH” as SEQ ID NO: 157 and “SDLGV” as SEQ ID NO: 5552.

FIGS. 27A-27B: Variant CTCFs are able to bind the engineered A3 variant binding site and recover MYC expression. CTCF variants selected to bind to the A3 variant binding site sequence were expressed in the A3_4.K562 cell line. Cells were analyzed for MYC expression and CTCF occupancy on the DNA 72 hours post nucleofection. Residues of ZF helix of the variant and wild-type (indicated by (wt) are listed in the legend. A3 binding site sequence and interacting recognition helix of the CTCF zinc finger array is also diagramed. FIG. 27A. Endogenous MYC levels are recovered to wild-type levels in the A3_4 cell line when CTCF variants are expressed. Endogenous MYC expression levels were quantified by RT-qPCR and are relative to reduced endogenous MYC levels of A3_4 cell line. Endogenous MYC levels from the exoMYC.K562 cell line without any alterations to the CTCF binding site is shown as a positive control (separated by dashed lines). FIG. 27B. CTCF variants are able to bind to the introduced variant binding site in A3_4 cell line while the wild-type CTCF does not. CTCF Ab specific enrichment captures both wild-type and variant CTCF while HA Ab will only detect HA-tagged CTCF (transiently expressed). exoMYC.K562 is included as a control for ChIP-qPCR and is separated by dashed line. exoMYC.K52 has the native sequence at the CTCF binding site upstream of MYC and should demonstrate wild-type CTCF binding. The exogenously expressed CTCFs (variant and wild-type) are HA tagged and expressed in the A3_4 cell line. ChIP-qPCR was performed to investigate CTCF binding to the variant CTCF site replacing the wild-type site ˜2 kb upstream of MYC (MYC site). An endogenous A3 site elsewhere in the genome and a region with no known CTCF binding served as a positive and negative control respectively. The variant CTCFs are able to bind to the variant site as indicated by enrichment with both CTCF and HA antibody above the negative control, while the wild-type CTCF does not bind. FIGS. 27A-B disclose “CAGGGGAGGAAC” as SEQ ID NO: 5562, “DTYKLKR” as SEQ ID NO: 3, “GNLKR” as SEQ ID NO: 119, “GNLVR” as SEQ ID NO: 117, “SNLRR” as SEQ ID NO: 116, “ANLRR” as SEQ ID NO: 69, “GNLRR” as SEQ ID NO: 118, “NNLRR” as SEQ ID NO: 121, “TNLRR” as SEQ ID NO: 68, “SKLKR” as SEQ ID NO: 3470, “EHMNR” as SEQ ID NO: 126, “EHMRR” as SEQ ID NO: 34, “EHMKR” as SEQ ID NO: 123, “THMKR” as SEQ ID NO: 33, “EHMAR” as SEQ ID NO: 127, “GTMKM” as SEQ ID NO: 1255, “DNLLA” as SEQ ID NO: 100, “DNLLV” as SEQ ID NO: 129, “DNLQA” as SEQ ID NO: 133, “DNLLT” as SEQ ID NO: 128, “DNLAT” as SEQ ID NO: 132, “DNLQT” as SEQ ID NO: 130, “DNLMA” as SEQ ID NO: 102, “DNLMT” as SEQ ID NO: 135 and “SDLGV” as SEQ ID NO: 5552.

FIG. 28: Variant CTCFs recover MYC expression of the Other 10 variant binding site cell line. CTCF variants selected to bind to the Other variant binding site sequence were expressed in the Other 10.K562 cell line. Cells were analyzed for MYC expression 72 hours post nucleofection. Residues of ZF helix of the variant and wild-type CTCFs (indicated by (wt) are listed in the legend. Other binding site sequence and interacting recognition helix of the CTCF zinc finger array is also diagramed. A. Endogenous MYC levels are recovered to wild-type levels in the Other 10 cell line when CTCF variants are expressed. Endogenous MYC expression levels were quantified by RT-qPCR and are relative to reduced endogenous MYC levels of Other 10 cell line. Endogenous MYC levels from the exoMYC.K562 cell line without any alterations to the CTCF binding site is shown as a positive control (separated by dashed lines). FIG. 28 discloses “RKSDLGV” as SEQ ID NO: 5, “CGTGGTGCGACC” as SEQ ID NO: 5574, “TKLRL” as SEQ ID NO: 160, “THLKK” as SEQ ID NO: 163, “GHLRK” as SEQ ID NO: 162, “TKLKL” as SEQ ID NO: 161, “AHLRK” as SEQ ID NO: 60, “AHLKK” as SEQ ID NO: 159, “SKLKR” as SEQ ID NO: 3470, “SRLKE” as SEQ ID NO: 44, “TRLKE” as SEQ ID NO: 165, “YKLKR” as SEQ ID NO: 5553, “RRDT” as SEQ ID NO: 169, “RPDT” as SEQ ID NO: 38, “RNDT” as SEQ ID NO: 172, “RADN” as SEQ ID NO: 167, “RHDT” as SEQ ID NO: 40 and “QSGT” as SEQ ID NO: 1582.

FIG. 29: Variant CTCFs recover MYC expression of the Aother_2 variant binding site cell line. CTCF variants selected to bind to the Aother variant binding site sequence were expressed in the Aother_2.K562 cell line. Cells were analyzed for MYC expression 72 hours post nucleofection. Residues of ZF helix of the variant and wild-type CTCFs (indicated by (wt) are listed in the legend. Aother binding site sequence and interacting recognition helix of the CTCF zinc finger array is also diagramed. A. Endogenous MYC levels are recovered to wild-type levels in the Aother_2 cell line when CTCF variants are expressed. Endogenous MYC expression levels were quantified by RT-qPCR and are relative to reduced endogenous MYC levels of Aother_2 cell line. Endogenous MYC levels from the exoMYC.K562 cell line without any alterations to the CTCF binding site is shown as a positive control (separated by dashed lines). FIG. 29 discloses “CGTGGTGCGAAC” as SEQ ID NO: 5575, “AKLRK” as SEQ ID NO: 89, “AKLRV” as SEQ ID NO: 61, “SKLRL” as SEQ ID NO: 92, “SKLKR” as SEQ ID NO: 3470, “NRLKV” as SEQ ID NO: 94, “SRLKE” as SEQ ID NO: 44, “YKLKR” as SEQ ID NO: 5553, “RTET” as SEQ ID NO: 98, “RPDT” as SEQ ID NO: 38, “RADV” as SEQ ID NO: 99, “QSGT” as SEQ ID NO: 1582, “SNLLV” as SEQ ID NO: 101, “DNLMA” as SEQ ID NO: 102, “DNLRV” as SEQ ID NO: 103 and “SDLGV” as SEQ ID NO: 5552.

DETAILED DESCRIPTION

To date, there are no engineered CTCF variants available that are designed to bind to mutant CBSs with higher affinity than wild-type CTCF. Therefore, there is a need for engineered CTCF variants that can bind to mutant CBSs with higher affinity than wild-type CTCF.
The present disclosure is based, at least in part, on the discovery that CTCF variants with alterations in the zinc finger array can be engineered to recognize CBSs that harbor one or more point mutations, i.e., mutant CBSs.

CTCF

CCCTC-binding factor (CTCF) is a multi-domain protein that acts as an essential genome organizer by maintaining higher-order chromatin structure while also having a role in cell differentiation and the promotion or repression of gene expression. CTCF maintains topologically associated domains (TADs) spanning megabases of the genome as well as smaller scale Sub-TADs leading to fine-tuned gene insulation or gene activation within gene clusters. In addition, CTCF has been found to regulate mRNA splicing by influencing the rate of transcription and more recently been implicated in promoting homologous recombination repair at double-strand breaks. Wild type CTCF binds throughout the genome via an 11 finger zinc finger array that recognizes canonical CTCF binding sites (CBSs).
Wild-type CTCF ZF arrays comprise the following sequences at ZFs 3-6 positions −1 to +6:

	ZF3 positions −1 to +6:
	(SEQ ID NO: 1)
	TSGELVR

	ZF4 positions −1 to +6:
	(SEQ ID NO: 2)
	EVSKLKR

	ZF5 positions −1 to +6:
	(SEQ ID NO: 3)
	DTYKLKR

	ZF6 positions −1 to +6:
	(SEQ ID NO: 4)
	QSGTMKM

	ZF7 positions −1 to +6:
	(SEQ ID NO: 5)
	RKSDLGV

A wild-type CTCF has an amino acid sequence that has greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% sequence identity as compared to the amino acid sequence shown below:

(SEQ ID NO: 190)

MEGDAVEAIVEESETFIKGKERKTYQRRREGGQEEDACHLPQNQTDGGEV

VQDVNSSVQMVMMEQLDPTLLQMKTEVMEGTVAPEAEAAVDDTQIITLQV

VNMEEQPINIGELQLVQVPVPVTVPVATTSVEELQGAYENEVSKEGLAES

EPMICHTLPLPEGFQVVKVGANGEVETLEQGELPPQEDPSWQKDPDYQPP

AKKTKKTKKSKLRYTEEGKDVDVSVYDFEEEQQEGLLSEVNAEKVVGNMK

PPKPTKIKKKGVKKTFQCELCSYTCPRRSNLDRHMKSHTDERPHKCHLCG

RAFRTVTLLRNHLNTHTGTRPHKCPDCDMAFVTSGELVRHRRYKHTHEKP

FKCSMCDYASVEVSKLKRHIRSHTGERPFQCSLCSYASRDTYKLKRHMRT

HSGEKPYECYICHARFTQSGTMKMHILQKHTENVAKFHCPHCDTVIARKS

DLGVHLRKQHSYIEQGKKCRYCDAVFHERYALIQHQKSHKNEKRFKCDQC

DYACRQERHMEVIHKRTHTGEKPYACSHCDKTFRQKQLLDMHFKRYHDPN

FVPAAFVCSKCGKTFTRRNTMARHADNCAGPDGVEGENGGETKKSKRGRK

RKMRSKKEDSSDSENAEPDLDDNEDEEEPAVEIEPEPEPQPVTPAPPPAK

KRRGRPPGRTNQPKQNQPTAIIQVEDQNTGAIENIIVEVKKEPDAEPAEG

EEEEAQPAATDAPNGDLTPEMILSMMDR

For the purpose of comparing two different nucleic acid or polypeptide sequences, one sequence (test sequence) may be described to be a specific percentage identical to another sequence (comparison sequence). The percentage identity can be determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated into various BLAST programs. The percentage identity can be determined by the “BLAST 2 Sequences” tool, which is available at the National Center for Biotechnology Information (NCBI) website. See Tatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). For pairwise DNA-DNA comparison, the BLASTN program is used with default parameters (e.g., Match: 1; Mismatch: −2; Open gap: 5 penalties; extension gap: 2 penalties; gap x_dropoff: 50; expect: 10; and word size: 11, with filter). For pairwise protein-protein sequence comparison, the BLASTP program can be employed using default parameters (e.g., Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter). Percent identity of two sequences is calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence. When BLAST is used to compare two sequences, it aligns the sequences and yields the percent identity over defined, aligned regions. If the two sequences are aligned across their entire length, the percent identity yielded by the BLAST is the percent identity of the two sequences. If BLAST does not align the two sequences over their entire length, then the number of identical amino acids or nucleotides in the unaligned regions of the test sequence and comparison sequence is considered to be zero and the percent identity is calculated by adding the number of identical amino acids or nucleotides in the aligned regions and dividing that number by the length of the comparison sequence. Various versions of the BLAST programs can be used to compare sequences, e.g., BLAST 2.1.2 or BLAST+ 2.2.22.

CTCF Binding Sites (CBSs)

The CBS is typically 40 bp in length with a highly conserved 15 bp core sequence (or core motif). Sequence flanking the core sequence is significantly less well conserved, but still important for CTCF binding at sites throughout the genome (FIG. 1).
Wild type CTCF binds to a “consensus CBS motif” contains the following core sequence:
(SEQ ID NO: 191)

5′-NCDNHNGRNGDNNNN-3′.
In one embodiment, the consensus CBS motif contains the following core sequence: 5′-CCAGCAGGGGGCGCT-3′ (SEQ ID NO:6). Other core sequences that are known in the art.
It is not known if the nucleotides flanking the core sequence are bound by the 11 finger ZF array present within CTCF. Co-crystal structures of the 11-finger Zinc Finger (ZF) array bound to a consensus CTCF Binding Sequence (CBS) suggests that only ZFs 3-7 of the 11-finger ZF array appear to bind directly to the highly conserved core sequence while ZFs 8-11 and 1-2 do not appear to mediate sequence-specific contacts. Progressive truncations of the ZF array suggest that ZFs 8-11 and ZFs 1-2 may improve DNA-binding of CTCF to CBSs and DNaseI foot printing, as well as ChIP-Seq and ChIP-Exo data, suggests that ZFs 9-11 may make important protein-DNA contacts (Rhee and Pugh, Cell (2011); Nakahashi et al., Cell Reports (2013)). Interestingly, the co-crystal structure of the CTCF Z array bound to a CBS only contains zinc finger 2-9 with the other fingers not visible in the structure, consistent with the idea that zinc fingers interacting with flanking regions of the motif may not make stable contacts with the DNA (Hashimoto, et al., Molecular Cell (2017)). Thus, it remains unclear what impact all 11 fingers of the array have on DNA binding activity of CTCF and if all zinc fingers, or a subset, contact the DNA.
CTCF binding is sensitive to changes in the conserved 15 bp core motif of the CBS, where, in mice, single nucleotide changes at certain positions can lead to loss of CTCF binding (Nakahashi et al., Cell Reports (2013)). CTCF binding sites have been reported to be mutational hotspots in cancer with cancer-associated mutations localized to the core sequence of the CTCF binding site in primary samples from gastrointestinal cancer patients and with accompanying atypical gene expression profiles of oncogenic and tumor suppressor genes (Guo et al., Nature Communications (2018)). Small deletions of CTCF binding sites have also been shown to lead to loss of expression of genes such as MYC and PTGS2, which both play a role in cancer development (Schuijers et al., Cell Reports (2018); Kang et al., Oncogene (2015)).
Methods described herein can be used to select and generate engineered CTCF variants comprising a plurality of zinc fingers, where the selected polypeptide has at least one amino acid residue in at least one zinc finger that differs in sequence from a wild-type CTCF, and where the engineered CTCF variant binds to a DNA sequence of interest (e.g., CBS harboring at least one mutation in the consensus CBS sequence) but does not bind to a consensus CBS. Using methods of the present invention, a scaffold polypeptide is re-engineered into a new scaffold-based zinc-finger polypeptide that has different structural and functional features, such that the new polypeptide binds to a sequence of interest but does not bind to a naturally occurring DNA binding site of the scaffold protein.
The term “zinc finger” or “Zf” refers to a polypeptide having DNA binding domains that are stabilized by zinc. The individual DNA binding domains are typically referred to as “fingers.” A Zf protein has at least one finger, preferably 2 fingers, 3 fingers, or 6 fingers. A Zf protein having two or more Zfs is referred to as a “multi-finger” or “multi-Zf” protein. Each finger typically comprises an approximately 30 amino acid, zinc-chelating, DNA-binding domain. An exemplary motif characterizing one class of these proteins is -Cys-(X) (2-4)-Cys-(X) (12)-His-(X) (3-5)-His (SEQ ID NO:7), where X is any amino acid, which is known as the “C(2)H(2)class.” A single Zf of this class typically consists of an alpha helix containing the two invariant histidine residues co-ordinated with zinc along with the two cysteine residues.
The term “bind to” or “binding” with respect to a nucleic acid binding factor and its target nucleic acid, e.g., CTCF (variant or wild-type) and CBS, refers to sequence-dependent binding of the nucleic acid binding factor to the target nucleic acid sequence of a nucleic acid through intermolecular interactions, e.g., ionic, covalent, London dispersion, dipole-dipole, or hydrogen bonding, in such a way that the binding allows the nucleic acid binding factor to mediate a biologically significant function, e.g., transcriptional activation, recruitment of other proteins to the binding site, and/or alteration of chromatic structure. Such binding can result in modulation of expression of genes, such as activation, overexpression, suppression, or inactivation of gene expression.
The term “does not bind to” with respect to a nucleic acid binding factor and its target nucleic acid, e.g., CTCF (variant or wild-type) and CBS, refers to the lack of sequence-specific binding of the nucleic acid binding factor to a nucleic acid through intermolecular interactions, e.g., ionic, covalent, London dispersion, dipole-dipole, or hydrogen bonding, as a result of the lack of presence of a target sequence in the nucleic acid (e.g., due to one or more point-mutations in the CBS). Such non-binding does not allow the nucleic acid binding factor to mediate a biologically significant function, e.g., transcriptional activation, DNA modification, DNA cleavage, recruitment of other proteins to the binding site, and/or alteration of chromatic structure.
Each finger within a Zf protein binds to from about two to about five base pairs within a DNA sequence. Typically a single Zf within a Zf protein binds to a three or four base pair “subsite” within a DNA sequence. Accordingly, a “subsite” is a DNA sequence that is bound by a single zinc finger. A “multi-subsite” is a DNA sequence that is bound by more than one zinc finger, and comprises at least 4 bp, preferably 6 bp or more. A multi-Zf protein binds at least two, and typically three, four, five, six or more subsites, i.e., one for each finger of the protein.

Compositions and Methods

Described herein are engineered CTCF variants that can bind to mutant CBSs with higher affinity than wild-type CTCF. The engineered CTCF variants can be used in regulating genes that are under the control of mutant CBSs (CBSs having at least one nucleic acid that is different in sequence from the nucleic acid sequence of a consensus CBS). The CTCF variants have at least one amino acid residue in at least one zinc finger that differs in sequence from the amino acid sequence of a wild-type CTCF.
Exemplary engineered CTCF variants include those that contain:
(1) the amino acid sequence DHLQT (SEQ ID NO:8), EHLNV (SEQ ID NO:9), AHLQV (SEQ ID NO:10), EHLRE (SEQ ID NO:11), DHLQV (SEQ ID NO:12), EHLKV (SEQ ID NO:13), DHLQV (SEQ ID NO:14), EHLVV (SEQ ID NO:15), DHLRT (SEQ ID NO:16), DHLAT (SEQ ID NO:17), or DHLQT (SEQ ID NO:18) at ZF7 positions +2 to +6;
(2) the amino acid sequence DHLQT (SEQ ID NO:19), EHLNV (SEQ ID NO:20), AHLQV (SEQ ID NO:21), EHLRE (SEQ ID NO:22), DHLQV (SEQ ID NO:23), EHLKV (SEQ ID NO:24), DHLQV (SEQ ID NO:25), EHLVV (SEQ ID NO:26), DHLRT (SEQ ID NO:27), DHLAT (SEQ ID NO:28), or DHLQT (SEQ ID NO:29) at ZF7 positions +2 to +6;
(3) the amino acid sequence NAMKR (SEQ ID NO:30), EHMGR (SEQ ID NO:31), DHIVINR (SEQ ID NO:32), THMKR (SEQ ID NO:33), EHMRR (SEQ ID NO:34), or THIVINR (SEQ ID NO:35) at ZF6 positions +2 to +6;
(4) the amino acid sequence MNES (SEQ ID NO:36), HRES (SEQ ID NO:37), RPDT (SEQ ID NO:38), RTDI (SEQ ID NO:39), or RHDT (SEQ ID NO:40) at ZF6 positions −1 to +3;
(5) the amino acid sequence HGLKV (SEQ ID NO:41), HRLKE (SEQ ID NO:42), HALKV (SEQ ID NO:43), SRLKE (SEQ ID NO:44), DGLRV (SEQ ID NO:45), HTLKV (SEQ ID NO:46), or NRLKE (SEQ ID NO:47) at ZF5 positions +2 to +6;
(6) the amino acid sequence ATLKR (SEQ ID NO:48), QALRR (SEQ ID NO:49), GGLVR (SEQ ID NO:50), HGLIR (SEQ ID NO:51), ANLSR (SEQ ID NO:52), TGLTR (SEQ ID NO:53), HGLVR (SEQ ID NO:54), GGLTR(SEQ ID NO:55), HTLRR(SEQ ID NO:56), TVLKR(SEQ ID NO:57), ADLKR (SEQ ID NO:58), or HGLRR (SEQ ID NO:59) at ZF5 positions +2 to +6;
(7) the amino acid sequence AHLRK (SEQ ID NO:60), AKLRV (SEQ ID NO:61), GGLGL (SEQ ID NO:62), AKLRI (SEQ ID NO:63), TKLKV (SEQ ID NO:64), or SKLRV (SEQ ID NO:65) at ZF4 positions +2 to +6;
(8) the amino acid sequence ATLRR (SEQ ID NO:66), RRLDR (SEQ ID NO:67), TNLRR (SEQ ID NO:68), ANLRR (SEQ ID NO:69), GNLTR (SEQ ID NO:70), AMLKR (SEQ ID NO:71), HMLTR (SEQ ID NO:72), AMLRR (SEQ ID NO:73), or TMLRR (SEQ ID NO:74) at ZF4 positions +2 to +6;
(9) the amino acid sequence QQLIV (SEQ ID NO:75), SQLIV (SEQ ID NO:76), QQLLV (SEQ ID NO:77), GELVV (SEQ ID NO:78), QQLLI (SEQ ID NO:79), GQLIV (SEQ ID NO:80), GQLTV (SEQ ID NO:81), TELII (SEQ ID NO:82), QGLLV (SEQ ID NO:83), QQLLT (SEQ ID NO:84), GQLLT (SEQ ID NO:85), GELLT (SEQ ID NO:86), or QQLLI (SEQ ID NO:87) at ZF3 positions +2 to +6;
(10) the amino acid sequence AKLKK (SEQ ID NO:88), AKLRK (SEQ ID NO:89), AHLRV (SEQ ID NO:90), AKLRV (SEQ ID NO:91), or SKLRL (SEQ ID NO:92) at ZF4 positions +2 to +6; the amino acid sequence ERLRV (SEQ ID NO:93), NRLKV (SEQ ID NO:94), SRLKE (SEQ ID NO:95), or NRLKV (SEQ ID NO:96) at ZF5 positions +2 to +6; the amino acid sequence RPDT (SEQ ID NO:97), RTET (SEQ ID NO:98), or RADV (SEQ ID NO:99) at ZF6 positions −1 to +3; and the amino acid sequence DNLLA (SEQ ID NO:100), SNLLV (SEQ ID NO:101), DNLMA (SEQ ID NO:102), or DNLRV (SEQ ID NO:103) at ZF7 positions +2 to +6;
(11) the amino acid sequence GHLKK (SEQ ID NO:104), AHLRK (SEQ ID NO:105), or GKLRI (SEQ ID NO:106) at ZF4 positions +2 to +6; the amino acid sequence SRLKE (SEQ ID NO:107), DALRR (SEQ ID NO:108), DGLKR (SEQ ID NO:109), or TRLRE (SEQ ID NO:110) at ZF5 positions +2 to +6; the amino acid sequence at RPDT (SEQ ID NO:111) or RTEN (SEQ ID NO:112) at ZF6 positions −1 to +3; and the amino acid sequence EHLKV (SEQ ID NO:113), DHLLA (SEQ ID NO:114), or HHLDV (SEQ ID NO:115) at ZF7 positions +2 to +6;
(12) the amino acid sequence SNLRR (SEQ ID NO:116), GNLVR (SEQ ID NO:117), GNLRR (SEQ ID NO:118), GNLKR (SEQ ID NO:119), ANLRR (SEQ ID NO:120), NNLRR (SEQ ID NO:121), or TNLRR (SEQ ID NO:122) at ZF4 positions +2 to +6; the amino acid sequence EHMKR (SEQ ID NO:123), EHMRR (SEQ ID NO:124), THMKR (SEQ ID NO:125), EHMNR (SEQ ID NO:126), or EHMAR (SEQ ID NO:127) at ZF6 positions +2 to +6; and the amino acid sequence DNLLT (SEQ ID NO:128), DNLLV (SEQ ID NO:129), DNLQT (SEQ ID NO:130), DNLLA (SEQ ID NO:131), DNLAT (SEQ ID NO:132), DNLQA (SEQ ID NO:133), DNLMA (SEQ ID NO:134), or DNLMT (SEQ ID NO:135) at ZF7 positions +2 to +6;
(13) the amino acid sequence GNLVR (SEQ ID NO:136), GNLRR (SEQ ID NO:137), GNLAR (SEQ ID NO:138), GNLMR (SEQ ID NO:139), ANLRR (SEQ ID NO:140), SNLRR (SEQ ID NO:141), or NNLRR (SEQ ID NO:142) at ZF4 positions +2 to +6; the amino acid sequence EHMNR (SEQ ID NO:143), EHMKR (SEQ ID NO:144), EHMRR (SEQ ID NO:145), SHMNR (SEQ ID NO:146), SHMRR (SEQ ID NO:147), THMKR (SEQ ID NO:148), or DHMNR (SEQ ID NO:149) at ZF6 positions +2 to +6; and the amino acid sequence EHLKV (SEQ ID NO:150), EHLAE (SEQ ID NO:151), STLNE (SEQ ID NO:152), DHLQV (SEQ ID NO:153), EHLNV (SEQ ID NO:154), DHLNT (SEQ ID NO:155), EHLQA (SEQ ID NO:156), or HHLMH (SEQ ID NO:157) at ZF7 positions +2 to +6; or
(14) the amino acid sequence GHLKK (SEQ ID NO:158), AHLKK (SEQ ID NO:159), TKLRL (SEQ ID NO:160), TKLKL (SEQ ID NO:161), GHLRK (SEQ ID NO:162), THLKK (SEQ ID NO:163), or AHLRK (SEQ ID NO:164) at ZF4 positions +2 to +6; the amino acid sequence TRLKE (SEQ ID NO:165) or SRLKE (SEQ ID NO:166) at ZF5 positions +2 to +6; and the amino acid sequence RADN (SEQ ID NO:167), RHDT (SEQ ID NO:168), RRDT (SEQ ID NO:169), RPDT (SEQ ID NO:170), RTSS (SEQ ID NO:171), or RNDT (SEQ ID NO:172) at ZF6 positions −1 to +3.
In some embodiments, the engineered CTCF variants contain two or more combinations of the above-listed amino acid sequences.
In one embodiment of the present disclosure, mutations at certain positions within the consensus CBS substantially reduced binding by the wild-type CTCF zinc finger array in a bacterial two-hybrid system that was used to select for variants from randomized libraries that are capable of recognizing the mutated CBS sequence. Combining fingers together can be used to generate variant CTCF zinc finger arrays capable of recognizing CBSs harboring multiple point mutations. In some embodiments of the present disclosure, CTCF proteins harboring these zinc finger array variants are used to restore CTCF binding activity at sites bearing one or more mutations within a CBS (i.e., non-canonical CBSs). In some embodiments of the present disclosure, CTCF variants capable of recognizing alternative non-CBS sites in the genome. In some embodiments, such CTCF variants can be used to create artificial TADs and/or enhancer-promoter loops that can purposefully insulate genes and/or perturb the higher order structure of the genome and thereby alter expression of certain target genes of interest.

Diagnosis and Treatment of Diseases

The engineered CTCF variants described herein can be used for treating diseases where aberrant gene regulation due to mutant CBS is an underlying factor. The engineered CTCF variants described herein can, for example, bind to mutant CBSs that do not bind wild-type CTCFs, thereby altering or restoring gene regulation that can reverse or slow down progression of diseases. CTCF binding has been shown to regulate expression of oncogenes, such as MYC. Mutations accumulated in CTCF binding sites and loss of wild-type CTCF binding are associated to dysregulation of oncogenes and increased risk of carcinogenesis. Transcriptional dysregulation of MYC is one of the most frequent events in aggressive tumor cells and the dysregulation is a result of mutations in CTCF binding site disrupting enhancer-promoter loop. Engineered CTCF variants can bind to the mutated sites and restore normal gene expression levels, reducing risk of cancer development. In another case, Fragile X Syndrome is the result of a duplication in a repetitive region and the loss of FMR1 expression. Duplication of a repeat region in the X chromosome disrupts a CTCF binding site, leading to the loss of an enhancer-promoter loop driving the expression of FMR1. The engineered CTCF variants could restore the enhancer-promoter loop, leading to restoration of FMR1 expression. Human Papilloma Virus (HPV) and other integrating viruses (such as HIV) are often silenced by CTCF-mediated insulation of the viral genome from nearby enhancers. In the case of HPV18, there is a CTCF binding site in the promoter region of the viral genome. HPV18 that have mutations in the CTCF binding site are not silenced because these sequence mutations in the binding site can no longer be recognized by CTCF. Engineered CTCF variants would be able to bind to the mutated HPV integrated genomes and restore the insulating loop.

Kits

Also provided herein are kits comprising the engineered CTCF variant, and/or nucleic acids encoding an engineered CTCF variant as described herein and instructions for use.

Other Applications for the Engineered CTCF Variants

The engineered CTCF variants described herein can be used in a number of other applications, some of which are disclosed herein.
In some embodiments, the engineered CTCF variant, or nucleic acids encoding such engineered CTCF variant can be used to further elucidate the complex interactions of CTCF and other chromatin organization proteins. The structural maintenance of chromosomes is tightly regulated within cells and CTCF plays a major role. It still remains unclear how higher order structures are inherited across cell division and maintained through cell differentiation, the use of CTCF variants can help clarify that role. CTCF variants might be used to investigate how loops are formed across the genome and to modify or restore normal genomic architecture in a manner that impacts endogenous gene expression for research and therapeutic applications. They might also be used to re-establish ancestral CTCF binding sites so that we may better understand the evolutionary implications of TAD-based genome organization and epigenetic regulation of gene expression or to create alternative genomic architectures that impact endogenous gene expression for research and therapeutic applications.

Examples

Materials and Methods

The following materials and methods were used in the examples set forth below.

Construction of B2H Reporter Assay Components

The zinc-finger bacterial expression plasmid contained the CTCF zinc finger array (or variants) fused to gal11P. The amino-terminal end of all or part of the CTCF 11-finger zinc finger array was fused to the carboxy-terminal end of gal11P with a Flag tag linker between them. The zinc finger expression plasmid contains a Kanamycin resistance gene. The second plasmid, known as the bacterial reporter plasmid, contained CTCF binding site sequence that was introduced via BsaI restriction digest followed by T4 mediated ligation of annealed oligos containing the CTCF binding site. The reporter plasmid contained bacterial lac promoter that promoted the expression of lacZ when the CTCF binding site was bound. The reporter plasmid also has a Chloramphenicol resistance gene.

Bacterial-Two-Hybrid (B2H) Randomized Library Construction

Complimentary oligos were synthesized by IDT with ‘VNS’ or ‘NNS’ variation introduced in the sequence by design. Oligos were annealed and ligated into the zinc finger expression plasmid (previously digested with XbaI and BamHI) using T4 ligase. Ligation reaction was purified using Qiagen Minelute column and the purified substrate was electro-transformed into electro-competent XL1blue E. coli strain. After 1 hour recover in SOC at 37° C., the transformation was inoculated into 150 mL Luria broth (LB) with 50 ug/mL of Kanamycin. After the culture reached a OD600 of 0.400-0.600 (about 10 hours growth at 37° C.) the culture was spun down and the library was harvested using Qiagen Maxiprep kit.

Bacterial-Two-Hybrid (B2H) Reporter Assay

600 ng of gal11P-zinc finger expression plasmid and 600 ng of reporter plasmid with CTCF binding site of interest were chemically transformed into 150 uL of Δλ E. coli strain with an alpha N-terminal domain of RNA polymerase (α-NTD)-Gal4 fusion. Plasmid and cell mixture was incubated on ice for 30 minutes, heat shocked at 42° C. for 1 minute, recovered on ice for 2 minutes, followed by recovery in 500 uL of Luria Broth for 1 hour. Post-recovery, transformation was plated on Kanamycin (50 ug/mL), Chloramphenicol (12.5 ug/uL) selective LB agar plates. After 14-16 hours of growth at 37° C., colonies were picked and grown overnight in 1 mL of induction media (Luria broth with 50 ug/uL of Kanamycin, 12.5 ug/mL of Chloramphenicol, 10 ug/mL of ZnCl, and 500 ug/mL of IPTG). After 15-17 hours of growth, 25 uL of the overnight culture was sub-cultured into 1 mL of fresh induction media and grown for 2 hours at 37° C. or until cultures were between OD595 0.157-0.268 as measured by spectrophotometer. 100 uL of the subculture in then lysed for minimum of 15 minutes using 11 ul of a 1:10 mixture of lysozyme and PopCulture soap. 15 uL of the lysis mixture was then analyzed for fold activation of LacZ by previously described colorimetric ONPG assay. Binding was quantified by fold activation of LacZ. Fold activation was determined by calculating the fold increase of β-gal levels of a sample above the β-gal levels of the negative control (no zinc finger protein fused to gal11P).

Bacterial-Two-Hybrid (B2H) Selection Assay

Plasmids involved in the selection assay are the same as before with only one difference: The reporter plasmid is made to be a selective plasmid by swapping LacZ with BlaC, an antibiotic resistance gene for β-lactam ring class of antibiotics, such as Carbenicillin. Selections are carried out by constructing libraries of variants from a pool of oligos ligated into the zinc finger-gal11P expression plasmid. These are electro-transformed into electro-competent Δλ E. coli strain containing the selective plasmid with the CTCF binding site of interest. Cells are recovered in 1 mL of SOC for 1 hour at 37° C. followed by induction of selective plasmid for 3 additional hours at 37° C. in 4 mLs of induction media (previously described). After four total hours, transformations are plated on low stringency plates (LB agar with 50 ug/mL of Kanamycin, 12.5 ug/mL of chloramphenicol, 100 ug/mL of Carbenicillin, 10 ug/mL of zinc chloride, and 200 ug/mL, IPTG and 0.45 ug/mL of Clavulanic acid). Plates are grown overnight at 37° C. for 20-24 hours and then colonies are harvested off the surface with 2 mL of LB. 50 uL of the scrapped colonies are sub-cultured into 1 mL of terrific broth (TB) with 50 ug/mL of Kanamycin, and 12.5 ug/mL of Chloramphenicol and grown 14-16 hours at 37° C. The next day, plasmid is harvested from the overnight cultures and chemically transformed into chemically competent Δλ E. coli strain containing the same selective plasmid with the CTCF binding site of interest as before. The chemical transformation is performed as previously described with the addition of 2 hour growth in induction media following a 1 hour recovery at 37° C. After a total of 3 hours of growth, cells are plated on high stringency selective gradient plates. The high stringency gradient plates contains 50 ug/mL of Kanamycin, 12.5 ug/mL of Chloramphenicol, 100 ug/mL of Carbenicillin, 10 ug/mL of ZnCl, 200 ug/mL of IPTG with a gradient of Clavulanic acid starting from ˜1 up to 40 ug/mL in concentration. Plates were incubated 20-24 hours at 37° C. Colonies that grew on the gradient with the highest levels of Clavulanic acid were picked and grown in 1 mL of TB with 50 ug/mL of Kanamycin and grown overnight in order to harvest the plasmid. The variant plasmid was then Sanger sequenced as well as analyzed for binding activity in the B2H β-gal reporter assay.

High Stringency Gradient Plates

The high stringency gradient plates contains 50 ug/mL of Kanamycin, 12.5 ug/mL of Chloramphenicol, 100 ug/mL of Carbenicillin, 10 ug/mL of ZnCl, 200 ug/mL of IPTG with a gradient of Clavulanic acid starting from ˜1 to 40 ug/mL in concentration. To obtain a gradient of Clavulanic acid, rectangle plates are elevated using a pipette tip so as to have a ˜25° C. slope (enough of an angle so that the thin end of the wedge is only barely covered with LB agar). 20-25 mL of LB agar with 50 ug/mL of Kanamycin, 12.5 ug/mL of Chloramphenicol, 100 ug/mL of Carbenicillin, 10 ug/mL of ZnCl, 200 ug/mL of IPTG and 4 ug/mL of Clavulanic acid is added to the inclined plate to form the bottom wedge. Once solidified, the plates are laid flat and 20-25 mLs of LB agar with 50 ug/mL of Kanamycin, 12.5 ug/mL of Chloramphenicol, 100 ug/mL of Carbenicillin, 10 ug/mL of ZnCl, 200 ug/mL of IPTG (with no Clavulanic acid) is poured on top. This creates plates with a gradient of Clavulanic acid ranging from ˜1 ug/mL up to 4.0 ug/mL.

CTCF Binding Assay Using ChIP-qPCR

K562 cells were seeded 18-24 hours in advance of transfection at a density of 3×10⁵cells/mL. 3 million K562s per variant were transfected using Lonza Kit V using the provided optimized protocol and pooled in a 10 cm dish. 5 ug of plasmid expressing HA epitope tagged CTCF (wild-type or variant) expressed by a pCAG promoter was used for each 1 million cell reaction. 72 hours post transfection, approximately 10 million cells were crosslinked with 1% Formaldehyde at 37° C. for 10 mins. Reaction was quenched with 1.2 mL of 2.5M Glycine for 5 mins at 37° C. Cells were pelleted at 430 g for 10 mins and sonicated on SFX250 Branson sonifier for 5.5 mins, 32% Amplitude, 1.3 s off, 0.7 s on. The samples were then split in half, one precipitated overnight, rotating at 4° C. with antibody specific to CTCF and the other precipitated overnight with HA specific antibody. The next day, antibody bound chromatin complexes were incubated with G-dynabeads for 2 hours at 4° C., rotating. Beads were washed three times in 1 mL of ice-cold RIPA 150 Wash Buffer (0.1% SDS, 0.1% DOC, 1% Triton X-100, 1 mM EDTA, 10 mM Tris-HCl pH 8, 150 mM NaCl), three time in 1 mL of ice-cold RIPA 500 wash buffer (0.1% SDS, 0.1% DOC, 1% Triton X-100, 1 mM EDTA, 10 mM Tris-HCl pH 8, 500 mM NaCl), three times in 1 mL of ice-cold LiCl wash buffer (10 mM Tris-HCl pH8, 250 mM LiCl, 0.5% Triton X-100, 0.5% DOC), and once in 1 mL of ice-cold 10 mM Tris-HCl pH 8.5. The antibody chromatin complex was eluted from the beads in 100 uL of Elution Buffer (10 mM Tris-HCl pH 8, 0.1% SDS, 150 mM NaCl) with 5 mM DTT added fresh. Beads were incubated with elution buffer at 65° C. for 1 hour, shaking at 900 rpm. Beads were pelleted by magnet and supernatant was moved to a clean tube where, after cooling to room temp, 1 uL of RNAse (Roche 11119915001) was added to the sample and incubated at 37° C. for 30 mins at 600 rpm. 3 uL of Proteinase K [20 mg/mL] was added to samples and incubated overnight at 65° C. (Lifetech #100005393). The next day, 100 uL of SPRI beads with 160 uL of PEG/NaCl (20% PEG, 2.5M NaCl) were added to samples, vortexed and incubated at room temp for 5 minutes before pelleting beads on a magnet. Pellet was washed twice with 80% ethanol and air dried for 5 minutes before final elution in 150 uL of 10 mM Tris-HCl pH 8. 3 uL of recovered supernatant was mixed with 5 uL of SYBR qPCR master mix and 2 uL of primer mix for quantification of fragment enrichment over 1% input untreated by antibody by Real Time-qPCR.

Generation of Variant Binding Site Cell Lines

Cell lines with the variant binding site introduced at the CTCF binding site ˜2 kb upstream of MYC TSS were generated by nucleofecting exoMYC.K562 with SpCas9-P2A-GFP, gRNA targeting the CTCF binding site, and one of 6 distinct ssODNs as HDR templates to introduce the 6 different variant binding sites. exoMYC.K562 is K562 cell line transduced with exogenous MYC construct expressed off of PGK promoter. This was necessary as any reduction of endogenous MYC expression can impact the survival of K562 cells. GFP+ cells were sorted at a high dilution into a 96 well plate for single-cell clonal expansion. Once expanded, gDNA and RNA was extracted to genotype and phenotype the clonal cell population. Clonal lines that had a reduction of endogenous MYC and also appeared homozygous at the target site for the desired HDR event were used in the study.

Quantifying MYC Expression by RT-qPCR

Three million K562 cells genome edited to harbor the variant binding site upstream of MYC were nucleofected with 5 ug of plasmid expressing a variant CTCF following the Lonza Kit V protocol. 72 hours post nucleofection, 1 million cells were isolated for RNA extraction following the NucleoSpin RNA Plus RNA isolation protocol. The RNA was converted to cDNA via Thermo High-Capacity RNA-to-cDNA Kit. 3 uL of 1:20 dilution of cDNA was mixed with 5 uL of Thermo Fast SYBRgreen Master Mix and run on RT-qPCR machine following standard PCR amplification protocol.

Results

Single Nucleotide Substitution at CBS Affecting CTCF Binding Efficiency

We reasoned we could use a bacterial two-hybrid (B2H) system to evolve the zinc finger array of CTCF to bind to mutated CBSs bearing single or multiple sequence changes that disrupt wild-type CTCF binding (Wright et al. Nature Protocols (2006); Sander et al., Nature Methods (2010); Maeder et al. Molecular Cell. (2008)). We used a previously described bacterial-two-hybrid (B2H) system to systematically define the impact of single nucleotide substitutions within a previously defined consensus CBS site (Joung et al., PNAS (2000)). In the B2H system, the binding of a DNA-binding zinc finger array to a target site of interest can be configured to result in increased transcription of a reporter gene (e.g., beta-galactosidase or an antibiotic resistance gene) (FIG. 2). To do this, two fusions are expressed in an E. coli cell bearing a reporter construct. The first fusion consists of a zinc finger array fused to a fragment of the yeast Gal11P protein, which interacts with a fragment of the yeast Gal4 fusion. The second fusion consists of a fusion of the N-terminal domain of the E. coli RNA polymerase alpha subunit to the yeast Gal4 fragment (the α-Gal4 fusion). The reporter construct consists of a weak E. coli promoter that drives expression of the reporter gene of interest with a binding site for the zinc finger array positioned upstream of the promoter. Binding of the zinc finger-Gal11P fusion to the zinc finger binding site results in recruitment of RNA polymerase complexes harboring the alpha-Gal4 fusion, resulting in increased transcription of the reporter gene. If the reporter gene is lacZ, which encodes for β-galactosidase (β-gal), the level of beta-gal expression can be easily quantified using a well-established colorimetric ONPG-based assay (FIG. 2).
In this B2H reporter assay, we determined the entire zinc finger array (ZF1-11) and the full CTCF binding site (CBS), not just the 15 bp consensus CBS sequence, was required for optimal expression of the lacZ gene (FIG. 3), which mimics observed CTCF binding requirements in human cells10,11. After optimizing positioning of the CBS site relative to the transcription start site, we then systematically introduce point mutations into the CBS and tested their impact on lacZ expression. These results demonstrated that mutation of nucleotides outside the 15 bp core sequence had little impact on lacZ expression. By contrast, binding, however certain sequences at certain positions within the core sequence resulted in no or reduced binding (FIG. 4). Our results closely match ChIP-Seq data for CTCF binding sites in human cells and reflect other studies in the literature in which point mutations in the CTCF core lead to loss of CTCF binding. Taken together, these results strongly suggest that binding activity of the CTCF zinc finger array in the B2H system mimics the binding activity of intact CTCF protein in human cells.
Although most sequence changes in the flanking regions of the binding site had little impact on binding efficiency, certain alterations appeared to slightly improve the fold-activation of lacZ expression. Therefore, we tested whether a more “optimized” CBS bearing the “best” nucleotides as defined in the B2H assay might lead to higher-fold activation of lacZ expression but we did not observe any higher activity compared with the original consensus sequence (derived from Nakahashi et al. ChIP-seq data) (FIG. 5).
Generation of Engineered CTCF Variants that Bind to Mutated CBSs with Single Altered Nucleotide
Next, we sought to determine if we could use the B2H system to select for CTCF zinc finger array variants capable of recognizing mutated CBSs not recognized by the wild-type CTCF zinc finger array. To do this, we modified the B2H reporter construct, replacing the lacZ gene with the blaC gene (FIG. 6), which encodes beta-lactamase and therefore confers resistance to beta-lactam antibiotics (e.g., carbenicillin). This modification enables us to select for cells that express a CTCF zinc finger array variant that can efficiently bind a mutant CBS positioned upstream of the weak promoter driving blaC expression. Increasingly higher levels of blaC expression can be selected for by using media containing carbenicillin and increasingly higher concentrations of the beta-lactamase inhibitor clavulanic acid. Gradients of clavulanic acid can be created within a single agar plate (FIG. 6; see Materials and Methods), thereby enabling sampling of cells at various concentrations of the inhibitor.
With this modified B2H selection system, we first sought to identify CTCF zinc finger array variants that can bind to CBSs bearing single point mutations that abolish binding by the wild-type CTCF zinc finger array in this system. In an initial set of selection experiments, we sought to identify CTCF zinc finger array variants that could bind to mutant CBSs bearing mutations of the C that is contacted by an aspartic acid (D) present at the third position (+3) of the alpha-helical recognition helix of ZF7 (shown by previously published co-crystal structures cited above). We created a randomized library of CTCF zinc finger array variants in which the codon encoding the ZF7 +3 position was randomized using a degenerate NNS codon (where N=G, A, C, or T and S=G or C). We then used the B2H selection system to interrogate this library to identify variants capable of recognizing CBSs bearing C to T, C to G, and C to A substitutions at the position contacted by ZF+3. Selections were initially performed on low stringency plates with clavulanic acid gradients ranging from 0 to 0.45 ug/ml) and surviving colonies harvested and plasmids encoding the variant zinc finger arrays were purified. This selected subset of variants was then subjected to high stringency selection in the B2H system on plates with carbenicillin and gradients of clavulanic acid ranging from 0 to 4 ug/ml). Plasmids encoding variant zinc finger arrays were purified from colonies that grew on the end of the gradient plate with highest concentration of clavulanic acid, sequenced, and then tested in the B2H reporter assay by beta-galactosidase assay.
As can be seen in FIGS. 7A-C, we obtained CTCF zinc finger array variants that showed preferential binding activity (as judged by the B2H reporter assay) for the mutated CBS compared with the original consensus CBS. These clones also showed selection for a particular amino acid at the ZF7 +3 position: for the C to T site, a threonine (T) was selected, for the C to A site, an asparagine (N) was selected, and for the C to G site a histidine (H) was selected. The identities of these amino acids is consistent with what might be expected to recognize the mutant nucleotide based on previous zinc finger selections using the Zif268 zinc finger array. However, although we successfully selected for mutants that had altered binding activity, in most cases, the binding activity of the variant for the mutated CBS was not as strong (as judged by the B2H reporter assay) as that of the wild-type CTCF zinc finger array for the consensus CBS (FIGS. 7A-C).
Based on our previous experience with re-engineering the DNA-binding specificities of the Zif268 zinc finger array, we hypothesized that obtaining stronger binding variants might require alteration of amino acids flanking the +3 position in ZF7. To test this idea, we created a larger library of variants in which we randomized positions +2, +3, +5 and +6 of ZF7 using degenerate VNS codons (where V=G, A, or C). Position +4 of ZF7 was not altered because it faces the internal core of the ZF domain and is not expected to make contacts to the DNA. We then performed B2H selections as described above using this library to identify variants that could identify a mutant CBS with a C to G mutation at the position contacted by ZF7 +3 in the wild-type CTCF zinc finger array. These selections identified variants that showed stronger binding activity for the mutant CBS and showed some degree of consensus in the identities of amino acids selected (FIG. 8).
Based on this success, we generated additional randomized libraries in which randomized positions −1, +1, +2, and +3 or +2, +3, +5 and +6 for ZF7, ZF6, ZF5, ZF4, and ZF3. We then performed selections as described above using these libraries against various matched mutant CBSs harboring nucleotide substitutions at positions expected to be contacted by residues randomized in the libraries (FIGS. 9-16). Analysis of variants from individual surviving colonies at the most selective end of the high stringency selection plates showed that many of these selections yielded variants with high activity for the mutant CBS of interest and sequencing of these clones showed that there was generally a degree of consensus in the amino acid sequences suggesting that selection was successfully occurring (FIGS. 9-16).
Generation of Engineered CTCF Variants that Bind to Mutated CBSs with Multiple Altered Nucleotides
Having successfully identified CTCF zinc finger variants that could recognize CBSs with a single altered nucleotide position, we next sought to identify variants that could recognize CBSs bearing multiple mutated nucleotides. To do this, we sought to recombine ZF variants each selected to bind to different “subsites” within the CBS that bear individual mutations. However, because of well-known context-dependent effects that exist between ZFs in a multi-finger array, we undertook a strategy in which we recombined together pools of selected ZF variants (rather than a single variant) for any given altered subsite to identify the combinations of mutated ZFs that best work together to recognize a CBS bearing multiple mutations. To isolate pools of ZF variants for various mutated CBS subsites, we harvested all remaining clones from the high stringency selection plates we performed with the CBS sites bearing single mutations (depicted in FIGS. 9-16). Deep sequencing of the various selected clones in these pools yielded a variety of sequences with some degree of consensus within each selection as expected (Table 1).
We then recombined pools of variants for ZFs 4, 5, 6, and 7 to create CTCF zinc finger arrays that harbored various altered recognition helices for these positions and then performed B2H selections (see Materials and Methods) against five different mutated CBSs bearing combinations of various nucleotide substitutions in subsites for ZFs 4, 5, 6, and 7 (FIGS. 17-21). Sequencing of clones from these selections showed that certain recognition helix sequences for each finger were selected multiple times, suggesting that the selections were identifying combinations that work well together. Importantly, for all five of the multiply mutated CBSs, several of the CTCF zinc finger array variants identified showed good binding activity on the site for which they were selected as judged by B2H assay (FIGS. 17-21). In addition, for four of the five mutant CBS sites, we were able to identify variants that not only bind to the mutant CBS but also fail to bind to the original unmutated (consensus) CBS. Thus, we conclude that using our approach described here we are able to identify CTCF ZF array variants capable of recognizing multiply mutated CBSs that are not efficient bound by the original wild-type CTCF zinc finger array.

Binding Specificity of Engineered CTCF Variants to Mutant and Wild-Type CBSs in Human Cells

Having successfully engineered variants that can recognize CBSs with multiple sequence changes across the motif, we next wanted to investigate whether the variants can bind to these same mutant binding sites in a human cell context while not binding to wild-type CBSs. First, we found a collection of sites in the human genome that matched the 15 bp core sequence for each of the five mutated binding sites that we had selected CTCF variants to bind (described in FIG. 17-21). We then looked at two variant binding sites with sequence that matched one of the five mutated binding sites (sequence depicted in FIG. 20) as well as known CBSs to determine if endogenous CTCF could bind to the wild-type CBS and not bind to the variant binding sites as the B2H reporter assay would suggest (FIG. 20). Human K562s, an erythroleukemia cell line, were harvested and analyzed by ChIP-qPCR using CTCF specific antibody to detect CTCF-DNA binding. Wild-type CTCF showed no detectable binding to two different target sites that matched the mutated CBS but showed great enrichment for wild-type CTCF binding site, supporting the results of the B2H reporter assay (FIG. 22). Next, we wanted to see if overexpressed, exogenous, 3×HA tagged wild-type CTCF delivered by plasmid transfection in K562s had the same binding profile observed with endogenous CTCF. Wild-type K562s were transfected with 3×HA-CTCF and 72 hours later were harvested and processed for ChIP-qPCR analysis with HA specific antibodies. Exogenous wild-type 3×HA-CTCF could bind to the wild-type CBSs and could not bind to the variant binding sites, same as endogenous wild-type CTCF, suggesting overexpression of CTCF by plasmid delivery reflects biologically relevant behavior (FIG. 23A). Based on these results, we next examined the ability of a variant CTCF to bind to the variant binding sites native to the human genome. The variant chosen was one pulled out from selection in the B2H selection assay and shown to bind to the variant site with the same sequence as variant site 1 and 2, used in FIGS. 22-23B, by the B2H reporter assay. K562s were transfected with the 3×HA-tagged CTCF variant and the same sites as before were examined for binding activity by ChIP-qPCR. Variant specific HA enrichment was present at the variant binding sites and lacking at the wild-type sites suggesting we successfully evolved a variant that can specifically bind to mutant CBS with as few as three nucleotide changes without binding native CBSs (FIG. 23B).

Gene Expression Regulation by Engineered CTCF Variants Via Looping

CTCF has the capacity to alter gene expression through CTCF-Cohesin mediated looping of the genome. We were curious to see if the variant CTCFs could reproduce the gene regulatory capacity of wild-type CTCF when bound to the endogenous variant binding sites. To investigate gene expression changes, we focused on genes within a 1 Mb region of the variant binding sites. Eleven genes were identified within 1 Mb region for Variant site 1.1 and 1.2 and another 10 genes were identified for Variant site 2.1 and 2.2. K562s were nucleofected with variant CTCFs fused to GFP that had the capacity to bind to Variant site 1 and Variant site 2. 72 hours post nucleofection, RNA was isolated from GFP+ cells and gene expression levels were compared to RNA extracted from K562s nucleofected with a wild-type CTCF control. Of the 11 genes for Variant site 1.1 and 1.2, 6 genes showed a change in gene expression relative to cells nucleofected with the wild-type CTCF control (JJ388) (FIG. 24A). 2 of the 10 genes identified for Variant site 2.1 and 2.2 had altered gene expression levels relative to wild-type control (FIG. 24B). This data suggests that not only do the variant CTCF proteins bind to their target sequence in human cells, but it also reproduces the biological role of native CTCF to regulate gene expression possibly through the formation of loops or sub-TADs.
Next we wanted to demonstrate that the CTCF variants could replicate the biological function of wild-type CTCF at a known CTCF binding site that creates an enhancer-promoter loop. MYC expression is maintained by a loop formed between a CTCF binding site ˜2 kb upstream of the transcriptional start site (TSS) of MYC and a CTCF binding site ˜1 kb downstream of the MYC TSS14. When CTCF Is bound to both sites, cohesin links both CTCFs via the CTCF's cohesin-interaction domain, creating a loop that maintains the expression of MYC. If one or both of the CTCF binding sites is disrupted the CTCF-mediated loop is lost and there is a reduction in MYC expression 14. Five cell lines were generated containing the 5 different variant binding site sequences (defined in FIG. 25) at the CTCF binding site ˜2 kb upstream of the MYC TSS. This was done in K562 background transduced with a lentiviral construct expressing exogenous MYC via phosphoglycerate kinase (PGK) promoter (exoMYC.K562) to compensate for any reduced cell fitness that reduction of endogenous MYC expression may cause. An additional sixth cell line was generated where point mutations to the CTCF binding site were made that should have no affect on wild-type CTCF binding as indicated by results from the B2H reporter assay. RNA was isolated from the clonal cell lines homozygous for the variant binding sites and endogenous MYC gene expression levels were assayed by reverse transcriptase Real Time qPCR (RT-qPCR). Each of the isolated cell lines with the variant CTCF binding site demonstrated a reduced level of MYC expression suggesting that the CTCF-mediated loop is disrupted (FIG. 25).
Based on this result, we wanted to see if expression of the variant CTCFs in these modified cell lines could bind to the engineered sites and restore MYC expression. HA tagged wild-type CTCF and HA tagged CTCF variants were expressed in the cell line that contained their matching variant binding site. Variants selected to bind to the G3 variant binding site were expressed in the G3_3 cell line, A3 variants in the A3_4 cell line, etc. HA-tagged wild-type CTCF was also tested in each of the variant cell lines for binding and for recovery of endogenous MYC expression. The level of endogenous MYC expression in exoMYC.K562 served as wild-type control as there is no alteration to the CTCF binding site upstream of the MYC TSS. CTCF variants expressed in the engineered cell lines recovered endogenous MYC expression while expression of wild-type CTCF in these cell lines failed to recover MYC expression (FIGS. 26A-29). The same samples were analyzed for occupancy of the variant binding sites by wild-type CTCF or the variant CTCFs by ChIP-qPCR enriching for CTCF-bound DNA fragments with CTCF or HA antibody. Wild-type CTCF had a reduced occupancy of the variant binding sites, consistent with continued reduction of MYC expression, while variant CTCF proteins could bind to the variant site they were selected for as well as rescue MYC expression (FIG. 26-29). Together, this data suggests that we have evolved CTCF variants that can bind to novel sequences and still interact with cohesin to form loops that maintain gene expression profiles.

Tables

Amino acid sequence of variants selected for on different CTCF binding sites. All amino acids sequences are listed from N to C terminal. Colonies growing on the highest stringency of selection were scrapped off, pooled, and plasmid encoding for the zinc finger was isolated and deep sequenced. The number of reads reflects how prominent the variant was in the population pooled from selections performed in triplicate.

TABLE 1

ZF7 selection on C:G change at
nt 2 of core motif in CBS.
Sequences reflect position
2 through 6.

SEQ
ID		#
NO:	Sequence	reads

8	DHLQT	2981

15	EHLVV	2413

155	DHLNT	1517

16	DHLRT	1442

13	EHLKV	1434

192	KDLVV	1357

193	DHLQA	1114

194	DHLLV	1076

195	DHLLT	881

196	EHLTV	803

197	STLME	786

17	DHLAT	777

9	EHLNV	736

12	DHLQV	574

198	DHLKT	541

199	EHLKE	517

200	DHLLE	506

201	EHLRV	503

202	STLRE	498

203	DHLMV	431

204	DHLKV	427

205	DHLRV	394

206	DHLNV	389

114	DHLLA	380

207	DHLKE	368

208	DHLNE	330

11	EHLRE	330

209	STLLE	323

210	DHLMA	305

211	KDLTV	296

212	DHLVT	284

213	AHLNV	278

214	AHLTV	268

215	HTLME	245

216	DHLRA	237

217	DHLAV	221

218	HHLAE	221

219	GHLMD	207

220	DHLST	199

221	EHLMV	197

222	AHLVV	196

223	EHLAV	192

224	HTLAE	187

225	STLQE	181

226	DHLAE	167

227	AHLQE	163

228	SSLNE	158

229	GHLNV	155

230	EHLVE	144

231	DHLME	143

232	DHLRE	134

233	AHLNA	120

234	HTLVE	120

235	STLKE	112

236	EHLQV	107

237	GTLME	106

238	HHLAV	102

239	HSLME	101

240	HSLTE	97

241	EHLMA	97

242	DHLHT	94

10	AHLQV	94

243	DHLTV	93

244	EHLIV	90

245	SGLNE	89

246	AHLLV	85

247	EHLLV	84

248	VKLKI	83

249	DHLQE	80

250	HTLTE	77

251	STLHE	76

252	DHLVV	76

253	AGLAL	70

254	STLND	69

255	DHLKA	68

256	KDLTQ	66

257	DKLMN	66

258	GTLRE	66

259	GHLTV	66

260	RLLTA	65

261	SSLRE	63

262	HTLKE	62

263	GHLAV	60

264	RLLAQ	58

265	KDLAV	57

266	EHLQE	57

267	SHLNV	57

268	AGLPI	57

269	TTLME	56

90	AHLRV	56

270	AHLMV	55

271	EHLME	55

272	EHLQT	55

273	EVLNR	55

274	HHLVV	54

275	KDLSV	54

276	RHLVM	53

277	THLNE	50

278	RDLRT	49

279	LLLGS	49

280	MVLGN	48

281	KTLIE	47

282	AHLGV	46

283	SGLLA	46

284	DHLHV	45

285	EHLNT	45

286	STLLQ	44

287	AHLKV	44

288	AHLAV	42

289	TNLID	41

290	GTLNE	41

291	QVLTQ	40

292	SSLME	39

293	GHLVE	38

294	HSLLE	38

295	SGLLE	38

296	GGLLE	36

297	STLRV	36

298	HTLAD	35

299	SHLME	35

300	DHLAI	35

301	EHLLA	35

302	HNLLL	34

303	PHLVV	34

304	KALGT	33

305	PHLVI	31

306	VLLII	30

307	HHLRE	29

308	GALRM	29

309	RGLHE	29

310	AHLLE	28

311	EHLKA	28

312	DTLLV	27

313	EHLRT	26

314	SSLRD	24

156	EHLQA	23

315	EHLAT	23

316	SGLGE	22

317	ATLQE	22

318	DHLSA	22

101	SNLLV	22

319	SHLLV	21

320	KDLMV	21

321	DHLQQ	20

322	ATLME	20

323	GHLQA	20

324	RTLTE	20

325	RRLAH	20

326	DTLQA	20

327	GHLEV	19

328	HQLKL	19

329	EHLLT	19

330	DGLRT	18

331	THLRP	18

132	DNLAT	18

332	EHLNA	17

333	STLVV	17

135	DNLMT	17

334	DTLLA	17

335	STLDE	16

336	KDLVA	15

337	AHLHA	15

338	KDLQV	15

339	HHLTV	15

340	SGLLD	15

341	ANLME	14

129	DNLLV	14

342	EHLKT	13

343	GSLAI	13

344	EHLSV	13

345	EHLNE	13

346	EHLVI	13

347	KDLKV	13

348	EGLGT	13

130	DNLQT	12

349	STLMS	12

350	AHLMM	12

351	IKLDG	12

352	VLLGA	12

353	PGLSA	12

354	AELNR	12

355	HQLVI	12

356	GHLVV	12

357	PHLLV	11

358	PRLAL	11

359	DHLNA	11

360	KDLDV	11

361	AHLHV	11

362	RVLGG	11

363	AHLQA	11

364	RQLRT	10

365	AHLQT	10

100	DNLLA	10

151	EHLAE	10

366	EHLAM	10

367	DRLSI	10

368	GGLGA	10

369	GHLNT	10

370	AHLRT	10

371	DTLRV	18

372	MSLRG	9

373	DHLTI	9

374	THLIV	9

375	DTLMA	9

376	MKLQE	9

377	TALGT	9

378	GHLLV	9

379	GQLAI	8

380	ANLES	8

381	AHLNT	8

382	EHLLE	8

383	SNLTV	8

384	STLLV	8

385	STLMV	8

386	GTLVS	7

387	DNLKT	7

388	GHLQT	7

128	DNLLT	7

389	EHLVT	7

390	GALRE	7

391	SSLAE	7

392	DTLRQ	7

393	KALLG	7

394	AMLNP	6

395	DTLHQ	6

396	DNLLQ	6

397	EHLAH	6

398	AHLKE	6

399	ATLAE	6

400	EHLMD	6

401	STLHM	6

402	DTLAV	6

403	DHLVE	6

404	PTLGE	6

405	KGLPL	6

406	DTLLQ	6

407	AHLNE	6

408	AHLAE	6

409	GHLKV	6

410	SGLQV	5

411	HHLLV	5

412	EPLLP	5

413	DNLAV	5

414	AHLLT	5

415	AHLST	5

133	DNLQA	5

416	DNLRT	5

417	DTLAL	5

418	DTLQV	5

419	EHLRA	5

420	SNLQV	5

421	KDLRV	5

422	DTLAT	5

423	DTLRA	5

424	QHLRV	4

425	SSLLE	4

426	SNLMV	4

427	SDLGG	4

428	DNLHT	4

429	DNLTA	4

430	DTLMV	4

431	EHLST	4

432	DTLSV	4

102	DNLMA	4

433	EHLVM	4

434	STLAE	4

435	KDLAE	4

436	SSLNV	4

437	SSLLV	4

438	AHLKT	4

439	AHLRE	4

440	KDLLV	4

TABLE 2

ZF7 selection on C:T change at
nt 2 of core motif in CBS.
Sequences reflect position
2 through 6.

SEQ
ID		Read
NO:	Sequence	#

312	DTLLV	3772

334	DTLLA	1720

406	DTLLQ	1681

326	DTLQA	1340

371	DTLRV	1048

418	DTLQV	715

423	DTLRA	643

375	DTLMA	620

430	DTLMV	538

402	DTLAV	451

422	DTLAT	406

441	DSLLV	373

432	DTLSV	359

442	DTLLM	339

392	DTLRQ	334

443	DTLLI	306

444	DTLTQ	300

434	STLAE	269

445	DTLAA	268

395	DTLHQ	246

446	DTLSA	227

447	DTLKA	216

384	STLLV	213

448	STLQQ	201

449	DTLQQ	200

450	DTLLL	194

451	DTLMQ	189

225	STLQE	189

452	DTLNA	180

453	STLLA	176

454	DTLKV	163

455	STLNA	162

456	DTLRE	161

457	DTLTA	152

458	DTLQD	146

459	DTLVA	137

460	DTLLS	123

461	STLTQ	122

462	DSLLA	116

463	DTLRT	116

464	DTLQI	115

465	DTLMN	114

466	STLSE	114

467	SSLQV	112

468	TNLAV	109

469	DTLVV	108

470	DTLHA	107

471	DTLMT	107

437	SSLLV	107

209	STLLE	107

472	DSLRV	106

473	DTLAE	105

474	STLNV	105

475	DTLRN	101

476	DTLNV	100

477	DTLRD	99

478	DSLAV	94

479	DTLVQ	94

480	DTLQE	93

481	STLLD	92

482	DTLTH	89

483	SSLND	88

484	STLTV	88

385	STLMV	87

485	DTLML	86

286	STLLQ	85

202	STLRE	85

486	STLQA	84

487	DTLLD	83

488	DTLKQ	82

489	DTLLT	81

417	DTLAL	76

490	DTLII	75

491	DTLLN	75

492	DSLLQ	73

493	STLEQ	73

494	DTLGV	71

495	DVLRE	67

496	STLSA	66

497	DSLSV	65

498	DTLLE	63

499	STLAA	63

500	DTLKI	62

501	DTLKM	62

502	DTLQN	60

197	STLME	60

503	TTLMT	60

504	TTLAE	59

505	STLTE	58

506	VELVQ	57

507	TTLNQ	56

508	DTLMI	54

509	TTLMD	54

510	STLMA	51

511	DVLLA	50

512	DVLLT	49

235	STLKE	49

513	TTLNE	49

514	MTLPT	48

292	SSLME	48

251	STLHE	48

515	HTLVV	47

269	TTLME	46

516	ATLTQ	45

517	STLAS	45

333	STLVV	44

425	SSLLE	43

518	SSLVE	42

519	DALQA	41

520	DVLDA	41

521	GSLMQ	41

522	DTLTM	40

523	STLAQ	39

524	STLMI	38

525	DTLAM	37

526	DTLHT	37

527	DTLQL	37

528	DSLKQ	36

529	DSLRA	36

530	STLHV	35

531	STLMQ	35

532	DGLMA	34

533	DTLRL	34

534	SSLLT	34

535	DSLQA	33

536	DTLRI	33

537	STLGE	33

538	DALKE	32

539	STLRA	31

540	DTLHH	30

541	DTLRG	30

542	DTLRM	30

543	DVLMT	30

544	DTLEI	29

228	SSLNE	29

545	DTLHV	28

546	GTLDE	28

547	SSLAV	28

548	STLKQ	28

549	DTLMD	27

550	GTLQT	27

551	SSLVQ	27

297	STLRV	27

552	LMLMG	25

553	STLRQ	25

554	STLTA	25

8	DHLQT	24

555	DSLVA	23

556	SSLRV	23

557	DSLRE	22

558	GRLQD	22

559	MALQD	22

560	STLLH	21

561	STLVQ	21

562	VRLTA	21

563	AVLGD	20

564	PILVT	20

565	STLDD	20

566	DSLMI	19

567	STLID	19

568	TKLDT	19

569	ATLVA	18

570	DTLIA	18

571	DTLTE	18

572	GTLNH	17

573	STLAI	17

282	AHLGV	16

129	DNLLV	16

574	DQLVQ	16

575	MPLIL	16

576	TTLHQ	16

577	TTLQV	16

578	ATLLE	15

579	DVLHE	15

580	ETLRA	15

581	KVLRS	15

101	SNLLV	15

135	DNLMT	14

582	DSLRQ	14

583	DTLAN	14

584	GTLNV	14

585	HNLMV	14

586	QTLQA	14

587	RQLTT	14

588	DTLSI	13

589	DRLVG	12

590	ETLRQ	12

591	SSLGE	12

592	SSLVV	12

193	DHLQA	11

128	DNLLT	11

593	DTLME	11

594	DTLTV	11

595	DTLVG	11

596	ETLKA	11

597	GVLSQ	11

598	LALMR	11

599	RTLVE	11

600	TTLLI	11

601	TTLNV	11

602	DTLSE	10

391	SSLAE	10

603	STLAV	10

TABLE 3

ZF7 selection on C:A change at
nt 2 of core motif in CBS.
Sequences reflect position
2 through 6.

SEQ
ID		#
NO:	Sequence	read

100	DNLLA	2659

101	SNLLV	2616

135	DNLMT	2555

130	DNLQT	1983

129	DNLLV	1945

128	DNLLT	1922

132	DNLAT	1457

604	DNLRA	1117

102	DNLMA	1038

605	DNLMV	901

606	DNLQV	845

607	DNLQQ	841

396	DNLLQ	813

387	DNLKT	582

133	DNLQA	571

420	SNLQV	565

608	DNLRQ	494

426	SNLMV	459

383	SNLTV	458

609	DNLNT	412

428	DNLHT	389

610	SNLVV	349

611	SNLQQ	334

429	DNLTA	323

612	DNLLS	322

413	DNLAV	316

416	DNLRT	309

613	DNLTT	300

614	DNLAA	295

615	SNLLA	295

616	SNLLQ	278

617	SNLAV	257

618	DNLNA	240

619	DNLGT	240

103	DNLRV	239

620	DNLKA	167

621	DNLMQ	156

622	DNLKV	148

623	SNLNV	132

624	SNLMA	128

625	SVLQD	113

626	DNLQS	110

627	DNLSA	105

628	DNLAQ	103

629	DNLMS	98

630	DNLSQ	95

631	DNLNV	87

632	DNLGV	87

633	SNLLT	87

634	DNLIA	83

635	DNLNQ	83

636	SNLQT	80

637	SNLRV	79

638	SNLIV	79

639	DNLSV	74

640	SNLQA	60

641	SNLLL	57

642	SNLDV	56

643	DNLVQ	54

644	SNLLI	54

645	TGLAL	52

646	SNLMQ	51

647	DQLKI	40

648	GDLGT	40

649	SNLKV	39

650	VPLVD	38

651	DNLRI	37

652	DNLLI	37

653	TNLDV	36

654	HDLKI	35

655	DNLVV	35

312	DTLLV	32

656	DNLTV	31

657	DNLVT	31

658	SNLAQ	30

659	DNLIV	28

660	SNLMT	27

465	DTLMN	25

661	SNLTQ	23

662	EILRI	23

663	IGLEA	22

664	HRLGG	22

8	DHLQT	21

665	DNLST	20

666	MRLHV	19

667	SNLTT	18

668	SNLGV	16

669	SNLAT	16

15	EHLVV	16

670	ANLMV	14

671	HVLVG	14

672	SNLRA	13

673	HNLQL	12

674	DNLVA	12

675	SNLTA	12

676	KGLRM	12

334	DTLLA	12

677	PMLGV	11

678	GVLVA	11

679	DNLQD	11

680	MKLGT	11

406	DTLLQ	11

TABLE 4

ZF7 selection on A:T change at
nt 3 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		#
NO:	Sequence	Reads

173	RKHD	4641

175	RKAD	1938

174	RRSD	1299

681	RRHD	868

682	RKTD	182

683	NVSM	146

684	RQSD	76

685	RKND	69

686	SENV	69

687	VDHR	60

688	AQIV	58

689	KTPH	56

690	PKIV	51

691	GAEP	42

692	MLVE	40

693	VVGN	40

694	KGPE	36

695	GKVM	33

696	TEPG	33

697	TPHN	32

698	MPGG	31

699	DLEK	28

700	GTDN	27

701	ISRL	25

702	ATGL	21

703	ASNP	19

704	GAPT	17

705	HSPN	17

706	RPVA	16

177	RKDD	6

707	MLVD	4

708	RHRK	3

709	RKHV	3

710	RKQD	3

711	RKSD	3

712	DHHT	2

713	GKHD	2

714	MKAD	2

715	RKAE	2

716	RRAD	2

717	APIG	1

718	AQNR	1

719	DMDA	1

720	EAPM	1

721	EEMM	1

722	EPIR	1

723	GALE	1

724	GENV	1

725	GKAD	1

726	GKVD	1

727	GPLA	1

728	GRIE	1

729	IEKL	1

730	KAAS	1

731	KEEH	1

732	LKVD	1

733	LLVE	1

734	LMTQ	1

735	MASL	1

736	MGIG	1

737	MPGD	1

738	MSLG	1

739	NDMT	1

740	NMHT	1

741	NRIV	1

742	PENA	1

743	QKHD	1

744	QVPD	1

745	RASD	1

746	REHD	1

747	RGHD	1

748	RKHA	1

749	RKHY	1

750	RKLD	1

751	RKPD	1

752	RKVD	1

753	RKYD	1

754	RMSD	1

755	RRLD	1

756	RRND	1

757	RRRD	1

758	RRSG	1

759	RWHD	1

760	SHRL	1

761	SQHV	1

762	SSHD	1

763	TTHV	1

764	VHHV	1

765	WKAD	1

766	WKHD	1

TABLE 5

ZF7 selection on A:G change at
nt 3 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		Read
NO:	Sequence	#

174	RRSD	2997

173	RKHD	2731

175	RKAD	1867

177	RKDD	667

682	RKTD	475

767	HADA	411

710	RKQD	376

768	RKWD	296

745	RASD	265

681	RRHD	169

685	RKND	126

754	RMSD	40

769	RKGD	5

743	QKHD	3

757	RRRD	3

711	RKSD	3

752	RKVD	2

180	QALL	2

753	RKYD	2

756	RRND	2

720	EAPM	1

770	RRCD	1

771	MLPA	1

772	RATD	1

773	RKDV	1

774	KKPV	1

775	GEHG	1

776	HPVR	1

777	RQHD	1

778	RMMQ	1

779	RRGD	1

780	GREV	1

781	REQD	1

782	DRDM	1

783	SKHD	1

784	RLSD	1

785	VPTV	1

786	HKWD	1

787	KKND	1

788	RRSE	1

749	RKHY	1

789	READ	1

790	RNTD	1

791	MVRA	1

792	RKED	1

793	KTMG	1

794	NEPN	1

795	RGSD	1

796	RKRD	1

797	RWSD	1

798	TPLP	1

799	RKAN	1

800	RKAY	1

801	QLPL	1

709	RKHV	1

802	QGTS	1

803	DTMV	1

804	LKWD	1

805	MNTL	1

806	HADV	1

697	TPHN	1

750	RKLD	1

807	GRAH	1

704	GAPT	1

808	MKHD	1

809	HEDA	1

712	DHHT	1

810	RMLS	1

811	WRSD	1

812	DDAT	1

735	MASL	1

730	KAAS	1

TABLE 6

ZF7 selection on A:C change at
nt 3 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		Read
NO:	Sequence	#

173	RKHD	9

813	DTEN	6

775	GEHG	5

814	STKN	5

815	NIEI	5

801	QLPL	4

780	GREV	4

712	DHHT	4

782	DRDM	4

816	MVIN	4

817	VPDT	4

818	NIVP	4

819	MVPS	4

820	PNHP	4

821	KTDV	4

794	NEPN	3

760	SHRL	3

736	MGIG	3

822	HIKM	3

823	ILQI	3

741	NRIV	3

824	IVMQ	3

825	QTNS	3

826	ENMD	3

827	TVER	3

828	THDR	3

829	IRSP	3

771	MLPA	3

721	EEMM	2

830	ARIA	2

785	VPTV	2

831	EELI	2

832	KPLR	2

812	DDAT	2

833	NRLS	2

834	PTLR	2

835	MHIL	2

836	GGGP	2

837	MVEN	2

719	DMDA	2

838	IVAT	2

839	TLDR	2

840	MEPL	2

841	DTGV	2

842	TSRS	2

843	VLSI	2

844	STVQ	2

845	GPAQ	2

846	VEQP	2

847	MTKK	2

848	PLIM	2

802	QGTS	2

849	AMTV	2

850	SPMR	2

851	EPNV	2

735	MASL	2

852	MQIN	2

853	ALDE	2

728	GRIE	2

854	ALEH	2

855	REKD	2

856	ELLA	2

857	GVAR	2

858	VDTL	2

859	GHEN	2

730	KAAS	2

860	ELES	2

861	DPDT	2

862	SLEL	2

863	TMNV	2

764	VHHV	2

864	IQPV	2

865	MLQE	1

866	VMTV	1

867	MVEE	1

868	VARP...	1

869	KAIG	1

870	DRSM	1

871	KNSI	1

872	DDVS	1

873	KPQP	1

874	PHVP	1

875	DTLQ	1

876	KLGT	1

877	IDPH	1

878	HPNT	1

879	KSRG	1

880	RQMA	1

881	KKEN	1

882	QVLD	1

722	EPIR	1

883	RRQM	1

798	TPLP	1

884	ILKN	1

885	HQMK	1

179	ELLN	1

886	MDGG	1

887	AAGS	1

888	STVV	1

889	PARA	1

890	ALQG	1

891	SAPG	1

892	PVLN	1

742	PENA	1

893	TSLL	1

731	KEEH	1

894	HLDV	1

895	IHIR	1

896	SVTL	1

897	VKDR	1

898	KMTI	1

899	AGEM	1

900	GDSE	1

901	QPVK	1

902	KVEA	1

903	EQER	1

729	IEKL	1

984	GHHV	1

905	GMHL	1

906	RLRR	1

907	ATIR	1

908	RMDI	1

909	SVIH	1

910	MDIG	1

911	LART	1

912	RLMA	1

913	RQPP	1

914	MTMT	1

915	EDTR	1

739	NDMT	1

916	MRGR	1

917	ELHA	1

918	TNGQ	1

919	VNLT	1

920	MHIR	1

921	MLLQ	1

922	GRGE	1

923	NLRG	1

924	HIML	1

807	GRAH	1

805	MNTL	1

763	TTHV	1

793	KTMG	1

925	MTSV	1

926	RLSM	1

803	DTMV	1

720	EAPM	1

927	DMGM	1

928	MLMM	1

929	LMEM	1

930	QAVS	1

931	SRVL	1

932	DEDP	1

933	SGDR	1

934	MMNC	1

935	NIGM	1

936	MVQR	1

937	APHR	1

938	LDAG	1

939	RLAN	1

940	MKGS	1

941	KKLV	1

942	VNQE	1

943	ILKQ	1

944	PVIP	1

945	VESL	1

946	IKQN	1

947	EDNI	1

948	THRD	1

949	IPAG	1

950	GLNH	1

951	VDGR	1

181	PHRM	1

952	RTGA	1

953	VSPD	1

954	KVGD	1

TABLE 7

ZF6 selection on C:T change at
nt 5 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

955	GHMRR	29

956	GHMNR	23

34	EHMRR	23

957	THMRR	19

33	THMKR	17

126	EHMNR	17

958	GHMKR	12

127	EHMAR	11

959	EHMQR	10

147	SHMRR	10

960	SAMRR	9

961	ENMGR	8

962	SHMKR	8

35	THMNR	7

963	NHMRR	7

964	EGMRR	7

965	GNMGR	7

146	SHMNR	6

966	NGMRI	6

967	EGMAR	6

968	ESMRR	6

969	GHMSR	5

970	EGMHR	5

971	TAMRR	5

972	TNMQR	5

973	VNMRR	5

974	AHMKR	4

975	NGMTA	4

976	DGMRR	4

977	GHMTR	4

978	EHMSR	4

123	EHMKR	4

979	GSMRR	4

980	TNMLR	4

981	NHMKR	4

982	ENMLR	4

983	SPMGV	3

984	TNMGR	3

985	SSMAR	3

986	GGMRR	3

987	GGMKL	3

988	SGMVR	3

989	EHMHR	3

990	THMSR	3

991	GSMKI	3

992	EKMKE	3

993	NGMAR	3

994	QNMVR	3

995	DNMRR	3

996	ENMER	3

997	NSMRR	3

998	SGMKR	3

999	ANMQR	3

1000	GHMQR	3

1001	ANMGR	3

1002	DNMVR	3

1003	QAMRE	2

1004	GNMSR	2

1005	ESMQR	2

1006	TPMKV	2

1007	SNMGR	2

1008	GAMRI	2

1009	ANMNR	2

1010	DNMMR	2

1011	GSMKM	2

31	EHMGR	2

1012	GNMAQ	2

1013	EGMKG	2

1014	SSMKI	2

1015	TSMRR	2

1016	DGMKR	2

1017	DNMAR	2

1018	SSMRR	2

1019	GNMMR	2

185	NAMRG	2

1020	THMKL	2

1021	ENMAR	2

1022	NNMVR	2

1023	TGMKR	2

1024	TAMKR	2

1025	AHMNR	2

1026	QNMGR	2

1027	TNMVR	2

1028	NHMNR	2

1029	EHMTR	2

1030	GNMIR	2

1031	SGMRR	2

1032	NHMSR	2

1033	GGMRL	2

1034	SPMKV	2

1035	TNMRR	2

1036	GNMRE	2

1037	ENMMR	2

1038	THMER	1

1039	QKMRT	1

1040	GAMRR	1

1041	TPMEV	1

1042	GGMRE	1

1043	GDMDR	1

1044	GAMRA	1

1045	PNMSR	1

1046	EGMGR	1

1047	EGTHR	1

1048	QSMRE	1

1049	THMKG	1

1050	NNMGR	1

1051	GHMNS	1

1052	IDMKG	1

1053	ESMTR	1

1054	SHMKI	1

1055	HNMMR	1

184	SNMVR	1

1056	TAMKV	1

1057	DSMKR	1

1058	SNMAR	1

1059	ESMGR	1

1060	EAMRR	1

1861	GNMVR	1

1062	ANMRR	1

1063	DGMKI	1

1064	SHMHR	1

1065	GAMKE	1

1066	ESMRE	1

1067	GSMLR	1

1068	THMEV	1

1069	TSMGR	1

1070	EAMSK	1

1071	NAMRQ	1

1072	EGMRT	1

1073	SHMQR	1

1074	NGMKR	1

1075	ESMKE	1

1076	ANMHR	1

1077	DHTKR	1

1078	NGMRE	1

1079	GSMRA	1

1080	EGMNQ	1

1081	GGMRM	1

1082	PNMKR	1

1083	NGMKI	1

1084	SNMLR	1

1085	SNMRR	1

1086	SHMTR	1

1087	TGMRR	1

1088	SGMRI	1

1089	DNMGR	1

183	EGMTR	1

TABLE 8

ZF6 selection on C:A change at
nt 5 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

965	GNMGR	873

968	ESMRR	784

964	EGMRR	772

967	EGMAR	672

970	EGMHR	648

994	QNMVR	597

980	TNMLR	556

998	SGMKR	486

975	NGMTA	479

979	GSMRR	453

1003	QAMRE	452

961	ENMGR	434

960	SAMRR	431

993	NGMAR	401

1079	GSMRA	390

996	ENMER	389

1007	SNMGR	378

1046	EGMGR	376

1017	DNMAR	368

1063	DGMKI	347

999	ANMQR	342

1040	GAMRR	322

973	VNMRR	297

997	NSMRR	295

1005	ESMQR	293

1018	SSMRR	289

1087	TGMRR	289

1009	ANMNR	279

1044	GAMRA	275

183	EGMTR	273

126	EHMNR	265

1004	GNMSR	263

971	TAMRR	260

972	TNMQR	257

1010	DNMMR	253

976	DGMRR	241

1026	QNMGR	240

1082	PNMKR	228

1089	DNMGR	226

1090	ETMRR	225

1091	DNMKI	224

1014	SSMKI	224

995	DNMRR	221

1053	ESMTR	214

1042	GGMRE	214

984	TNMGR	211

1031	SGMRR	204

986	GGMRR	203

1022	NNMVR	201

1092	TNMER	197

1083	NGMKI	195

1021	ENMAR	194

1059	ESMGR	194

1019	GNMMR	193

1036	GNMRE	193

1002	DNMVR	187

1093	TNMAR	186

34	EHMRR	182

1066	ESMRE	181

1027	TNMVR	181

1015	TSMRR	175

988	SGMVR	173

1024	TAMKR	170

1030	GNMIR	169

985	SSMAR	163

991	GSMKI	159

1094	EHMKQ	149

982	ENMLR	149

1016	DGMKR	144

1012	GNMAQ	139

1095	SGMQR	138

1084	SNMLR	133

1061	GNMVR	130

1001	ANMGR	129

1096	HNMRR	129

1050	NNMGR	128

1081	GGMRM	127

1033	GGMRL	124

1097	QNMER	124

1057	DSMKR	122

1035	TNMRR	122

1008	GAMRI	115

1058	SNMAR	115

1056	TAMKV	114

1098	VSMKR	113

966	NGMRI	112

1099	TNMMR	110

1013	EGMKG	109

1071	NAMRQ	108

123	EHMKR	107

1032	NHMSR	106

1100	GAMRM	102

1070	EAMSK	100

1101	TAMNQ	99

1102	ESMSR	96

1103	GGMNQ	95

1048	QSMRE	95

185	NAMRG	92

1104	GGMKR	89

184	SNMVR	84

1105	ESMRL	83

1075	ESMKE	81

1106	SAMRE	80

1107	GGMQM	76

1023	TGMKR	73

1037	ENMMR	69

1108	NSMKM	69

1109	ESMKN	66

1072	EGMRT	64

987	GGMKL	64

1110	TNMSR	63

1111	DAMRV	61

1112	GNMER	60

1113	GAMRE	59

182	GNMAR	54

1114	EGMRK	53

1011	GSMKM	50

1115	SGMAR	58

TABLE 9

ZF6 selection on C:G change at
nt 5 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		#
NO:	Sequence	Read

34	EHMRR	3207

955	GHMRR	2397

957	THMRR	2025

956	GHMNR	1880

33	THMKR	1415

35	THMNR	1341

958	GHMKR	1208

978	EHMSR	1038

127	EHMAR	927

962	SHMKR	771

959	EHMQR	764

126	EHMNR	676

146	SHMNR	646

147	SHMRR	579

123	EHMKR	511

1029	EHMTR	460

963	NHMRR	436

992	EKMKE	381

32	DHMNR	374

981	NHMKR	342

983	SPMGV	322

977	GHMTR	318

1028	NHMNR	285

1116	DHMKR	264

969	GHMSR	258

1025	AHMNR	247

989	EHMHR	232

974	AHMKR	227

31	EHMGR	210

1117	GHMHR	129

1118	THMKV	129

1020	THMKL	117

1006	TPMKV	110

1000	GHMQR	105

1119	DHMRR	105

990	THMSR	97

1120	AHMRR	92

1121	EKMRE	86

1122	GHMAR	84

1074	NGMKR	81

1123	VHMNR	77

1052	IDMKG	72

1124	NHMTR	65

1032	NHMSR	64

964	EGMRR	57

1125	THMTR	57

1126	GHMKI	56

1073	SHMQR	52

1127	EHMVR	43

1086	SHMTR	43

1128	TKMKE	42

1129	EHMER	38

1130	THMKT	37

1043	GDMDR	36

1131	NGMRR	35

1132	EPMLM	34

1133	GHMVR	31

1134	THMRT	29

968	ESMRR	28

1135	PHMKR	26

1136	EHMRQ	24

1137	EHMRT	23

1138	DHMSR	22

1039	QKMRT	22

1139	ETMMI	21

1034	SPMKV	21

1140	SHMKL	21

1141	TPMKL	21

1142	GHMKM	20

965	GNMGR	19

1143	RQMLI	19

1144	GHMRM	18

1145	EGMKR	17

1146	EHMKA	17

1147	QIMPL	17

1148	SHMKV	16

1149	SGMNR	16

1150	THMAR	16

1151	QGMKR	15

960	SAMRR	14

1152	TKMEG	14

1153	RPMGR	14

1154	VHMRR	13

1155	THMRV	13

1068	THMEV	12

1156	NHMKS	11

1049	THMKG	11

1157	AAMST	11

980	TNMLR	11

996	ENMER	10

1158	GKMRD	10

1159	THMEL	10

998	SGMKR	10

1160	TPMRV	10

1161	SPMRV	10

1104	GGMKR	10

967	EGMAR	10

1162	THMGV	9

971	TAMRR	9

995	DNMRR	9

966	NGMRI	9

961	ENMGR	9

1163	MGMGR	8

973	VNMRR	8

1164	GKPSM	8

975	NGMTA	8

1165	SHMRV	8

1166	SPMNR	8

1167	SAMNR	8

1168	SHMSR	8

1169	NGMPR	8

972	TNMQR	8

1170	SPMRR	8

994	QNMVR	8

970	EGMHR	8

1017	DNMAR	7

1026	QNMGR	7

1171	GHMGV	7

1172	THMRL	7

979	GSMRR	7

1173	QHMKR	7

1174	THMGR	7

976	DGMRR	7

1175	THMQR	6

1038	THMER	6

1021	ENMAR	6

1176	RHMKR	6

1018	SSMRR	6

1177	EHMRV	6

1178	KHMKR	6

1179	QHMNR	6

1180	RAMKV	6

993	NGMAR	6

984	TNMGR	6

1002	DNMVR	6

1066	ESMRE	6

1181	GHMRV	6

982	ENMLR	6

185	NAMRG	5

1014	SSMKI	5

1182	TPMGV	5

1040	GAMRR	5

1183	GHMKV	5

1184	RHMNR	5

1009	ANMNR	5

1185	TPMEL	5

1022	NNMVR	5

988	SGMVR	5

1186	SPMKL	5

1187	SPMKR	5

1035	TNMRR	5

1082	PNMKR	5

1188	LAMEE	5

1044	GAMRA	5

1100	GAMRM	5

1046	EGMGR	5

1033	GGMRL	5

1189	PGMMS	5

986	GGMRR	5

991	GSMKI	5

1089	DNMGR	5

183	EGMTR	4

1190	SHMEV	4

1004	GNMSR	4

1191	GMMLT	4

1003	QAMRE	4

997	NSMRR	4

1087	TGMRR	4

1192	TPMKG	4

1041	TPMEV	4

1193	THMHR	4

1194	SHMGV	4

1063	DGMKI	4

1016	DGMKR	4

1195	THMKS	4

1196	THMRG	4

1197	GHMKT	4

1015	TSMRR	4

1019	GNMMR	4

999	ANMQR	4

1079	GSMRA	4

1036	GNMRE	4

1083	NGMKI	4

1008	GAMRI	4

1050	NNMGR	4

1198	THMRS	4

1013	EGMKG	4

1199	NHMQR	4

1007	SNMGR	4

1200	SHMAR	3

1061	GNMVR	3

1201	EAMKR	3

1202	GSMRE	3

1203	SPMEL	3

1204	AHMAR	3

1057	DSMKR	3

1205	PPMMV	3

1027	TNMVR	3

1096	HNMRR	3

1206	KHMNR	3

1030	GNMIR	3

1084	SNMLR	3

1207	TPMKR	3

1208	QSMKR	3

1209	RHMRR	3

1075	ESMKE	3

1210	DHMQR	3

1056	TAMKV	3

1211	AHMSR	3

1212	EHMRS	3

1213	AHMTR	3

1214	GHINR	3

1048	QSMRE	3

1093	TNMAR	3

1215	EYMRR	3

1216	GQMNR	3

1217	GHMKE	3

1011	GSMKM	3

1064	SHMHR	3

1059	ESMGR	3

1005	ESMQR	3

1051	GHMNS	3

1058	SNMAR	3

1012	GNMAQ	3

1023	TGMKR	3

1031	SGMRR	3

1001	ANMGR	3

987	GGMKL	3

1218	EHMMR	2

1219	SHMRL	2

1072	EGMRT	2

1107	GGMQM	2

1220	GGMKA	2

1070	EAMSK	2

1221	EHMPR	2

1222	AHMKS	2

1223	AHMQR	2

1224	GHTRR	2

1225	GHMKG	2

1226	EPMKV	2

1227	EHMAK	2

1228	GYMNR	2

1229	THMSS	2

1230	GDMNR	2

1231	GHMRT	2

1094	EHMKQ	2

1232	QRMGV	2

1233	GSMRQ	2

1234	DHMTR	2

1235	VEMER	2

1236	SPMEV	2

1237	GPMKV	2

1238	TPMER	2

1239	EHMDR	2

1240	EHVRR	2

1091	DNMKI	2

1241	GGMAR	2

1242	HHMKR	2

1243	GHMRS	2

1244	EYMAR	2

1245	KHMRR	2

1246	EHMSS	2

1247	TPMRL	2

1248	GHMSL	2

1249	VHMKR	2

1250	GHTNR	2

1251	GPMRT	2

1081	GGMRM	2

1092	TNMER	2

1109	ESMKN	2

1252	EQMRR	2

1053	ESMTR	2

1253	EHMKS	2

1254	THMKM	2

1065	GAMKE	2

1024	TAMKR	2

1010	DNMMR	2

985	SSMAR	2

1037	ENMMR	2

1255	GTMKM	1

1256	VHRIR	1

1257	DHMNK	1

1258	TPMNM	1

1259	RQMII	1

1260	EHMRW	1

1261	SPMRL	1

1262	GVMRA	1

1263	GHMQV	1

1264	GPMKL	1

1265	IDMKR	1

1266	PGMMG	1

1267	KHMER	1

1268	TPMNV	1

1269	EHVQR	1

1270	ENMKE	1

1271	DHMKM	1

1272	SHMNQ	1

1108	NSMKM	1

1273	GLMKR	1

1274	APMNL	1

1275	RHMSR	1

1276	EHMRG	1

1277	DWMRR	1

1278	GHMRH	1

1279	QNMHR	1

1280	CHMRR	1

1281	ERMRR	1

1282	EHMKE	1

1283	EPMKR	1

1284	AHINR	1

1285	SHMRT	1

1286	PHMNR	1

1287	AHMKV	1

1288	THMGM	1

1289	NGMKM	1

1290	EKMKR	1

1291	EHMIR	1

1292	NNMHR	1

1293	GNMNR	1

1294	KRMQR	1

1295	EKMRR	1

1296	TQMKQ	1

1297	EHMKV	1

1298	DHMKE	1

1299	EHTTR	1

1300	SPMRM	1

1301	GKMNR	1

1302	TNMKR	1

1303	THKRR	1

1304	SQTNR	1

1305	THLKR	1

1306	SHMQS	1

1307	THMSV	1

1308	THMRH	1

1309	DPMKV	1

1310	PHMMS	1

1311	SHVKR	1

1102	ESMSR	1

1312	SHMGL	1

1313	TDMVA	1

1314	PQMMS	1

1315	KHMQR	1

1316	EHMQL	1

1317	EHISR	1

1318	SHMKK	1

1319	EQMTR	1

1320	TPMRG	1

1321	GHISR	1

1322	GPMGV	1

1323	GYMRR	1

1324	GHMTV	1

1325	APMIM	1

1326	THINR	1

1327	DHMMS	1

1328	GHMKL	1

1329	EKMEE	1

1330	DPMRM	1

1331	SHMKT	1

1332	SPMGL	1

1333	SPMGE	1

1334	DHISR	1

1335	TPMKQ	1

1336	GHMKW	1

1337	EHMCR	1

1338	NNMKR	1

1339	ESMKR	1

1340	TEMLI	1

1341	SHMKM	1

1342	EHVNR	1

1343	GHMER	1

1344	NHMDR	1

1345	GHMWR	1

1346	THMKI	1

1347	QKMKE	1

1348	THMNK	1

1349	AHMKQ	1

1350	DHMGR	1

1351	EGMKW	1

1352	TQMKE	1

1353	TRMRR	1

1354	AHMGR	1

1355	TRMKR	1

1356	KNLTR	1

1357	PEMMS	1

1358	EHLTL	1

1359	RHMKV	1

1360	PGMIR	1

1361	THTKR	1

1362	EHIRR	1

1363	THMPR	1

1364	GKMKQ	1

1365	GPMRV	1

1366	AHVNR	1

1367	EPMSR	1

1368	PRMMV	1

1369	ELMSR	1

1090	ETMRR	1

1370	SNMNR	1

1371	TSMKT	1

1372	GNMHR	1

1373	TQMRR	1

1374	SHMKG	1

1375	DHMRT	1

1376	EHMRE	1

1377	SQLNR	1

1378	SHMGR	1

1379	GHKNR	1

1380	THMNL	1

1381	GYMKR	1

1382	SNMKV	1

1383	GHMRC	1

1384	NHMRV	1

1385	SGMKT	1

1386	EHLRR	1

1387	VPMRR	1

1388	DLMKR	1

1389	TSMKL	1

1390	APMTV	1

1105	ESMRL	1

1391	EHMLM	1

1392	EKMNR	1

1393	THRRR	1

1111	DAMRV	1

1394	ERMNR	1

1395	NHMHR	1

1396	DLMNR	1

1397	GQMQR	1

1398	RGMMI	1

1399	TQMKR	1

1400	EHMGV	1

1401	AHMTQ	1

1402	TPMMV	1

1403	GHKRR	1

1404	GPMER	1

1405	EPMQV	1

1101	TAMNQ	1

1406	GDMRR	1

1407	EHLKR	1

1408	DHMKK	1

1409	GDIDR	1

1410	GHMKK	1

1411	TQMMI	1

1412	SGMKA	1

1413	TPMRM	1

1414	SPMKG	1

1415	KQLNR	1

1416	NHMKT	1

1417	TKMRE	1

1098	VSMKR	1

1418	EHMAV	1

1419	EHMNS	1

1420	DHMHR	1

1421	AHMVR	1

1422	GRMRR	1

1423	GHMNV	1

1424	GHMNL	1

1425	GHVSR	1

1426	GQMHR	1

1427	EKMAR	1

1428	NHMGL	1

1429	EHMKG	1

1430	EPMAL	1

1431	AHLTR	1

1432	KHMTR	1

1433	GHMTM	1

1434	EPMSG	1

1435	NHMNM	1

1436	GQMKR	1

1437	TPMEG	1

1438	KHMRV	1

1439	SLMKR	1

1440	DGMRN	1

1441	RQMHI	1

1442	EPMRV	1

1113	GAMRE	1

1443	SHMRM	1

1444	EQMAR	1

1445	SHMRS	1

1446	EHMQV	1

1447	EPMPM	1

1448	IDMNR	1

1449	TKMKQ	1

1450	RQMLS	1

1451	ATMML	1

1452	PQMMI	1

1453	NAMKI	1

1454	GHMQS	1

1455	EAMKK	1

1456	THMRK	1

1457	PHMRR	1

1458	GHMKA	1

1459	AHMNH	1

1460	EYMSR	1

1461	EHMAW	1

1462	NHMGR	1

1463	GHMKS	1

1464	EHMRL	1

1465	ENMTR	1

1099	TNMMR	1

1466	QAMRV	1

1467	EHMQP	1

1468	THMSM	1

1469	IDMKE	1

1047	EGTHR	1

1055	HNMMR	1

1045	PNMSR	1

184	SNMVR	1

1062	ANMRR	1

1042	GGMRE	1

1060	EAMRR	1

1067	GSMLR	1

1054	SHMKI	1

1076	ANMHR	1

1069	TSMGR	1

1077	DHTKR	1

1078	NGMRE	1

1071	NAMRQ	1

1080	EGMNQ	1

1085	SNMRR	1

1088	SGMRI	1

TABLE 10

ZF6 selection on A:C change at
nt 6 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		Read
NO:	Sequence	#

37	HRES	6362

36	MNES	5959

1470	VKES	3337

1471	LRDS	2986

1472	HLES	1799

1473	TRES	1285

1474	MREA	648

1475	VRET	601

1476	MRET	284

1477	LLES	222

1478	MRTS	192

1479	ERKS	122

1480	IKES	111

38	RPDT	95

1481	VRVT	61

1482	RNES	51

1483	HVES	41

98	RTET	40

1484	LSHT	33

1485	RPES	33

1486	SRES	32

1487	ENKA	25

167	RADN	24

1488	TREN	23

1489	DSPQ	21

1490	RRES	20

1491	RGEN	17

1492	VRES	17

1493	HRDS	15

1494	HREA	15

1495	LRDT	15

1496	RVES	15

1497	EKKS	14

1498	GRES	13

1499	RMES	13

1500	LRES	12

1501	RTDN	12

1502	HADH	12

1503	VNES	12

1504	ANES	12

112	RTEN	12

1505	RNEH	11

1506	MNET	11

1507	RLDT	11

99	RADV	10

1508	RLET	9

1509	HRET	9

	HMR...	9

1510	NRES	8

1511	TGEA	8

1512	TGES	8

1513	RHET	8

1514	MRES	7

172	RNDT	7

1515	LVES	7

1516	VGSS	7

40	RHDT	7

1517	RIDT	7

1518	VREA	6

1519	HMES	6

1520	ERKN	5

1521	RPEA	5

1522	TPPI	5

1523	RREA	5

1524	RQEN	5

1525	VKDS	4

1526	RKES	4

1527	MLGL...	4

1528	DRPN	4

1529	RKEA	4

1530	VMLGL...	4

1531	TRDS	4

1532	HLET	4

1533	HLDS	4

1534	PPAT	4

1535	ENAS	4

1536	VKET	4

1537	GREA	4

1538	TREA	4

H...	4

1539	IRDS	3

1540	MNDS	3

1541	LLDS	3

1542	RTES	3

1543	RPET	3

1544	IDVH	3

1545	RTEH	3

1546	TRET	3

1547	HGES	3

1548	TMES	3

1549	LRVS	2

1550	PREA	2

1551	EGKN	2

1552	TSES	2

1553	VKFGHIFCVL

	L*NV...	2

1554	YRES	2

1555	MKES	2

39	RTDI	2

1556	MNEG	2

1557	MIES	2

1558	QRES	2

1559	MMEA	2

1560	MNER	2

	RGS	2

171	RTSS	2

1561	RNAS	2

1562	RTDT	2

1563	TRVS	1

1564	TFNV	1

1565	VRVS	1

1566	FRDS	1

1567	IKER	1

1568	RLEN	1

1569	IKET	1

1570	HRVS	1

1571	DRKG	1

1572	VKEC	1

1573	MSEA	1

1574	LRDR	1

1575	INES	1

1576	MSES	1

1577	NLES	1

1578	LQDS	1

1579	HAPT	1

	HRR...	1

1580	HRKA	1

1581	LRGS	1

1582	QSGT	1

1583	HUES	1

1584	ETGS	1

	SGT...	1

1585	MLGF...	1

1586	MNGS	1

1587	MRED	1

1588	TKES	1

1589	RPDH	1

1590	HRGS	1

1591	GNES	1

1592	LWDS	1

1593	MRDS	1

1594	IHES	1

1595	LRDG	1

1596	LRDC	1

1597	MYES	1

1598	RPNI	1

1599	EGRS	1

	TRR...	1

1600	RLES	1

1601	LGLPTGR...	1

1602	ARES	1

1603	HLGS	1

1604	HSES	1

1605	PRTS	1

1606	MNKS	1

1607	RRDS	1

1608	RREN	1

1609	QGES	1

1610	LREA	1

1611	LLET	1

1612	MREV	1

1613	VEES	1

1614	MNEA	1

1615	RNEN	1

1616	HWES	1

1617	RHEA	1

1618	MTES	1

1619	GRDS	1

1620	VSET	1

1621	MRKA	1

1622	EKES	1

1623	ERKG	1

	VKR...	1

1624	RNDH	1

1625	VPDA	1

	TGR...	1

1626	RKDA	1

1627	SPDT	1

1628	TTTL	1

1629	RKDS	1

1630	RRLT	1

1631	RTSN	1

	LRT...	1

1632	RQSA	1

1633	ARFT	1

1634	DRKS	1

169	RRDT	1

1635	RMDS	1

1636	HRKS	1

1637	GT113	1

1638	DKRN	1

1639	RPERE...	1

1640	SGDS	1

	TAG	1

	GR...	1

	T...	1

1582	...QSGT...	0

TABLE 11

ZF6 selection on A:G change at
nt 6 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		#
NO:	Sequence	Reads

38	RPDT	6216

1482	RNES	2750

98	RTET	1736

1485	RPES	1565

167	RADN	1412

112	RTEN	973

1499	RMTS	860

1507	RLDT	734

1490	RRES	690

1501	RTDN	588

1496	RVES	584

1505	RNEH	575

1517	RIDT	557

1521	RPEA	516

1491	RGEN	467

99	RADV	455

172	RNDT	452

1513	RHET	413

1529	RKEA	340

1508	RLET	297

1543	RPET	263

1523	RREA	252

40	RHDT	247

37	HRES	239

1526	RKES	231

1524	ROTN	199

1641	RGSA	186

171	RTSS	154

39	RTDI	152

1479	ERKS	123

36	MNES	104

1561	RNAS	90

1608	RREN	88

1642	RLDP	82

169	RRDT	80

1545	RTEH	80

1626	RKDA	63

1470	VKES	61

1643	RRET	53

1471	LRDS	44

1562	RTDT	36

1568	RLEN	35

1564	TFNV	29

1644	RADT	28

1472	HLES	28

1473	TRES	27

1645	RKET	24

1646	ATNM	23

1647	RREH	22

1648	RTDH	21

1632	RO5A	21

1542	RTES	20

1649	RNET	20

1650	RPDN	19

1651	THVP	19

1633	ARFT	18

1487	ENKA	18

1637	GTTP	17

1652	EASN	16

1653	RMTG	14

1654	RTAA	14

1589	RPDH	14

1627	SPDT	14

1489	DSPQ	14

1497	EKKS	13

1474	MREA	13

1655	RNEP	12

1656	VHDN	12

1657	RKEN	12

1658	RPYT	12

1659	ROTS	11

1660	RSGS	11

1661	RPDS	10

1475	VRET	10

1662	MTGN	7

1530	VMLGL...	7

1615	RNEN	7

1663	RGET	6

1664	RKGS	6

1600	RLES	5

1476	MRET	5

1624	RNDH	5

1665	RNDS	5

1666	STET	5

1537	GREA	5

1667	SNES	5

1668	RPDA	4

1669	RNER	4

1670	RPEN	4

1671	RVET	4

1672	RAET	4

1673	SHET	4

1674	RSDT	4

	Q...	4

1535	ENAS	3

1675	LPDT	3

1676	MMES	3

1677	SPES	3

1678	RMTN	3

1679	RVEI	3

1607	RRDS	3

1680	RMTT	3

1681	SADN	3

1682	RAES	3

1683	RPDV	3

1684	RTEA	3

1685	RHES	3

1686	ROTA	3

1478	MRTS	3

1520	ERKN	3

1687	RNRS	2

1688	RAEA	2

1689	RVDN	2

1690	RNEG	2

1691	RVEG	2

1692	RAEN	2

1693	RVDT	2

1694	RDDN	2

1695	RLEA	2

1696	RPNT	2

1697	RGES	2

1698	SPEA	2

1699	RTAG	2

1700	MKEA	2

1486	SRES	2

1701	WNES	2

1591	GNES	2

1629	RKDS	2

1628	TTTL	2

1702	RVEN	2

1635	RMDS	2

1703	RMEH	2

1630	RRLT	2

1704	RKEH	1

1705	ENRS	1

1706	RNKS	1

1707	RPGE...	1

1708	RKDT	1

1625	VPDA	1

1709	RGEA	1

1710	WIDT	1

1711	RNEY	1

1712	RADI	1

1713	RADY	1

1714	RTDD	1

1715	RVDS	1

1716	HTET	1

1717	HTEN	1

1718	SGEN	1

1719	RTST	1

1720	RAGR...	1

1721	SNAS	1

1722	RPGT	1

1723	RAEH	1

1724	MHDT	1

1725	REDN	1

1726	REEV	1

	RRR...	1

1727	RMEW	1

1728	RRER	1

1729	RLDN	1

	RPT...	1

1730	MVES	1

1510	NRES	1

1731	RIPA	1

1732	RMEA	1

1733	RHNT	1

1734	RNSS	1

1735	LPES	1

1736	SLDP	1

1737	STEN	1

1738	RPKS	1

	ATS...	1

1739	MIDT	1

1740	PPDT	1

1741	GLDA	1

1742	RPEGE...	1

1743	RHYT	1

1744	RTEI	1

1745	SPEN	1

	APR...	1

	LSL...	1

1746	RHEN	1

1747	REDV	1

1748	RLKT	1

1749	RIET	1

1750	RIES	1

1477	LLES	1

1751	RPDI	1

1752	MNDT	1

1753	RLYT	1

1504	ANES	1

1754	RAYN	1

1755	RADS	1

1756	KNES	1

1757	RVSA	1

1758	RPED	1

1759	RGEH	1

1728	RRER...	1

1760	LTET	1

1761	LADN	1

	GTR...	1

1762	RPER...	1

1763	MLGLPGTR...	1

1764	RPDP	1

1765	QADV	1

1599	EGRS	1

	RGR...	1

1766	MADV	1

1767	HTDN	1

1768	RKEV	1

1769	RADA	1

1770	RDAS	1

1771	MLDT	1

1772	RPGS	1

1773	RTEY	1

1774	SLDT	1

1775	RWES	1

1776	ERKA	1

1777	RIYT	1

1778	TPVP	1

1779	RQDA	1

1780	RMER	1

1631	RTSN	1

	LRT...	1

1559	MMEA	1

1481	VRVT	1

1634	DRKS	1

1488	TREN	1

1636	HRKS	1

1500	LRES	1

1639	RPERE...	1

1638	DKRN	1

1781	VGTV	1

1582	...QSGT...	0

TABLE 12

ZF6 selection on A:C change at
nt 6 of core motif in CBS.
Sequences reflect position
−1 to 3.

SEQ
ID		#
NO:	Sequence	Reads

37	HRES	7487

1479	ERKS	7125

1489	DSPQ	876

1487	ENKA	801

1497	EKKS	508

1473	TRES	141

38	RPDT	126

1520	ERKN	120

1537	GREA	112

1535	ENAS	103

1471	LRDS	95

36	MNES	89

1504	ANES	84

1571	DRKG	73

1634	DRKS	72

1599	EGRS	69

1584	ETGS	67

1482	RNES	60

1470	VKES	57

1486	SRES	50

98	RTET	42

1625	VPDA	39

1630	RRLT	37

167	RADN	30

1485	RPES	30

1782	ERGG	27

1472	HLES	25

1638	DKRN	25

112	RTEN	21

1628	TTTL	19

1636	HRKS	19

1490	RRES	19

1499	RMTS	18

1551	EGKN	17

1623	ERKG	16

1491	RGEN	16

1705	ENRS	15

1498	GRES	15

1501	RTDN	15

1507	RLDT	13

1496	RVES	13

1517	RIDT	13

1510	NRES	13

1505	RNEH	12

1783	EKGT	11

1513	RHET	11

1474	WIREA	10

1543	RPET	9

	QGK	9

1519	HNIES	9

1475	VRET	9

99	RADV	9

	HMR...	9

1784	ERNS	8

1524	ROTN	8

172	RNDT	8

40	RHDT	8

1493	HRDS	7

171	RTSS	7

1529	RKEA	7

1785	ENNS	6

1776	ERKA	6

1523	RREA	5

	RGS	5

	QEK...	5

1478	WIRTS	5

1500	LRES	4

1526	RKES	4

1786	HREN	4

1521	RPEA	4

1547	HGES	4

39	RTDI	4

1508	RLET	4

1477	LLES	3

1626	RKDA	3

1476	WIRET	3

1590	HRGS	3

1787	ERKR	3

1561	RNAS	3

1788	ERKI	3

1789	ERRS	2

1642	RLDP	2

1604	HSES	2

1790	YSPQ	2

1791	EGKS	2

1792	HRER	2

	QVK...	2

1793	DRKA	2

1794	ESGN	2

	QG...	2

1795	ERES	2

1796	HKES	2

1797	ESKS	2

1558	QRES	2

1798	WKS	2

1627	SPDT	2

169	RRDT	2

1527	MLGL	2

1633	ARFT	2

1562	RTDT	2

1799	KRKS	1

1652	EASN	1

1800	TGDA	1

1801	NRKS	1

	RGK	1

1802	EKNS	1

	HRE...	1

1803	QGKS	1

1662	WITGN	1

1804	DSPD.	1

	TGE...	1

1805	VRKS	1

1509	HRET	1

1806	ENKV	1

1568	RLEN	1

1732	RMTA	1

1494	HREA	1

1692	RAEN	1

1774	SLDT	1

	R...	1

1512	TGES	1

1644	RADT	1

	QAK...	1

1807	DIPQ	1

	QGT...	1

1808	ERKC	1

1809	HSPQ	1

1542	RTES	1

1538	TREA	1

1810	RTAT	1

	QGR...	1

1811	TRKS	1

1812	GRKS	1

1813	ESKA	1

	ERK...	1

1554	YRES	1

1814	EKRN	1

	MGK...	1

1815	DSPH	1

1816	ERNG	1

1817	VSPQ	1

	QWK...	1

1818	EKKC	1

1601	LGLPTGR...	1

1819	ERNN	1

1643	RRET	1

1820	TNES	1

1821	HRKN	1

	RLF...	1

1822	DKSN	1

1823	DRNS	1

	KRN	1

1824	ERMS	1

1608	RREN	1

1825	+IAS	1

1826	HREC	1

1827	ERKT	1

1828	ETGN	1

1632	RQSA	1

1631	RTSN	1

1635	RMDS	1

1545	RTEH	1

1559	MMEA	1

1629	RKDS	1

	LRT...	1

1481	VRVT	1

1488	TREN	1

1639	RPERE...	1

1637	GTTP	1

1640	SGDS	1

1582	...QSGT...	0

TABLE 13

ZF5 selection on G:T change at
nt 7 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		#
NO:	Sequence	Read

165	TRLKE	2129

42	HRLKE	1938

44	SRLKE	1530

110	TRLRE	1078

1829	HRLRE	1073

47	NRLKE	1015

1830	QRLRE	769

1831	DALKR	700

109	DGLKR	681

1832	SRLRE	534

43	HALKV	389

94	NRLKV	381

93	ERLRV	375

1833	DGLKK	374

41	HGLKV	335

1834	HRLKV	315

1835	ERLRM	295

1836	QRLKE	243

1837	DGLVR	235

46	HTLKV	233

1838	NRLRE	195

1839	ARLRE	168

108	DALRR	168

1840	ERLRQ	141

1841	ARLKE	135

1842	TRLRD	125

1843	DGLRR	118

1844	SRLNE	118

1845	TGLKV	92

1846	HRLSE	91

1847	HRLNE	78

1848	SHLKV	75

1849	TTLKV	75

1850	HRLGE	68

1851	STLKV	66

1852	DGLKV	65

1853	DGLRK	61

1854	HRLTE	60

1855	DRLKV	59

1856	HSLKV	56

45	DGLRV	47

1857	SRLKV	45

1858	QRLKV	44

1859	HGLTV	43

1860	HRLME	43

1861	RLLPN	42

1862	ERLKV	41

1863	NRLRV	35

1864	TRLKV	34

1865	DGLKE	29

454	DTLKV	29

1866	HGLRV	29

1867	SALKT	28

1868	HRLAE	25

1869	ERUS	23

1870	DGLTR	22

1871	DALVR	21

1872	HRLKR	21

1873	ERLRE	20

1874	HQLKV	20

1875	TTLKQ	18

1876	SRLKR	17

1877	DRLKQ	16

1878	HRLRV	16

1879	TRLKR	16

1880	TRLNE	16

1881	NRLKQ	15

1882	TRLKD	14

1883	TRLRV	14

1884	EALKR	13

1885	HTLKQ	13

1886	NALKV	13

1887	SALKV	13

1888	SRLKD	13

1889	DGLRE	12

1890	ERLKE	12

488	DTLKQ	11

1891	HKLKV	11

1892	GTLKV	10

1893	ERLRR	9

1894	HALKT	9

1895	HGLKE	9

1896	HHLVQ	9

1897	NGLKV	9

538	DALKE	8

1898	DALKV	8

1899	HALKE	8

1900	HHLKQ	8

1901	HHLKV	8

1902	TRLKK	8

1903	DRLRT	7

1904	DRLRV	7

371	DTLRV	7

1905	HRLKK	7

262	HTLKE	7

1906	NRLKK	7

235	STLKE	7

1907	SRLIE	6

1908	TRLME	6

1909	ATLKV	5

1910	HGLVV	5

1911	HRLRM	5

1912	HRLRQ	5

1913	HTLKA	5

1914	NRLRD	5

1915	TGLKE	5

1916	TGLKT	5

1917	TRLRQ	5

1918	TTLM	5

1919	TTLRV	5

1920	DRLKE	4

1921	HRLKA	4

1922	HRLKD	4

1923	HSLKE	4

1924	NRLM	4

1925	NRLKR	4

1926	STLKA	4

548	STLKQ	4

1927	TRLKA	4

1928	TRLKQ	4

1929	TRLRR	4

447	DTLKA	3

1930	HALKR	3

1931	HGLKA	3

1932	HGLKR	3

1933	HPEG...	3

1934	HRLK...	3

1935	HRLRK	3

1936	HTLRV	3

1937	NTLKQ	3

1938	QRLRV	3

1939	SRLME	3

1940	SRPKE	3

1941	TQLKV	3

1942	TRLQE	3

1943	TRLR...	3

1944	ARLKR	2

1945	ARLKV	2

1946	ARLR...	2

1947	ARLRV	2

1948	ARLVR	2

1949	DALKK	2

1950	DALRV	2

1951	DAPKR	2

1952	DRLRE	2

1953	EGLKV	2

1954	ERLLV	2

1955	ERLRA	2

1956	ERMRM	2

1957	GGLKV	2

1958	GGLVT	2

1959	HALRE	2

1960	HGLRE	2

1961	HHLKE	2

1962	HILKA	2

1963	HRLQE	2

1964	HRLRR	2

1965	KRLKE	2

1966	KTLKQ	2

1967	NALKE	2

1968	NRLNE	2

1969	NTLKV	2

1970	QRLKR	2

1971	QRLRQ	2

1972	QSLIA	2

1973	QTLKV	2

1974	RKLRS	2

1975	RRLRE	2

1976	SALKE	2

1977	SRLKK	2

1978	SRLRK	2

1979	SRLRV	2

297	STLRV	2

1980	TMLKE	2

1981	TRLKG	2

1982	TRLRM	2

1983	TRLTE	2

1984	TRRKE	2

1985	AALKR	1

1986	AGLKR	1

1987	AGLKV	1

1988	AGLVR	1

1989	ARLGE	1

1990	ARLME	1

1991	ARLNE	1

1992	ARLRD	1

1993	ARLRM	1

1994	CRLKE	1

1995	DALDR	1

1996	DALKT	1

1997	DALKW	1

1998	DALRK	1

1999	DALTV	1

2000	DELKR	1

2001	DELPG	1

2002	DGLK...	1

2003	DGLKG	1

2004	DGLKW	1

2005	DGLLR	1

2006	DGLRQ	1

2007	DGLTV	1

2008	DGLVW	1

1016	DGMKR	1

2009	DKLKQ	1

2010	DKLRQ	1

2011	DRLRK	1

2012	DTHAG...	1

2013	DTLKT	1

2014	DVLKK	1

2015	EAAG...	1

2016	EHLRQ	1

2017	ELLKV	1

2018	EPLRV	1

2019	ERLCV	1

2020	ERLKK	1

1893	ERLRR...	1

2021	ERLVR	1

2022	ERLWE	1

2023	ERPRM	1

2024	ERPRV	1

2025	ERQRM	1

2026	GGLKQ	1

2027	GGLKR	1

2028	GMLKV	1

2029	GRLKE	1

2030	GTLKQ	1

2031	HALKA	1

2032	HALKG	1

2033	HALPV	1

2034	HAPEV	1

2035	HGLKK	1

2036	HGLKQ	1

2037	HGLMV	1

2038	HGLPV	1

2039	HGLRD	1

54	HGLVR	1

2040	HGQKE	1

2041	HGRKV	1

2042	HGRRG	1

2043	HHLRV	1

2044	HILIA	1

2045	HKLKE	1

2046	HKLRV	1

2047	HMLKR	1

2048	HMLRE	1

2049	HNLKV	1

2050	HPLKV	1

2051	HQLKE	1

2052	HQLRE	1

2053	HQLRV	1

	HR*A...	1

2054	HRGCG...	1

2055	HRLDE	1

2056	HRLIE	1

2057	HRLKF	1

2058	HRLKG	1

2059	HRLKL	1

2060	HRLMV	1

2061	HRLN...	1

2062	HRLR...	1

2063	HRLRA	1

2064	HRLS...	1

2065	HRLVR	1

2066	HRMRE	1

2067	HRPKE	1

2068	HRPNE	1

2069	HRQRE	1

2070	HRRKE	1

2071	HRRME	1

2072	HRRRE	1

2073	HRVRE	1

2074	HSACG...	1

2075	HSLNV	1

2076	HSLRV	1

2077	HTLAQ	1

2078	HTLNV	1

2079	HTMKV	1

2080	HVLKV	1

2081	HWLRE	1

2082	KGLKQ	1

2083	MHLRS	1

2084	MRLRE	1

2085	MRLRM	1

2086	NALKR	1

2087	NGLKE	1

2088	NLLRE	1

2089	NMLKE	1

2090	NMLNV	1

2091	NPLRE	1

2092	NRFKE	1

2093	NRLIE	1

2094	NRLKA	1

2095	NRLKF	1

2096	NRLKL	1

2097	NRLKT	1

2098	NRLME	1

2099	NRLND	1

2100	NRLNV	1

2101	NRLQE	1

2102	NRLR...	1

2103	NRLRM	1

2104	NRLRQ	1

2105	NRMKE	1

2106	NRPKE	1

2107	NRPKV	1

2108	NRQKE	1

2109	NSLKE	1

2110	NTLTV	1

2111	PRLKE	1

2112	PRLLP	1

2113	PRLRE	1

2114	PRLTE	1

2115	QAEG...	1

2116	QRLIS	1

2117	QRLKK	1

2118	QRLME	1

2119	QRLRG	1

2120	QRLRM	1

2121	QRLTE	1

2122	QTA*R...	1

2123	QTAW...	1

2124	QTG*S...	1

	R...	1

2125	RGLKV	1

2126	RRLGD	1

2127	RRLKE	1

2128	RRLNE	1

2129	RRLTK	1

2130	SALKK	1

2131	SALKR	1

2132	SCLKE	1

2133	SGLAM	1

2134	SGLAV	1

2135	SGLKV	1

2136	SHLKE	1

2137	SKLKV	1

649	SNLKV	1

2138	SQLKV	1

2139	SRLIG	1

2140	SRLK...	1

2141	SRLKA	1

2142	SRLKG	1

2143	SRLQE	1

2144	SRLR...	1

2145	SRLRA	1

2146	SRLRM	1

2147	SRLRQ	1

2148	SRLTE	1

2149	SRQRE	1

2150	SSLKE	1

2151	SSLKV	1

2152	SSQRE	1

2153	STLKR	1

	TAG...	1

2154	TGLKG	1

2155	TGLKQ	1

2156	TGLKS	1

2157	TGLRV	1

2158	TGRRG	1

2159	TLLRE	1

2160	TMQKE	1

2161	TRL*L	1

2162	TRLAE	1

2163	TRLE...	1

2164	TRLEE	1

2165	TRLGE	1

2166	TRLK...	1

2167	TRLKY	1

2168	TRLRG	1

2169	TRLRK	1

2170	TRLSE	1

2171	TRPKE	1

2172	TRQRD	1

2173	TRRRD	1

2174	TRVRE	1

2175	TSLRE	1

2176	TTLKA	1

2177	TTLKE	1

2178	TTLKL	1

2179	TTLKT	1

2180	TTPRG	1

2181	TTRKQ	1

2182	TWLRE	1

2183	VRRKV	1

2184	YGLKR	1

2185	YRLKE	1

2186	YTLKV	1

TABLE 14

ZF5 selection on G:C change at
nt 7 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

44	SRLKE	2533

165	TRLKE	2146

42	HRLKE	1984

47	NRLKE	1528

1829	HRLRE	1001

1832	SRLRE	799

110	TRLRE	625

46	HTLKV	499

41	HGLKV	320

1830	QRLRE	299

1851	STLKV	249

1841	ARLKE	238

1836	QRLKE	135

235	STLKE	126

1849	TTLKV	102

447	DTLKA	95

1891	HKLKV	87

454	DTLKV	84

43	HALKV	82

1962	HILKA	80

1845	TGLKV	80

1839	ARLRE	78

1850	HRLGE	75

1838	NRLRE	75

1854	HRLTE	61

1861	RLLPN	55

1852	DGLKV	50

1834	HRLKV	46

1856	HSLKV	43

1931	HGLKA	37

94	NRLKV	30

1901	HHLKV	27

1972	QSLIA	26

371	DTLRV	25

1864	TRLKV	25

2177	TTLKE	25

262	HTLKE	24

1888	SRLKD	23

1948	ARLVR	20

2187	SKLKE	20

1855	DRLKV	19

93	ERLRV	19

1857	SRLKV	19

1831	DALKR	18

109	DGLKR	18

2029	GRLKE	18

1892	GTLKV	18

1842	TRLRD	17

1913	HTLKA	16

1868	HRLAE	15

488	DTLKQ	14

1895	HGLKE	14

2188	HILKT	14

1974	RKLRS	14

2133	SGLAM	12

1875	TTLKQ	12

1926	STLKA	11

1833	DGLKK	10

2126	RRLGD	10

1882	TRLKD	10

2189	TSLKV	10

1837	DGLVR	9

1835	ERLRM	9

1961	HHLKE	9

1896	HHLVQ	9

1847	HRLNE	9

1885	HTLKQ	9

1880	TRLNE	9

2190	HRLHE	8

1848	SHLKV	8

2191	SKLRM	8

45	DGLRV	7

1862	ERLKV	7

2192	GTLRV	7

1921	HRLKA	7

2193	HTLKS	7

1844	SRLNE	7

1915	TGLKE	7

108	DALRR	6

2194	HGLKT	6

1859	HGLTV	6

2045	HKLKE	6

1860	HRLME	6

1887	SALKV	6

1909	ATLKV	5

2195	DTLKE	5

2196	GILND	5

2135	SGLKV	5

2141	SRLKA	5

1871	DALVR	4

2197	ETLKV	4

1846	HRLSE	4

1923	HSLKE	4

1936	HTLRV	4

1969	NTLKV	4

1858	QRLKV	4

2140	SRLK...	4

2198	THLKE	4

1928	TRLKQ	4

1945	ARLKV	3

1853	DGLRK	3

1843	DGLRR	3

1840	ERLRQ	3

1957	GGLKV	3

1960	HGLRE	3

1900	HHLKQ	3

1965	KRLKE	3

2199	NALRV	3

1897	NGLKV	3

2200	NRLGE	3

1906	NRLKK	3

1975	RRLRE	3

2132	SCLKE	3

2137	SKLKV	3

2201	SRLRD	3

1979	SRLRV	3

548	STLKQ	3

1927	TRLKA	3

1942	TRLQE	3

2186	YTLKV	3

2202	APLLR	2

2009	DKLKQ	2

2203	DKLKV	2

1920	DRLKE	2

1873	ERLRE	2

1899	HALKE	2

2043	HHLRV	2

2051	HQLKE	2

2204	HRLEE	2

1878	HRLRV	2

2205	HTLKG	2

1966	KTLKQ	2

2206	MVLVV	2

2094	NRLKA	2

2207	NRLKD	2

1881	NRLKQ	2

2101	NRLQE	2

2108	NRQKE	2

2208	NTLKA	2

1938	QRLRV	2

1973	QTLKV	2

2127	RRLKE	2

2209	SRLKQ	2

2151	SSLKV	2

553	STLRQ	2

297	STLRV	2

1983	TRLTE	2

2175	TSLRE	2

1987	AGLKV	1

2210	AQMKE	1

1991	ARLNE	1

1992	ARLRD	1

2211	ARRRE	1

2212	CRLM...	1

2213	CRLMV	1

538	DALKE	1

1898	DALKV	1

2001	DELPG	1

1865	DGLKE	1

2010	DKLRQ	1

2214	DRLKA	1

2215	DRLKT	1

1952	DRLRE	1

1903	DRLRT	1

2013	DTLKT	1

2216	DTPKA	1

1869	ERLIS	1

1893	ERLRR...	1

2023	ERPRM	1

2026	GGLKQ	1

2028	GMLKV	1

2217	GRLKA	1

2218	GRLKV	1

2030	GTLKQ	1

2219	GVLKE	1

2220	GVLTG	1

2221	HALDV	1

2031	HALKA	1

2222	HELKV	1

2223	HGLEA	1

2036	HGLKQ	1

2224	HGLRG	1

2225	HGMKA	1

2226	HGPKV	1

2044	HILIA	1

2227	HILKE	1

2228	HILKV	1

2229	HILNA	1

2230	HKLKG	1

2231	HKLKQ	1

2046	HKLRV	1

2048	HMLRE	1

1933	HPEG...	1

2232	HPLKE	1

1874	HQLKV	1

2233	HRLGV	1

1922	HRLKD	1

2058	HRLKG	1

2059	HRLKL	1

1872	HRLKR	1

2234	HRLLE	1

2235	HRLQG	1

2063	HRLRA	1

2236	HRLRS	1

2237	HRLTV	1

2065	HRLVR	1

2066	HRMRE	1

2072	HRRRE	1

2238	HSG*G...	1

2239	HSLKQ	1

2240	HSLRE	1

2241	HSVKA	1

2242	HTG*R...	1

2077	HTLAQ	1

2243	HTLEV	1

215	HTLME	1

2244	HTLMV	1

2245	HTLQE	1

2246	HTLRQ	1

2080	HVLKV	1

2247	IRLKE	1

2248	IRQEE	1

2082	KGLKQ	1

2249	KRLKV	1

2250	LRLKK	1

2251	NKLKE	1

2252	NKLKG	1

2092	NRFKE	1

2253	NRLAE	1

2254	NRLEE	1

1925	NRLKR	1

2255	NRLKS	1

2097	NRLKT	1

1914	NRLRD	1

2256	NRLRG	1

1863	NRLRV	1

2257	NRLTE	1

2109	NSLKE	1

1937	NTLKQ	1

2258	PAEG...	1

2259	PPPPE	1

2113	PRLRE	1

2115	QAEG...	1

2260	QGRRE	1

2261	QRLEE	1

2119	QRLRG	1

2262	QSLGR	1

2134	SGLAV	1

2263	SKLK...	1

2264	SMLRE	1

2265	SRLAE	1

2266	SRLCE	1

2142	SRLKG	1

2267	SRLLE	1

2143	SRLQE	1

2145	SRLRA	1

1978	SRLRK	1

1940	SRPKE	1

2149	SRQRE	1

2268	SRRKE	1

2150	SSLKE	1

2152	SSQRE	1

539	STLRA	1

202	STLRE	1

2155	TGLKQ	1

2269	TGLRE	1

2270	THLKV	1

2271	TILYE	1

2272	TLLKE	1

1981	TRLKG	1

1908	TRLME	1

1883	TRLRV	1

2273	TRLTV	1

2274	TRMGE	1

2275	TRMKQ	1

2176	TTLKA	1

1918	TTLKI	1

2178	TTLKL	1

2276	YTLKE	1

TABLE 15

ZF5 selection on G:A change at
nt 7 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

46	HTLKV	3934

41	HGLKV	2682

1851	STLKV	2167

1861	RLLPN	1887

1849	TTLKV	1471

43	HALKV	923

454	DTLKV	888

1875	TTLKQ	754

1891	HKLKV	571

1885	HTLKQ	513

1845	TGLKV	482

1892	GTLKV	473

488	DTLKQ	462

1852	DGLKV	443

1856	HSLKV	352

1896	HHLVQ	298

1901	HHLKV	259

1834	HRLKV	210

42	HRLKE	190

371	DTLRV	189

44	SRLKE	186

165	TRLKE	178

1887	SALKV	177

1909	ATLKV	155

1900	HHLKQ	149

1926	STLKA	140

1897	NGLKV	136

47	NRLKE	124

548	STLKQ	118

1973	QTLKV	112

1874	HQLKV	94

2135	SGLKV	91

1829	HRLRE	89

1936	HTLRV	88

297	STLRV	78

447	DTLKA	75

1957	GGLKV	75

1928	TRLKQ	75

1966	KTLKQ	69

2277	HTL*A	66

1913	HTLKA	64

1832	SRLRE	61

110	TRLRE	58

1937	NTLKQ	56

2278	SKLKQ	55

1830	QRLRE	53

2203	DKLKV	51

1919	TTLRV	48

2151	SSLKV	43

1848	SHLKV	42

2030	GTLKQ	40

1864	TRLKV	40

2270	THLKV	38

1969	NTLKV	37

553	STLRQ	35

2279	HALRV	34

1931	HGLKA	33

2009	DKLKQ	32

109	DGLKR	29

1953	EGLKV	29

2197	ETLKV	29

2280	GILKV	28

1855	DRLKV	26

1866	HGLRV	24

2281	SVLKQ	23

1831	DALKR	22

93	ERLRV	22

2282	GQLHV	21

2283	TTLRQ	21

45	DGLRV	20

2284	DTLKN	20

2179	TTLKT	20

2285	GVLKV	17

2010	DKLRQ	16

2286	GTLKA	16

2026	GGLKQ	15

2036	HGLKQ	15

2043	HHLRV	15

94	NRLKV	15

2192	GTLRV	14

262	HTLKE	14

2287	SVLKV	14

2155	TGLKQ	14

1835	ERLRM	13

1838	NRLRE	13

2137	SKLKV	13

649	SNLKV	13

2288	TVLKV	13

1841	ARLKE	12

1839	ARLRE	12

1833	DGLKK	12

2289	HHLRQ	12

2205	HTLKG	12

2080	HVLKV	12

1917	TRLRQ	12

2290	NTLRQ	11

2134	SGLAV	11

108	DALRR	10

2291	QTLKQ	10

2292	RTLKQ	10

235	STLKE	10

1987	AGLKV	9

2013	DTLKT	9

274	HHLVV	9

2049	HNLKV	9

1836	QRLKE	9

2293	STLKG	9

2294	TVLKQ	9

1837	DGLVR	8

2295	GGLVV	8

2296	HGLQV	8

1850	HRLGE	8

1854	HRLTE	8

2246	HTLRQ	8

1857	SRLKV	8

2297	DTLKG	7

2298	GGLTV	7

2299	GVLKA	7

2031	HALKA	7

2194	HGLKT	7

2176	TTLKA	7

2300	GTLRQ	6

2301	HALKQ	6

1844	SRLNE	6

2302	STLKT	6

1842	TRLRD	6

2303	ATLKA	5

2304	ATLKQ	5

2305	DGLKQ	5

1843	DGLRR	5

1862	ERLKV	5

2306	GTLNA	5

2307	GVLKN	5

1895	HGLKE	5

1910	HGLVV	5

2308	TTLKG	5

1853	DGLRK	4

1840	ERLRQ	4

2309	ETLRV	4

2310	HGLKG	4

2311	HGLNV	4

1859	HGLTV	4

1961	HHLKE	4

1846	HRLSE	4

1886	NALKV	4

484	STLTV	4

2312	VGLGE	4

2186	YTLKV	4

2313	AGLAT	3

1948	ARLVR	3

2314	D*LPG	3

2003	DGLKG	3

2315	DKLRV	3

1899	HALKE	3

1860	HRLME	3

2239	HSLKQ	3

2078	HTLNV	3

2079	HTMKV	3

2316	HTQKV	3

2262	QSLGR	3

1974	RKLRS	3

474	STLNV	3

2177	TTLKE	3

1871	DALVR	2

2001	DELPG	2

2317	DGLRA	2

2318	DVLKV	2

2319	GALRV	2

2320	GGLVQ	2

2321	GNLKV	2

2322	GPLKV	2

2323	GTLKG	2

2324	GVLKQ	2

2325	GVLRV	2

678	GVLVA	2

2032	HALKG	2

2326	HDLKV	2

2327	HGLEV	2

2226	HGPKV	2

2328	HHMVQ	2

1962	HILKA	2

2329	HKLKA	2

2045	HKLKE	2

2231	HKLKQ	2

1921	HRLKA	2

2330	HRLKQ	2

1847	HRLNE	2

2082	KGLKQ	2

2331	KTLKV	2

2332	PTLKV	2

1972	QSLIA	2

2333	RLLPY	2

2334	RLRPN	2

2335	RTLAQ	2

2336	RTLKV	2

2337	SALTV	2

2338	STLKL	2

1916	TGLKT	2

2339	TKLKQ	2

1918	TTLKI	2

2340	TTPKV	2

2341	AGLAS	1

2342	AGLKM	1

2343	APLKV	1

1945	ARLKV	1

1992	ARLRD	1

2344	ATLKG	1

538	DALKE	1

1898	DALKV	1

2345	DELRQ	1

2346	DGLKA	1

1865	DGLKE	1

2347	DGLKL	1

2348	DKLKG	1

1877	DRLKQ	1

1952	DRLRE	1

1904	DRLRV	1

2349	DSLKV	1

2195	DTLKE	1

2350	DTLNQ	1

326	DTLQA	1

423	DTLRA	1

533	DTLRL	1

2351	DTLWQ	1

2352	DTMKV	1

2353	EGLKQ	1

1955	ERLRA	1

1873	ERLRE	1

2023	ERPRM	1

2354	ETLKE	1

2355	ETRRV	1

2356	GGLAV	1

2357	GGLRG	1

2358	GGLRV	1

2359	GHLKA	1

2196	GILND	1

2028	GMLKV	1

2360	GPLRA	1

2361	GQQHV	1

2362	GTLQA	1

2363	GTPKV	1

2364	HALES	1

2365	HALKF	1

2366	HALMV	1

2033	HALPV	1

2367	HAMKV	1

2368	HARKV	1

2222	HELKV	1

2369	HGLKD	1

2370	HGLKL	1

2371	HGLKM	1

2372	HGLKW	1

2373	HGRKI	1

2041	HGRKV	1

2374	HHLAQ	1

2375	HHLGQ	1

2376	HHLMQ	1

2377	HHMKV	1

2044	HILIA	1

2228	HILKV	1

2230	HKLKG	1

2378	HKLKM	1

2379	HKLNV	1

2380	HKLQE	1

2046	HKLRV	1

2381	HMLNV	1

2382	HPLDV	1

2050	HPLKV	1

2383	HPLQV	1

2384	HQLKA	1

2385	HQLKG	1

2386	HQLKT	1

1868	HRLAE	1

2058	HRLKG	1

2059	HRLKL	1

1872	HRLKR	1

1912	HRLRQ	1

2065	HRLVR	1

2067	HRPKE	1

2387	HSLKA	1

1923	HSLKE	1

2388	HSLKG	1

2389	HSLKL	1

2241	HSVKA	1

2077	HTLAQ	1

2390	HTLAV	1

2243	HTLEV	1

2391	HTLKN	1

2244	HTLMV	1

2392	HTLNA	1

2393	HTLQV	1

250	HTLTE	1

2394	HTLTV	1

2395	HTPKV	1

2396	HTRKQ	1

2397	HVLKF	1

2398	HVMKV	1

2399	HWLKV	1

2400	KADTV	1

2401	KGLKG	1

2402	KRLKQ	1

2403	KTLAQ	1

2404	KTLRV	1

2405	KTLTQ	1

2406	LHLKV	1

2407	LTLKQ	1

2408	LTLKV	1

2409	MGLKV	1

2410	MPPK	1

2411	MRLKQ	1

2412	NAVTE	1

2413	NGLKG	1

2414	NGLKL	1

2415	NRLKG	1

1914	NRLRD	1

1863	NRLRV	1

2416	NTLRV	1

2417	PGLKV	1

2418	QGLKV	1

1858	QRLKV	1

1938	QRLRV	1

2419	QRQRV	1

2420	QTLKA	1

2421	QTLKG	1

2422	QTLKK	1

2423	QTLKM	1

2424	QTLMV	1

2125	RGLKV	1

2425	RHLVQ	1

2426	RLLPT	1

2427	RLLSN	1

2428	RLMPD	1

2429	RMLPN	1

2126	RRLGD	1

2430	RSLKV	1

2431	RTLKG	1

2432	SALKQ	1

2433	SALRQ	1

2434	SELKV	1

2435	SFLKV	1

2133	SGLAM	1

2436	SGLKQ	1

2437	SHLKQ	1

2438	SKLKA	1

2187	SKLKE	1

1888	SRLKD	1

2145	SRLRA	1

556	SSLRV	1

2152	SSQRE	1

2439	STLKK	1

2440	STLKM	1

385	STLMV	1

448	STLQQ	1

554	STLTA	1

2441	STMKA	1

2442	STMKV	1

2443	TALKV	1

2444	TGLKA	1

2445	TGLKD	1

1915	TGLKE	1

2154	TGLKG	1

2446	TGLMV	1

2198	THLKE	1

2447	THLKG	1

2448	THLKL	1

2449	THLKQ	1

2450	THLMV	1

64	TKLKV	1

2451	TPLQV	1

1882	TRLKD	1

1981	TRLKG	1

2452	TRLPQ	1

1942	TRLQE	1

2453	TTLEV	1

2454	TTLHV	1

507	TTLNQ	1

577	TTLQV	1

2455	TTLRG	1

2456	TTLYV	1

2457	TTMKV	1

2458	TVLRQ	1

2459	VGLGG	1

2460	VTLKV	1

TABLE 16

ZF5 selection on G: A change position 8 of the CBS
core motif. Sequences reflect position 2 to 6.

SEQ
ID
NO:	Sequence	# Read

2461	GGLRR	341

50	GGLVR	336

2462	TGLRR	274

2463	EGLRR	267

1843	DGLRR	232

2464	SGLRR	206

2465	AGLAR	179

2466	SGLAR	178

2467	GGLAR	177

55	GGLTR	168

2468	DGLAR	152

1986	AGLKR	148

2469	TGLAR	135

1837	DGLVR	129

2470	GGLQR	127

70	GNLTR	124

117	GNLVR	123

2471	HGLAR	123

2027	GGLKR	111

2472	TGLVR	108

2473	AGLTR	105

2474	SGLSR	102

2475	AGLRR	100

2476	GGLSR	94

59	HGLRR	91

54	HGLVR	87

2477	SGLTR	84

2478	NGLVR	80

2479	AGLQR	79

118	GNLRR	79

2480	AGLHR	76

2481	GNLER	76

2482	HNLLR	76

138	GNLAR	73

1870	DGLTR	72

2483	HALRR	69

2484	HGLQR	69

2485	NGLRR	69

2486	SGLVR	68

2487	SNLDR	67

68	TNLRR	66

2488	HGLTR	63

2489	SSLRR	63

108	DALRR	61

2490	EGLTR	61

2491	GGLER	61

109	DGLKR	60

2492	TGLQR	60

56	HTLRR	59

1985	AALKR	58

1988	AGLVR	55

2493	AGLIR	54

1932	HGLKR	54

2494	ANLVR	53

2495	EGLKR	53

2496	SNLLR	51

2497	EGLAR	50

2498	AGLSR	49

2499	DGLIR	48

2500	TGLKR	48

2501	SGLQR	46

2502	ETLKR	45

2503	HGLLR	45

2504	NGLQR	45

2505	TGLMR	45

69	ANLRR	43

2506	DNLVR	42

2507	TGLLR	42

2508	DGLMR	41

2509	ASLKR	39

2510	QGLRR	38

2511	TNLVR	38

2512	NGLTR	37

2513	SGLDR	37

2514	SGLHR	37

2515	TGLNR	37

2516	TGLSR	37

2517	GNLLR	36

2518	NNLVR	36

2519	TGLIR	36

2520	DMLRR	35

2521	GALKR	35

2522	GNLDR	35

2523	SALRR	35

2524	SNLAR	35

2525	SGLLR	34

2526	TNLNR	33

2527	AGLLR	31

2528	GGLIR	31

2529	DGLHR	30

2530	DTLRR	30

2531	HLLKR	30

2532	SALAR	30

2533	SMLAR	30

2534	VGLKR	30

2535	DNLLR	28

2536	GGLMR	28

2537	SGLMR	28

2538	AALRR	27

2539	ETLRR	27

2540	NGLAR	27

2157	TGLRV	27

53	TGLTR	27

2541	TNLQR	27

2542	ANLAR	26

2543	NNLAR	26

2544	SNLSR	26

2545	STLSR	26

2546	AALAR	25

2547	HALVR	25

2548	HGLSR	25

2549	SGLNR	25

2550	STLAR	25

2551	ANLIR	24

2552	DGLDR	24

2553	DGLSR	24

2554	GTLKR	24

1884	EALKR	23

2555	NGLSR	23

2556	SMLRR	23

2557	HNLHR	22

2558	HNLRR	22

2559	SGLKR	22

2560	TGLGR	22

2561	TNLMR	22

1871	DALVR	21

2562	GTLTR	21

2563	DGLNR	20

2564	SSLVR	20

2565	TGLER	20

2566	DTLKR	19

2567	GNLSR	19

51	HGLIR	19

2568	HSLVR	19

2569	AGLNR	18

2570	DALAR	18

2571	GGLHR	18

2572	NGLIR	18

2573	QGLTR	18

2574	QMLKR	18

2575	QNLRR	18

1845	TGLKV	18

2576	AILKR	17

119	GNLKR	17

139	GNLMR	17

2577	HNLTR	17

2578	HTLAR	17

2579	QGLKR	17

2580	SGLER	17

2581	SGLGR	17

2582	SNLVR	17

2583	EALRR	16

2584	GTLRR	16

2585	HGLGR	16

2586	HTLMR	16

2587	NTLRR	16

2588	TGLHR	16

2589	TSLRR	16

2590	TTLQR	16

2591	DNLKR	15

2592	GALTR	15

2593	QTLRR	15

2594	SGLIR	15

2595	TNLKR	15

2596	DGLGR	14

2597	DSLQR	14

2598	EGLNR	14

2599	ENLRR	14

2600	GSLRR	14

2601	NGLNR	14

2602	QALKR	14

2603	SALSR	14

2604	SSLGR	14

2605	VNLKR	14

66	ATLRR	13

2005	DGLLR	13

2606	EMLKR	13

2607	GALVR	13

2608	GNLGR	13

2609	GNLQR	13

2610	HALAR	13

2611	HSLIR	13

2612	HTLER	13

2613	HTLQR	13

2614	NGLER	13

2615	NGLMR	13

2616	QGLVR	13

2617	TALKR	13

2618	TTLMR	13

2619	VGLRR	13

2620	ANLKR	12

2621	ANLNR	12

2622	ATLTR	12

2623	DNLRR	12

2624	ENLKR	12

2625	GGLLR	12

2626	GTLVR	12

2627	HNLSR	12

2628	NTLKR	12

2629	SALER	12

2630	SSLTR	12

2631	TALVR	12

52	ANLSR	11

2632	DNLAR	11

2633	ENLSR	11

2634	ESLRR	11

2635	NALRR	11

2636	NGLKR	11

2637	NNLLR	11

2418	QGLKV	11

116	SNLRR	11

2638	STLRR	11

2639	VNLSR	11

2640	DMLKR	10

2641	GALRR	10

2642	GGLDR	10

2643	HGLMR	10

2644	HNLVR	10

2645	HQLIR	10

2086	NALKR	10

1969	NTLKV	10

2646	QNLQR	10

1887	SALKV	10

2647	SMLIR	10

2648	TALRV	10

2649	TNLAR	10

2650	TQLKR	10

1849	TTLKV	10

2651	TTLTR	10

2652	VGLQR	10

2653	AALSR	9

2654	ATLAR	9

2655	DALGR	9

2656	DTLNR	9

2657	EILKR	9

2658	ESLKR	9

2659	GGLNR	9

2660	GSLTR	9

2661	HNLAR	9

2662	MGLKR	9

2663	NGLHR	9

2664	NMLKR	9

2665	PNLKR	9

2666	SALTR	9

2667	SDLKR	9

2668	STLGR	9

2669	AGLER	8

2670	DILRR	8

2671	DMLNR	8

2672	DTLAR	8

2673	HALLR	8

2674	HALSR	8

2675	HNLGR	8

2676	NALVR	8

2677	SMLTR	8

2678	TALAR	8

2679	TNLER	8

2680	TNLGR	8

2681	TTLNR	8

2682	DALLR	7

2683	DSLAR	7

2684	GTLAR	7

2685	GTLLV	7

2686	HALIR	7

2687	HGLDR	7

2688	HGLER	7

2689	HTLLR	7

2690	NNLIR	7

2691	NNLMR	7

2692	QSLKR	7

2693	SALGR	7

2694	SALVR	7

2695	SNLMR	7

2696	SQLRR	7

2697	STLQR	7

2698	STLVR	7

2699	SVLKR	7

2189	TSLKV	7

2700	AALTR	6

2701	DSLKR	6

2702	DSLRR	6

2703	DTLMR	6

2704	EGLLR	6

2705	ENLAR	6

2706	GNLNR	6

2707	GTLQR	6

2708	HALDR	6

2709	HVLER	6

2710	IGLRR	6

2711	INLTR	6

2712	NMLRR	6

2713	QMLRR	6

2714	TNLHR	6

2715	TSLHR	6

2716	VGLAR	6

2717	AALQR	5

2718	AGLDR	5

48	ATLKR	5

1833	DGLKK	5

2719	DTLQR	5

2720	DVLKR	5

2721	GALSR	5

2722	GMLKR	5

2723	GTLSR	5

2724	HNLER	5

2725	NGLLV	5

2726	NNLTR	5

2727	QALAV	5

2728	QGLAR	5

2729	QNLHR	5

2730	SALMR	5

2731	SLLLR	5

2732	SVLAR	5

2733	SVLTR	5

2734	TALRR	5

74	TMLRR	5

2735	TQLRV	5

2736	TTLLR	5

2737	TTLRR	5

2738	AALNR	4

2739	ATLVR	4

2740	DALHR	4

2741	DALMR	4

2742	DGLER	4

2743	DGLQR	4

45	DGLRV	4

2744	DLLRR	4

1855	DRLKV	4

2745	GGLGR	4

2746	GNLHR	4

1892	GTLKV	4

2747	GTLNR	4

2748	HALHR	4

2749	HALMR	4

2750	HILTR	4

2751	HLLLR	4

2752	HNLQR	4

2753	HTLGR	4

2754	IGLTG	4

2755	NGLLR	4

2756	NSLRR	4

2757	PNLIR	4

2758	PNLRR	4

2759	SALIR	4

2760	SILGR	4

2761	SPLVR	4

2762	STLTR	4

2763	TALKT	4

2764	TALTR	4

2765	TGLDR	4

2766	TSLKR	4

2767	TTLVR	4

2768	VGLQN	4

2769	VNLRR	4

2770	AALVR	3

58	ADLKR	3

2771	ANLGR	3

2772	ATLSR	3

2773	DNLQR	3

2774	DNLTR	3

2775	DRLRR	3

2776	DTLVR	3

2777	EGLVR	3

2778	GALNR	3

2779	GDLKR	3

2780	GDLTR	3

62	GGLGL	3

2781	GSLQR	3

1930	HALKR	3

2782	HGLHR	3

1866	HGLRV	3

2783	HTLKR	3

2784	HVLKR	3

2785	NGLDR	3

2786	NMLAR	3

2787	NSLAR	3

2788	NTLAR	3

2789	QGLHR	3

2134	SGLAV	3

2790	SILTR	3

2791	SILVR	3

2792	SQLKR	3

2793	SSLQR	3

2794	TALHR	3

2795	TALNR	3

2796	TALSR	3

2797	AGLGR	2

2798	AGLMR	2

2799	ASLQR	2

2800	ASLVR	2

2801	ATLMR	2

2802	AVLKR	2

2803	DALNR	2

2804	DALQR	2

2805	DALSR	2

1853	DGLRK	2

2806	DHLHR	2

2807	DHLVR	2

2808	DNLSR	2

2809	DTLSR	2

2810	DTLTR	2

2811	DVLRR	2

2812	EGLIR	2

2813	EGLSR	2

2814	GAEE . . .	2

2815	GALQR	2

2319	GALRV	2

2816	GDLRR	2

2817	GDLVR	2

1957	GGLKV	2

2358	GGLRV	2

2818	GSLAR	2

2819	GSLKR	2

2820	HDLRR	2

2821	HGLNR	2

2822	HHLIR	2

2047	HMLKR	2

2823	HMLRR	2

2824	HQLVR	2

2825	HSLAR	2

2826	HSLHR	2

2827	HSLRR	2

46	HTLKV	2

2828	HTLNR	2

2829	HTLTR	2

2830	HTLVR	2

2831	IGLKR	2

2832	ITLKR	2

2833	MTLKR	2

2834	NALHR	2

2835	NALSR	2

2836	NGLGR	2

2837	NTLHR	2

2838	QDLKR	2

2839	QGLLR	2

2840	QNLLR	2

2841	QNLRW	2

2842	QSLRR	2

2843	QTLKR	2

2131	SALKR	2

2844	SALRV	2

2845	SSLAR	2

2846	SSLSR	2

2847	STLDR	2

2848	STLER	2

2849	STLHR	2

1851	STLKV	2

2850	STLMR	2

2851	TALGR	2

2852	TGLAT	2

2853	TGLSV	2

2854	TGLVT	2

2855	TNLKV	2

2856	TNLSR	2

2857	TTLAR	2

2858	TTLGR	2

2859	TTLIR	2

2860	TTLKR	2

2179	TTLKT	2

2861	TVLRM	2

2862	VQLAM	2

2863	VTLTR	2

	A*S . . .	1

2864	AALLR	1

2865	AALMR	1

2866	AAPER	1

2867	ADLRR	1

2868	AGLAW	1

2869	AGLRW	1

2870	AGLTS	1

2871	AILTR	1

71	AMLKR	1

2872	ANLPR	1

1944	ARLKR	1

2873	ARLQR	1

2874	ARLTR	1

2875	ASLRR	1

2876	ASLTR	1

2877	ATLDR	1

2878	ATLER	1

2879	ATLIR	1

2880	ATLLR	1

2881	ATLQR	1

2882	AVLRR	1

1831	DALKR	1

1950	DALRV	1

2883	DGLSV	1

2884	DILHR	1

2885	DQLRR	1

2886	DSLSR	1

2887	DTLAK	1

2888	DVLLR	1

2889	EALNR	1

2890	EALTR	1

1953	EGLKV	1

2891	EGLMR	1

2892	EGLQR	1

2893	EGLRL	1

2894	EGLRV	1

2895	EGVRR	1

2896	ELLRR	1

2897	ENLER	1

2898	ETLLR	1

2899	GALHR	1

2900	GGHRR	1

2901	GGLAG	1

2356	GGLAV	1

2902	GGLDV	1

2903	GGLGS	1

2904	GGLQE	1

2905	GGLVL	1

1958	GGLVT	1

2906	GGPSH	1

2907	GGPSR	1

2908	GGQRR	1

2909	GGVRR	1

2910	GGWR . . .	1

2911	GILER	1

2912	GKLRR	1

2913	GMLAR	1

2914	GNLIR	1

2915	GSLER	1

2916	GSLVR	1

2917	GTLER	1

2918	GTLGR	1

2919	GTLHR	1

2920	GTQVR	1

2921	GVLRR	1

2922	GVLTR	1

2923	HALGR	1

43	HALKV	1

2924	HDLAK	1

2925	HGAAR	1

2035	HGLKK	1

2371	HGLKM	1

41	HGLKV	1

2926	HGLSV	1

2927	HGLTW	1

2928	HGPAR	1

2929	HKLAR	1

2930	HNLLS	1

2931	HRLSR	1

2932	HSLNR	1

2933	HSLSR	1

2934	HTLHR	1

2935	HVLAR	1

2936	INLSR	1

2937	NALAR	1

2938	NHLVQ	1

2939	NTLIR	1

2940	NTLNR	1

2941	NTLQR	1

2942	NVLKR	1

2943	PALKR	1

2944	PGLLR	1

	PWS . . .	1

2945	QAAWG . . .	1

2946	QALAR	1

2947	QALTR	1

2948	QDLIR	1

2949	QTLAR	1

2950	QTLQR	1

2951	QVLRR	1

2952	RGLTR	1

2953	RGLVR	1

2954	SALDR	1

2955	SALMC	1

2956	SALNR	1

2957	SDLAR	1

2958	SDLQR	1

2959	SDLRR	1

2960	SGPRR	1

2961	SLLSD	1

2962	SMLHR	1

2963	SNLQR	1

2964	SSLIR	1

2965	SSLKR	1

2966	STLLR	1

2967	STLNR	1

2968	STLRK	1

2969	SVLGR	1

2970	SVLRR	1

2971	TALER	1

2972	TALRT	1

2973	TDLAR	1

2974	TDLRR	1

2975	TGLQV	1

2976	TGLVRR	1

2977	TGPAR	1

2978	TMLKR	1

2979	TNLPR	1

2980	TSLAR	1

2981	TSLGG	1

2982	TSLGR	1

2983	TSLQR	1

2984	TSLVR	1

2985	VALAR	1

2986	VALKR	1

2987	VALSR	1

2988	VGLKC	1

2989	VGLSR	1

2990	VGLTM	1

2991	VNLAR	1

2992	VNLIR	1

2993	VNLNR	1

2994	VTLGR	1

2995	VTLKR	1

2996	VTLMR	1

2997	VTLRR	1

2998	WGLER	1

TABLE 17

ZF5 selection on G: C change at nt 8 of core motif
in CBS. Sequences reflect position 2 to 6.

SEQ
ID
NO:	Sequence	# Read

1843	DGLRR	498

108	DALRR	388

2463	EGLRR	348

1871	DALVR	288

1837	DGLVR	262

2468	DGLAR	261

1986	AGLKR	257

1870	DGLTR	255

2462	TGLRR	237

2530	DTLRR	196

59	HGLRR	192

66	ATLRR	176

2539	ETLRR	149

2464	SGLRR	142

2584	GTLRR	136

50	GGLVR	132

2545	STLSR	132

2707	GTLQR	131

2553	DGLSR	127

2027	GGLKR	126

2684	GTLAR	123

2578	HTLAR	114

2486	SGLVR	111

2779	GDLKR	109

2593	QTLRR	107

2472	TGLVR	106

2668	STLGR	103

2776	DTLVR	102

2563	DGLNR	100

2811	DVLRR	100

2698	STLVR	100

2720	DVLKR	99

48	ATLKR	96

2461	GGLRR	93

2638	STLRR	93

2802	AVLKR	91

2816	GDLRR	90

2554	GTLKR	89

1932	HGLKR	89

56	HTLRR	89

2492	TGLQR	87

2559	SGLKR	86

2672	DTLAR	84

2654	ATLAR	83

2848	STLER	81

2737	TTLRR	80

2495	EGLKR	79

2562	GTLTR	79

2469	TGLAR	75

2529	DGLHR	74

54	HGLVR	74

2828	HTLNR	73

2967	STLNR	71

2489	SSLRR	69

2516	TGLSR	68

2772	ATLSR	67

2656	DTLNR	67

2788	NTLAR	66

58	ADLKR	65

2570	DALAR	65

2626	GTLVR	64

2719	DTLQR	62

2739	ATLVR	61

2478	NGLVR	61

109	DGLKR	59

2467	GGLAR	59

2568	HSLVR	59

2804	DALQR	58

2507	TGLLR	58

2640	DMLKR	57

55	GGLTR	56

2867	ADLRR	55

2474	SGLSR	55

2564	SSLVR	54

2500	TGLKR	53

2475	AGLRR	52

2550	STLAR	52

2783	HTLKR	51

2587	NTLRR	51

2857	TTLAR	51

2622	ATLTR	49

2817	GDLVR	49

2667	SDLKR	49

2767	TTLVR	49

2466	SGLAR	48

2847	STLDR	48

2850	STLMR	48

2515	TGLNR	48

2502	ETLKR	47

2970	SVLRR	47

2849	STLHR	46

2959	SDLRR	45

2699	SVLKR	44

2488	HGLTR	43

2702	DSLRR	42

2974	TDLRR	42

2471	HGLAR	40

2586	HTLMR	40

2477	SGLTR	40

2966	STLLR	40

2736	TTLLR	40

2636	NGLKR	39

2810	DTLTR	38

2598	EGLNR	37

2723	GTLSR	37

2978	TMLKR	37

2589	TSLRR	37

2801	ATLMR	36

2999	DALTR	36

2697	STLQR	36

2762	STLTR	36

2780	GDLTR	35

2476	GGLSR	35

51	HGLIR	35

2509	ASLKR	34

2630	SSLTR	34

1985	AALKR	33

3000	DALIR	33

2859	TTLIR	33

2490	EGLTR	32

2753	HTLGR	32

2613	HTLQR	32

2692	QSLKR	32

2701	DSLKR	31

2131	SALKR	31

2845	SSLAR	31

2618	TTLMR	31

2878	ATLER	30

2086	NALKR	30

2594	SGLIR	30

2556	SMLRR	30

3001	GVLKR	29

53	TGLTR	29

2497	EGLAR	28

2612	HTLER	28

2766	TSLKR	28

3002	GDLHR	27

2644	HNLVR	27

1936	HTLRV	27

2465	AGLAR	26

3003	GDLNR	26

2503	HGLLR	26

3004	SILKR	26

2858	TTLGR	26

2499	DGLIR	25

2732	SVLAR	25

2590	TTLQR	25

2473	AGLTR	24

1988	AGLVR	24

2805	DALSR	24

3005	DTLIR	24

2777	EGLVR	24

2579	QGLKR	24

2820	HDLRR	23

2784	HVLKR	23

3006	NTLTR	23

2957	SDLAR	23

2965	SSLKR	23

2973	TDLAR	23

2803	DALNR	22

3007	HTLIR	22

2628	NTLKR	22

2838	QDLKR	22

2860	TTLKR	22

3008	EVLRR	21

3009	GDLSR	21

3010	HVLRR	21

2837	NTLHR	21

3011	TDLTR	21

2681	TTLNR	21

1833	DGLKK	20

2520	DMLRR	20

2919	GTLHR	20

2833	MTLKR	20

2980	TSLAR	20

3012	ATLHR	19

3013	DSLVR	19

3014	GTLDR	19

2830	HTLVR	19

3015	NTLLR	19

2843	QTLKR	19

2634	ESLRR	18

3016	HDLQR	18

2821	HGLNR	18

2823	HMLRR	18

57	TVLKR	18

3017	ATLNR	17

2596	DGLGR	17

2485	NGLRR	17

2549	SGLNR	17

2501	SGLQR	17

3018	STLIR	16

2617	TALKR	16

2519	TGLIR	16

3019	TTLSR	16

3020	DILKR	15

3021	ETLNR	15

2916	GSLVR	15

3022	MDLKR	15

2504	NGLQR	15

2949	QTLAR	15

2964	SSLIR	15

2538	AALRR	14

2818	GSLAR	14

2484	HGLQR	14

2512	NGLTR	14

3023	QDLRR	14

2588	TGLHR	14

3024	TSLTR	14

71	AMLKR	13

3025	ATLGR	13

3026	GDLQR	13

2470	GGLQR	13

2819	GSLKR	13

3027	NTLVR	13

3028	SILRR	13

2582	SNLVR	13

2846	SSLSR	13

2995	VTLKR	13

2880	ATLLR	12

2597	DSLQR	12

2659	GGLNR	12

2548	HGLSR	12

2525	SGLLR	12

2792	SQLKR	12

2505	TGLMR	12

2982	TSLGR	12

2479	AGLQR	11

2670	DILRR	11

3029	DTLER	11

3030	DTLLR	11

2917	GTLER	11

2689	HTLLR	11

2540	NGLAR	11

2663	NGLHR	11

3031	SDLTR	11

3032	SMLKR	11

1849	TTLKV	11

2879	ATLIR	10

2722	GMLKR	10

2600	GSLRR	10

3033	GTLLR	10

2510	QGLRR	10

2480	AGLHR	9

2498	AGLSR	9

2740	DALHR	9

2005	DGLLR	9

3034	DTLGR	9

3035	GDLAR	9

1930	HALKR	9

2782	HGLHR	9

46	HTLKV	9

3036	HVLVR	9

2664	NMLKR	9

2939	NTLIR	9

3037	QDLAR	9

2560	TGLGR	9

2875	ASLRR	8

2881	ATLQR	8

3038	ETLAR	8

2592	GALTR	8

2607	GALVR	8

2547	HALVR	8

2643	HGLMR	8

3039	HILKR	8

3040	HMLVR	8

2827	HSLRR	8

3041	NTLSR	8

2948	QDLIR	8

3042	SDLVR	8

2537	SGLMR	8

2677	SMLTR	8

2189	TSLKV	8

2651	TTLTR	8

2700	AALTR	7

3043	ETLQR	7

2521	GALKR	7

2641	GALRR	7

2528	GGLIR	7

117	GNLVR	7

3044	HDLGR	7

3045	HDLTR	7

2826	HSLHR	7

2934	HTLHR	7

2942	NVLKR	7

2678	TALAR	7

3046	TDLKR	7

1845	TGLKV	7

3047	TSLNR	7

2983	TSLQR	7

3048	VDLKR	7

2014	DVLKK	6

3049	GILKR	6

2921	GVLRR	6

2610	HALAR	6

2483	HALRR	6

2531	HLLKR	6

3050	HNLKR	6

2834	NALHR	6

3051	QDLQR	6

2616	QGLVR	6

2532	SALAR	6

3052	SDLGR	6

2514	SGLHR	6

2302	STLKT	6

3053	TDLSR	6

2565	TGLER	6

2742	DGLER	5

3054	DILVR	5

2566	DTLKR	5

1884	EALKR	5

2657	EILKR	5

3055	GVLVG	5

3056	HSLTR	5

3057	HTLDR	5

2937	NALAR	5

2572	NGLIR	5

2555	NGLSR	5

3058	QQLQR	5

2523	SALRR	5

2694	SALVR	5

2513	SGLDR	5

2581	SGLGR	5

2496	SNLLR	5

3059	SVLLR	5

3060	TDLGR	5

3061	TDLQR	5

2534	VGLKR	5

2493	AGLIR	4

2576	AILKR	4

3062	ALLKR	4

2683	DSLAR	4

2886	DSLSR	4

3063	DTLRK	4

3064	ETLTR	4

3065	GELTR	4

70	GNLTR	4

2660	GSLTR	4

2918	GTLGR	4

2748	HALHR	4

3066	HDLNR	4

2482	HNLLR	4

3067	MTLRR	4

2615	NGLMR	4

3068	NTLER	4

2956	SALNR	4

2958	SDLQR	4

3069	SELKR	4

2580	SGLER	4

2604	SSLGR	4

3070	STLSM	4

3071	TDLMR	4

68	TNLRR	4

2650	TQLKR	4

3072	TSLLR	4

3073	TSLMR	4

2984	TSLVR	4

3074	TTLER	4

3075	TVLRR	4

2738	AALNR	3

3076	ADLTR	3

2669	AGLER	3

2542	ANLAR	3

69	ANLRR	3

2877	ATLDR	3

2741	DALMR	3

3077	DILTR	3

3078	DMLQR	3

2632	DNLAR	3

2591	DNLKR	3

2809	DTLSR	3

3079	DVLVR	3

2583	EALRR	3

2813	EGLSR	3

3080	ETLRK	3

2481	GNLER	3

3081	GTLMR	3

2747	GTLNR	3

3082	HAEG . . .	3

3083	HDLMR	3

3084	HMLQR	3

2577	HNLTR	3

3085	HSLKR	3

2829	HTLTR	3

2935	HVLAR	3

2835	NALSR	3

2518	NNLVR	3

3086	QSLNR	3

3087	SILAR	3

2962	SMLHR	3

297	STLRV	3

2733	SVLTR	3

3088	SVLVR	3

2734	TALRR	3

2981	TSLGG	3

2994	VTLGR	3

2546	AALAR	2

2864	AALLR	2

2770	AALVR	2

3089	ADLVR	2

2569	AGLNR	2

2494	ANLVR	2

3090	ASLAR	2

3091	ASLIR	2

2800	ASLVR	2

2655	DALGR	2

2552	DGLDR	2

2743	DGLQR	2

1853	DGLRK	2

2506	DNLVR	2

3092	DVLMR	2

3093	DVLQR	2

3094	EGLGR	2

3095	EGLHR	2

2892	EGLQR	2

2658	ESLKR	2

2536	GGLMR	2

138	GNLAR	2

139	GNLMR	2

3096	HDLSR	2

2687	HGLDR	2

2585	HGLGR	2

2371	HGLKM	2

3097	HILMR	2

2557	HNLHR	2

2627	HNLSR	2

2611	HSLIR	2

3098	HSLQR	2

3099	HVLHR	2

3100	IDLKR	2

2755	NGLLR	2

3101	NILVR	2

2943	PALKR	2

3102	PGLAR	2

3103	PTLMR	2

2573	QGLTR	2

2574	QMLKR	2

2842	QSLRR	2

3104	QTLSR	2

2759	SALIR	2

2603	SALSR	2

3105	SELRR	2

2487	SNLDR	2

116	SNLRR	2

2544	SNLSR	2

2696	SQLRR	2

2153	STLKR	2

2968	STLRK	2

3106	TDLHR	2

3107	TDLVR	2

3108	TGLKL	2

2157	TGLRV	2

3109	TMLNR	2

2649	TNLAR	2

2595	TNLKR	2

2511	TNLVR	2

3110	TSLIR	2

2176	TTLKA	2

3111	VDLRR	2

3112	VTLAR	2

3113	AALHR	1

2717	AALQR	1

2866	AAPER	1

3114	ADLNR	1

3115	ADLRV	1

2868	AGLAW	1

3116	AGLKK	1

2527	AGLLR	1

3117	AILRR	1

2621	ANLNR	1

3118	ASLKS	1

2799	ASLQR	1

2876	ASLTR	1

3119	ASMKR	1

3120	ATPVP	1

2882	AVLRR	1

3121	AVLTR	1

3122	CGLRR	1

3123	DAEA . . .	1

3124	DALER	1

1831	DALKR	1

2682	DALLR	1

3125	DALPR	1

3126	DARRR	1

3127	DDLNR	1

3128	DGAAE . . .	1

1852	DGLKV	1

3129	DGLWR	1

3130	DGPAR	1

3131	DGPKK	1

3132	DGRRR	1

3133	DGVRR	1

3134	DMLTR	1

2535	DNLLR	1

2808	DNLSR	1

3135	DSLNR	1

3136	DTLDR	1

371	DTLRV	1

3137	DVLRK	1

3138	DVLRS	1

3139	DVLSR	1

3140	DVQKR	1

3141	EALVR	1

2812	EGLIR	1

3142	EGLKM	1

2704	EGLLR	1

2891	EGLMR	1

3143	EGLQC	1

3144	EGLRS	1

2894	EGLRV	1

3145	EGRRR	1

2895	EGVRR	1

3146	EGWS . . .	1

2705	ENLAR	1

2633	ENLSR	1

3147	ESLAR	1

3148	ETGWG . . .	1

3149	ETLER	1

3150	ETLHR	1

3151	ETLVR	1

3152	ETRRR	1

3153	EVLKR	1

2814	GAEE . . .	1

3154	GALAR	1

2778	GALNR	1

3155	GDLYR	1

3156	GDPAP . . .	1

2642	GGLDR	1

2745	GGLGR	1

2904	GGLQE	1

3157	GGQTR	1

3158	GGVVR	1

3159	GHLQR	1

3160	GILRR	1

3161	GMLRR	1

2522	GNLDR	1

3162	GNLLL	1

2517	GNLLR	1

2609	GNLQR	1

3163	GNLVM	1

2685	GTLLV	1

2192	GTLRV	1

3164	GTLRW	1

3165	GTPHR	1

3166	GVLAR	1

3167	GVLNR	1

3168	GVLVR	1

3169	GWLSR	1

3170	HAEA . . .	1

43	HALKV	1

3171	HDLKR	1

3172	HELTR	1

3173	HGLRW	1

3174	HGMRR	1

3175	HILIR	1

3176	HLLNR	1

2661	HNLAR	1

3177	HPAP . . .	1

2645	HQLIR	1

2825	HSLAR	1

2933	HSLSR	1

3178	HTLNK	1

3179	HTLRA	1

3180	HTLRG	1

3181	HTLSR	1

2709	HVLER	1

3182	HWLLR	1

2710	IGLRR	1

2754	IGLTG	1

2711	INLTR	1

3183	ITLTR	1

3184	KGLPG	1

3185	MDVKG	1

3186	MTLIR	1

2635	NALRR	1

2676	NALVR	1

2614	NGLER	1

2938	NHLVQ	1

2786	NMLAR	1

2543	NNLAR	1

2637	NNLLR	1

2787	NSLAR	1

2940	NTLNR	1

2941	NTLQR	1

3187	P*MGS	1

3188	PALKP	1

3189	PGWAG	1

3190	PTLKR	1

3191	PTLRR	1

	PWS . . .	1

2602	QALKR	1

2947	QALTR	1

3192	QDLAT	1

3193	QDLVR	1

2728	QGLAR	1

2729	QNLHR	1

2646	QNLQR	1

2575	QNLRR	1

2841	QNLRW	1

3194	QPACV	1

3195	QTLHR	1

2950	QTLQR	1

3196	QTLTR	1

3197	RGLKR	1

3198	RPAA . . .	1

2336	RTLKV	1

3199	SALHR	1

1887	SALKV	1

2955	SALMC	1

2730	SALMR	1

3200	SDLKS	1

3201	SILKV	1

3202	SILNR	1

2791	SILVR	1

2533	SMLAR	1

3203	SMLLR	1

3204	SMLR	1

2524	SNLAR	1

3205	SNLHR	1

2963	SNLQR	1

3206	SPLHR	1

3207	SSLKW	1

3208	STPER	1

3209	STQVR	1

3210	SVLQR	1

3211	SVLSR	1

2795	TALNR	1

2631	TALVR	1

2765	TGLDR	1

3212	TGLKW	1

3213	TGLNV	1

3214	TGLQC	1

3215	TGLRQ	1

2977	TGPAR	1

3216	TGPNR	1

3217	TGQRR	1

74	TMLRR	1

2561	TNLMR	1

2526	TNLNR	1

3218	TRLVR	1

3219	TSLIS	1

3220	TTLDR	1

3221	TTLKK	1

3222	TTLRT	1

1919	TTLRV	1

2861	TVLRM	1

2985	VALAR	1

3223	VALRR	1

3224	VGLHR	1

3225	VGLNR	1

2652	VGLQR	1

2619	VGLRR	1

2990	VGLTM	1

2605	VNLKR	1

3226	YGLAR	1

3227	YGLVR	1

3228	YILRR	1

TABLE 18

ZF5 selection on G: T change at nt 8 of core motif
in CBS. Sequences reflect position 2 to 6.

SEQ
ID
NO:	Sequence	Read #

50	GGLVR	178

2538	AALRR	174

2607	GALVR	170

2462	TGLRR	162

2464	SGLRR	158

2461	GGLRR	152

2463	EGLRR	148

2475	AGLRR	143

2641	GALRR	126

56	HTLRR	125

2027	GGLKR	117

2700	AALTR	111

2473	AGLTR	108

2521	GALKR	104

2465	AGLAR	102

54	HGLVR	101

1932	HGLKR	99

2610	HALAR	97

1986	AGLKR	96

59	HGLRR	96

1985	AALKR	94

2466	SGLAR	93

66	ATLRR	90

2539	ETLRR	90

2471	HGLAR	90

2495	EGLKR	83

2477	SGLTR	82

2488	HGLTR	79

1843	DGLRR	77

2592	GALTR	75

2467	GGLAR	74

2483	HALRR	74

2523	SALRR	71

2486	SGLVR	70

2734	TALRR	69

3154	GALAR	66

2500	TGLKR	66

55	GGLTR	63

2694	SALVR	61

2875	ASLRR	57

108	DALRR	57

2530	DTLRR	52

2819	GSLKR	50

2748	HALHR	46

2568	HSLVR	46

2546	AALAR	45

2131	SALKR	45

2583	EALRR	44

2770	AALVR	42

1884	EALKR	42

2827	HSLRR	42

2532	SALAR	42

2666	SALTR	42

2489	SSLRR	41

2654	ATLAR	40

1930	HALKR	40

2587	NTLRR	40

2956	SALNR	40

2479	AGLQR	39

1837	DGLVR	38

2502	ETLKR	38

49	QALRR	38

2678	TALAR	36

2857	TTLAR	36

2737	TTLRR	36

2547	HALVR	35

2578	HTLAR	35

2476	GGLSR	34

2738	AALNR	33

2470	GGLQR	33

2564	SSLVR	33

2656	DTLNR	31

2600	GSLRR	31

2586	HTLMR	30

2559	SGLKR	30

2550	STLAR	30

2498	AGLSR	29

1988	AGLVR	29

2509	ASLKR	29

2684	GTLAR	29

3229	QALVR	29

2594	SGLIR	29

2545	STLSR	29

2472	TGLVR	29

2468	DGLAR	28

2701	DSLKR	28

2762	STLTR	28

2653	AALSR	27

2674	HALSR	27

2603	SALSR	27

2850	STLMR	26

2828	HTLNR	25

1870	DGLTR	24

51	HGLIR	24

2628	NTLKR	24

2589	TSLRR	24

2997	VTLRR	24

2569	AGLNR	23

2721	GALSR	23

2630	SSLTR	22

2480	AGLHR	21

2778	GALNR	21

2753	HTLGR	21

2593	QTLRR	21

53	TGLTR	21

2717	AALQR	20

2562	GTLTR	20

2643	HGLMR	20

2617	TALKR	20

2799	ASLQR	19

2739	ATLVR	19

1831	DALKR	19

2634	ESLRR	19

2659	GGLNR	19

2622	ATLTR	18

2528	GGLIR	18

2660	GSLTR	18

2554	GTLKR	18

2707	GTLQR	18

2636	NGLKR	18

2667	SDLKR	18

2698	STLVR	18

2584	GTLRR	17

2525	SGLLR	17

2493	AGLIR	16

2800	ASLVR	16

2818	GSLAR	16

2934	HTLHR	16

2549	SGLNR	16

2474	SGLSR	16

1871	DALVR	15

2916	GSLVR	15

2782	HGLHR	15

2878	ATLER	14

3098	HSLQR	14

2501	SGLQR	14

2519	TGLIR	14

2516	TGLSR	14

2858	TTLGR	14

2767	TTLVR	14

2995	VTLKR	14

2772	ATLSR	13

2702	DSLRR	13

2759	SALIR	13

2631	TALVR	13

2736	TTLLR	13

2864	AALLR	12

3230	HALTR	12

2616	QGLVR	12

2469	TGLAR	12

2880	ATLLR	11

2563	DGLNR	11

2626	GTLVR	11

2602	QALKR	11

3231	SALLR	11

3232	SSLHR	11

2967	STLNR	11

2492	TGLQR	11

2590	TTLQR	11

2876	ASLTR	10

109	DGLKR	10

2756	NSLRR	10

2692	QSLKR	10

2537	SGLMR	10

2849	STLHR	10

2638	STLRR	10

3113	AALHR	9

2879	ATLIR	9

3017	ATLNR	9

2672	DTLAR	9

2566	DTLKR	9

2484	HGLQR	9

2933	HSLSR	9

2943	PALKR	9

2964	SSLIR	9

2764	TALTR	9

2588	TGLHR	9

2881	ATLQR	8

3007	HTLIR	8

2829	HTLTR	8

2941	NTLQR	8

2579	QGLKR	8

2699	SVLKR	8

3047	TSLNR	8

3233	AALIR	7

2865	AALMR	7

2999	DALTR	7

2719	DTLQR	7

3234	GSLHR	7

2781	GSLQR	7

2548	HGLSR	7

2478	NGLVR	7

2965	SSLKR	7

2848	STLER	7

2795	TALNR	7

48	ATLKR	6

2802	AVLKR	6

3038	ETLAR	6

2503	HGLLR	6

2830	HTLVR	6

2784	HVLKR	6

3235	NALQR	6

2485	NGLRR	6

3236	NSLVR	6

2580	SGLER	6

2514	SGLHR	6

2860	TTLKR	6

3237	AALER	5

3238	AALGR	5

3025	ATLGR	5

2598	EGLNR	5

2904	GGLQE	5

70	GNLTR	5

2086	NALKR	5

2788	NTLAR	5

2843	QTLKR	5

2950	QTLQR	5

2505	TGLMR	5

2515	TGLNR	5

2980	TSLAR	5

2743	DGLQR	4

2703	DTLMR	4

2777	EGLVR	4

2745	GGLGR	4

2536	GGLMR	4

3239	GSLIR	4

3240	GSLNR	4

2673	HALLR	4

2783	HTLKR	4

46	HTLKV	4

2938	NHLVQ	4

2510	QGLRR	4

3241	QVLKR	4

3199	SALHR	4

2845	SSLAR	4

2668	STLGR	4

3018	STLIR	4

2966	STLLR	4

3242	TALQR	4

3073	TSLMR	4

3243	AALDR	3

2527	AGLLR	3

2542	ANLAR	3

69	ANLRR	3

3244	ASLSR	3

3012	ATLHR	3

2570	DALAR	3

2804	DALQR	3

2499	DGLIR	3

2553	DGLSR	3

2520	DMLRR	3

2497	EGLAR	3

2490	EGLTR	3

2658	ESLKR	3

2491	GGLER	3

2625	GGLLR	3

138	GNLAR	3

117	GNLVR	3

3245	GSLSR	3

3246	HALQR	3

2577	HNLTR	3

3085	HSLKR	3

2613	HTLQR	3

2832	ITLKR	3

2833	MTLKR	3

2787	NSLAR	3

3247	NSLSR	3

2940	NTLNR	3

2947	QALTR	3

2573	QGLTR	3

3195	QTLHR	3

3248	QTLVR	3

2730	SALMR	3

2496	SNLLR	3

2604	SSLGR	3

2847	STLDR	3

2970	SVLRR	3

2507	TGLLR	3

2561	TNLMR	3

68	TNLRR	3

3249	TSLER	3

2618	TTLMR	3

2534	VGLKR	3

2718	AGLDR	2

2669	AGLER	2

2797	AGLGR	2

3250	ASLMR	2

3251	ASLNR	2

2552	DGLDR	2

2529	DGLHR	2

2591	DNLKR	2

2535	DNLLR	2

2623	DNLRR	2

2506	DNLVR	2

2683	DSLAR	2

3030	DTLLR	2

2809	DTLSR	2

2810	DTLTR	2

2720	DVLKR	2

2811	DVLRR	2

2890	EALTR	2

3043	ETLQR	2

3252	GALDR	2

2779	GDLKR	2

2780	GDLTR	2

3253	GGPRR	2

2917	GTLER	2

3254	HALNR	2

2820	HDLRR	2

2687	HGLDR	2

2585	HGLGR	2

2821	HGLNR	2

2482	HNLLR	2

2826	HSLHR	2

3255	MPLTR	2

2834	NALHR	2

2540	NGLAR	2

2572	NGLIR	2

2755	NGLLR	2

2504	NGLQR	2

2512	NGLTR	2

2837	NTLHR	2

2939	NTLIR	2

2942	NVLKR	2

2948	QDLIR	2

2838	QDLKR	2

2842	QSLRR	2

3004	SILKR	2

2556	SMLRR	2

2793	SSLQR	2

2697	STLQR	2

2971	TALER	2

2851	TALGR	2

2157	TGLRV	2

2978	TMLKR	2

2511	TNLVR	2

2715	TSLHR	2

3019	TTLSR	2

2651	TTLTR	2

3256	AALTG	1

2866	AAPER	1

58	ADLKR	1

2868	AGLAW	1

3257	AGVIR	1

3258	AGVTR	1

71	AMLKR	1

2621	ANLNR	1

3090	ASLAR	1

3259	ASLRG	1

2801	ATLMR	1

3260	ATLRM	1

3261	ATPRR	1

3262	AVLAR	1

2882	AVLRR	1

3263	AVLVR	1

2803	DALNR	1

2596	DGLGR	1

1833	DGLKK	1

1853	DGLRK	1

3129	DGLWR	1

3264	DGPAA . . .	1

2640	DMLKR	1

2597	DSLQR	1

2776	DTLVR	1

2014	DVLKK	1

3265	EALHR	1

3266	EALSR	1

3095	EGLHR	1

2891	EGLMR	1

3267	EGLRG	1

2894	EGLRV	1

2705	ENLAR	1

2633	ENLSR	1

2814	GAEE . . .	1

3268	GALER	1

3269	GALGK	1

3270	GALIR	1

3271	GALKV	1

3272	GALMR	1

2815	GALQR	1

3273	GAPRR	1

3003	GDLNR	1

2817	GDLVR	1

2642	GGLDR	1

2571	GGLHR	1

3274	GGPAR	1

3275	GGPVR	1

3276	GGQVR	1

3277	GGVAR	1

3278	GGWP . . .	1

2913	GMLAR	1

2481	GNLER	1

139	GNLMR	1

2609	GNLQR	1

3279	GSLRV	1

2918	GTLGR	1

2919	GTLHR	1

3081	GTLMR	1

2747	GTLNR	1

2723	GTLSR	1

3280	HAAQ . . .	1

3281	HALAS	1

3282	HALER	1

3283	HALVH	1

3284	HAMRR	1

3285	HAQHR	1

3286	HGLTL	1

3287	HGLVM	1

2531	HLLKR	1

2661	HNLAR	1

2557	HNLHR	1

3050	HNLKR	1

2627	HNLSR	1

2644	HNLVR	1

3177	HPAP . . .	1

2645	HQLIR	1

3288	HSLGR	1

1936	HTLRV	1

2935	HVLAR	1

2710	IGLRR	1

2754	IGLTG	1

2711	INLTR	1

3184	KGLPG	1

3289	MPLQR	1

2937	NALAR	1

2663	NGLHR	1

2615	NGLMR	1

2555	NGLSR	1

2664	NMLKR	1

2543	NNLAR	1

2637	NNLLR	1

3006	NTLTR	1

	PWS . . .	1

3290	QAPWP . . .	1

3023	QDLRR	1

2728	QGLAR	1

2574	QMLKR	1

2729	QNLHR	1

2646	QNLQR	1

2841	QNLRW	1

3104	QTLSR	1

3291	RGLQR	1

2629	SALER	1

2693	SALGR	1

2955	SALMC	1

3292	SALQR	1

3293	SAQR . . .	1

3294	SARVR	1

2957	SDLAR	1

3295	SDLNR	1

2958	SDLQR	1

2959	SDLRR	1

3105	SELRR	1

3296	SGADA . . .	1

3297	SGLR . . .	1

3298	SGLVC	1

3299	SGPDP . . .	1

2533	SMLAR	1

2487	SNLDR	1

2963	SNLQR	1

2544	SNLSR	1

2696	SQLRR	1

3300	SSLPR	1

2302	STLKT	1

2968	STLRK	1

3301	STPSR	1

2733	SVLTR	1

3302	TALLR	1

3303	TAPTR	1

2973	TDLAR	1

2974	TDLRR	1

3304	TGLIK	1

2977	TGPAR	1

3217	TGQRR	1

2595	TNLKR	1

2526	TNLNR	1

2766	TSLKR	1

2983	TSLQR	1

2859	TTLIR	1

1849	TTLKV	1

2681	TTLNR	1

2861	TVLRM	1

3305	TWLRR	1

2985	VALAR	1

3306	VALQR	1

2652	VGLQR	1

2990	VGLTM	1

2605	VNLKR	1

3307	VSLKR	1

3308	VSLRR	1

3112	VTLAR	1

2994	VTLGR	1

TABLE 19

ZF4 selection on G: T change at nt 10 of core
motif in CBS. Sequences reflect position 2 to
6.

SEQ
ID
NO:	Sequence	Read #

60	AHLRK	4967

158	GHLKK	1446

3309	THLRA	1429

1386	EHLRR	1293

162	GHLRK	1082

3310	HHLTK	876

63	AKLRI	867

61	AKLRV	641

3311	AKLRL	625

3312	AKLKI	599

3313	SHLRK	566

159	AHLKK	560

163	THLKK	496

160	TKLRL	486

92	SKLRL	475

2137	SKLKV	466

161	TKLKL	466

3314	QHLRK	457

3315	AKLKL	443

3316	GHLVK	419

3317	GKLKI	302

3318	THLRK	268

3319	AKLKV	258

106	GKLRI	246

3320	GKLRL	224

3321	GHLRL	213

3322	TKLKI	199

3323	RSLGL	178

90	AHLRV	177

3324	AHLRL	153

3325	TKLRV	152

3326	SKLKI	146

3327	SHLVG	132

3328	GKLKL	116

64	TKLKV	108

3329	THLRT	107

3330	GHLRR	102

	*R . . .	92

3331	SHLRL	90

65	SKLRV	80

3332	GALV . . .	79

3333	GHLKM	75

3334	SKLRI	74

3335	GILS . . .	71

3336	SK*VL	63

3337	SKLVL	62

	TR . . .	61

3338	IRLGV	59

3339	MALGL	58

3340	EHLRK	54

3341	GHLRM	54

1407	EHLKR	50

3342	ITLM . . .	48

3343	AHLVK	40

3344	THLRL	40

3345	GKLKV	38

3346	GHLKL	34

3347	AHLRR	32

3348	GHLIK	30

3349	EHLVR	28

3350	GKLRV	27

3351	TALSM	26

3352	EHLQR	25

3353	EKLKV	25

3354	QHLVK	25

3355	TKLNL	25

3356	GHLRA	23

3357	GRLPK	21

	NGR . . .	21

3358	SKLKL	21

3359	THLTK	21

3360	RLLSG	20

3361	TKLRI	19

3362	AHLRI	18

409	GHLKV	16

3363	GHLRV	16

3364	GLLPG	16

3365	AKLRT	14

3366	RHLRV	14

3367	AALRK	11

3368	AHLHK	11

3369	GHLTK	11

3370	QHLRR	11

3371	RSHS . . .	11

3372	SHLNK	11

3373	AHLQK	10

3374	GHLMK	10

3375	SKLRT	10

287	AHLKV	9

3376	AHLRA	9

370	AHLRT	9

3377	EHLRL	9

3378	GHLKI	9

3379	SHLKL	9

3380	EHLKK	8

3381	GHLRT	8

3382	GKLKM	8

3383	HHLKK	8

3384	SKLTI	8

3385	THEKP . . .	8

	*G . . .	7

3386	AKLIL	7

3387	AKLTI	7

3388	HALAA	7

3389	TKLQV	7

3390	AKLRM	6

3391	EHLRI	6

3392	GHLAK	6

3393	GHLKR	6

3394	GKLTL	6

3395	SHLKK	6

3396	SHLRR	6

3397	AILKA	5

89	AKLRK	5

3398	AKLTL	5

3399	ASLTG	5

201	EHLRV	5

3400	EVLTM	5

3401	GHLKT	5

3402	NGRS . . .	5

3403	THLRR	5

3404	AHLKL	4

3405	GALVH	4

3406	GKLVL	4

3407	NGRSPV . . .	4

3408	QALSI	4

3409	SHLRT	4

	TRS . . .	4

3410	AALRL	3

3411	AHLMK	3

439	AHLRE	3

3412	AHLRQ	3

3413	AKLNL	3

3414	AKLRA	3

3415	APLRK	3

186	EKLRI	3

3416	GALMG	3

3417	GALTG	3

3418	GHLRG	3

3419	GHLTL	3

3420	GKLRK	3

3421	GKLTV	3

187	GKLVT	3

3422	HHLRK	3

3423	MGLVG	3

1848	SHLKV	3

3424	SHLRI	3

3425	SKLIL	3

3426	SKLMV	3

3427	SLLAG	3

3428	THLKI	3

3429	THLQK	3

3430	VPLAG	3

3431	AGLLG	2

3432	AHLKM	2

3433	AHLRN	2

3434	AHLTK	2

3435	AKLIV	2

3436	AKLKA	2

88	AKLKK	2

3437	AKLTV	2

3438	AKLVL	2

3439	AKSRI	2

3440	AMLMQ	2

3441	AQLRI	2

3442	DALR . . .	2

419	EHLRA	2

313	EHLRT	2

3443	EKLKL	2

3444	GGLQK	2

3445	GGLTM	2

	GH*R . . .	2

3446	GHLLR	2

3447	GHLRI	2

3448	GHLVG	2

3449	GHLVR	2

3450	GKLNL	2

2912	GKLRR	2

3451	GKLVP	2

3452	GLLGL	2

3453	GNLGM	2

3454	GVLQK	2

3455	HGLLP	2

2043	HHLRV	2

3456	HLLEN	2

3457	IGLQR	2

3458	KTLGV	2

3459	LSLLK	2

3460	MRLGE	2

3461	NSLTR	2

3462	NVLNK	2

3463	PHLRK	2

3464	PLLMP	2

3465	PRLRH	2

3466	QKLHL	2

3467	QKLNL	2

3468	SHLRV	2

3469	SKLHL	2

3470	SKLKR	2

3471	SKLNL	2

3472	SPLAE	2

3473	SVLML	2

	TH*R . . .	2

2448	THLKL	2

3474	THLRV	2

3475	TKLIL	2

3476	TKLMV	2

3477	TPLNI	2

3478	TRLQK	2

3024	TSLTR	2

3479	VGLGQ	2

3480	VHLRK	2

3481	AALES	1

3482	AALRI	1

3483	ADLRK	1

3484	AELLG	1

3485	AELRI	1

3486	AGLAA	1

1986	AGLKR	1

3487	AGLMD	1

3488	AHLGL	1

3489	AHLK . . .	1

3490	AHLKA	1

3491	AHLKI	1

438	AHLKT	1

3492	AHLNK	1

3493	AHLR . . .	1

3494	AHLSK	1

3495	AHLSP	1

214	AHLTV	1

3496	AHLWK	1

3497	AKFKI	1

3498	AKIKH	1

3499	AKIRI	1

3500	AKIRL	1

3501	AKIRV	1

3502	AKLHT	1

3503	AKLKE	1

3504	AKLKG	1

3505	AKLKM	1

3506	AKLMN	1

3507	AKLNI	1

3508	AKLQL	1

3509	AKLRG	1

3510	AKLRR	1

3511	AKLSM	1

3512	AKSRV	1

3513	AKVKL	1

3514	AKVRI	1

3515	ALLMA	1

3516	ALLRR	1

3517	AMLIM	1

3518	AMLKI	1

3519	AMLRG	1

3520	AMLRL	1

3521	ANLSN	1

3522	ANVAQ	1

3523	APLKK	1

3524	AQFRK	1

3525	AQLVD	1

3526	ARLAG	1

3527	ARLGT	1

3528	ARLRA	1

3529	ARLRK	1

3530	ASLRM	1

3531	ATLKL	1

3532	ATLRV	1

3533	C*LKI	1

3534	DELMR	1

3535	DELRV	1

3536	DGLES	1

2005	DGLLR	1

3537	DGLMD	1

3538	DGLVG	1

3539	DHLKK	1

3540	DHLRK	1

3541	DHLRR	1

3542	DKLRK	1

3543	DLLGV	1

3544	DLLLN	1

3545	DNLRE	1

3546	DPLAR	1

3547	DSLGE	1

3548	EALMA	1

3549	EDLVK	1

3550	EELGL	1

3551	EELMM	1

3267	EGLRG	1

3552	EGLVE	1

3553	EHLG . . .	1

3554	EHLHK	1

3555	EHLKL	1

3556	EHLKM	1

2016	EHLRQ	1

3557	EHLRS	1

3558	EHLSE	1

3559	EHLSR	1

3560	EHLTK	1

3561	EHLVK	1

3562	EQLGP	1

3563	ERLAA	1

3564	ERLGR	1

1893	ERLRR	1

3565	ESLMA	1

3566	ETLSH	1

3567	EVLGI	1

3568	FFLRV	1

3569	GALGR	1

3570	GALIM	1

3571	GDLSG	1

3572	GGLDL	1

3573	GGLDQ	1

1957	GGLKV	1

3574	GGLNM	1

3575	GGLPE	1

2295	GGLVV	1

3576	GHFKT	1

3577	GHFQN	1

3578	GHLK . . .	1

3579	GHLMN	1

3580	GHLMV	1

3159	GHLQR	1

3581	GHLR . . .	1

3582	GILAG	1

3583	GKLHE	1

3584	GKLKA	1

3585	GKLKF	1

3586	GKLKT	1

3587	GKLR . . .	1

3588	GKLRA	1

3589	GKLRM	1

3590	GKLVA	1

3591	GKLVV	1

3592	GLLGE	1

3593	GLLLD	1

3594	GLLMG	1

3595	GLLRG	1

3596	GMLGG	1

3597	GPLGV	1

3598	GPLRV	1

3599	GRLKI	1

3600	GRLKK	1

3601	GSLST	1

3602	GSLVK	1

2554	GTLKR	1

3603	GVLAG	1

3604	GVLLV	1

3605	GVLS . . .	1

3606	GYLRK	1

3607	HALRT	1

3608	HALVN	1

3609	HGLTG	1

3610	HHLAK	1

3611	HHLRR	1

3612	HIRS . . .	1

3613	HTHEK	1

3614	IELVQ	1

3615	IGLGL	1

3616	IKLRL	1

3617	IMLRE	1

3618	IMLVE	1

3619	IPLGD	1

3620	IQLRK	1

3621	IRLG . . .	1

3622	IRLGG	1

3623	IRLVV	1

3624	IVLAA	1

3625	KHLRA	1

3626	KHLRL	1

3627	KILPE	1

3628	KKLLE	1

3629	KMLPP	1

3630	KNLIK	1

3631	KSLMP	1

3632	LALGG	1

3633	LGLGA	1

3634	LGLVG	1

3635	LHLTK	1

	LQ . . .	1

3636	LRLIG	1

	LTE . . .	1

3637	LTLQR	1

3638	LVLRR	1

3639	MA*SHMK	1

3640	MALRL	1

3641	MALTR	1

3642	MGLDP	1

3643	MGLGE	1

3644	MGLQN	1

3645	MHLRM	1

3646	MKLEQ	1

3647	MLLRN	1

3648	MLLSH	1

3649	MLLVN	1

3650	MPLRA	1

3651	MQLGG	1

3652	MRLAR	1

3653	MRLMG	1

3654	MRLVG	1

3655	MSLER	1

3656	MTLPL	1

3657	MTLSD	1

3658	MVLAG	1

	NG . . .	1

2615	NGLMR	1

2504	NGLQR	1

3659	NKLRL	1

3660	NLAH	1

3661	NLLPT	1

3662	NRLES	1

3663	NRLGG	1

3664	NTLPK	1

3665	PGLHG	1

3666	PGLRA	1

3667	PHFTK	1

3668	PILLQ	1

3669	PKLGL	1

3670	PLLKS	1

3671	PQLTG	1

3672	PREAM	1

3673	PTLQR	1

3674	QELGR	1

3675	QGLPV	1

3676	QHLKK	1

3677	QHLQR	1

3678	QHLR . . .	1

3679	QHLRI	1

3680	QHLRL	1

3681	QHLTK	1

3682	QILLH	1

3683	QKLRI	1

3684	QNLHK	1

3685	QPLIK	1

3686	QQVTA . . .	1

3687	QTLAE	1

3688	QVTLA	1

3689	RALSA	1

	RGL . . .	1

3690	RGLGA	1

3691	RGLTA	1

2953	RGLVR	1

3692	RGLVV	1

3693	RHLRA	1

3694	RHLRE	1

3695	RHLRM	1

3696	RHLRR	1

3697	RILPR	1

3698	RKLIV	1

3699	RKLKL	1

3700	RLLGA	1

3701	RLLMP	1

3702	RLLRR	1

3703	RMLVP	1

3704	RRLEG	1

3705	RRLVN	1

3706	RTLML	1

3707	RTLTQ	1

3708	SDLHV	1

3709	SDLRK	1

2581	SGLGR	1

3710	SGLLV	1

2486	SGLVR	1

3711	SHLKM	1

3712	SHLRA	1

3713	SHLRE	1

3714	SHLRG	1

3715	SHLTK	1

3716	SHLTM	1

3717	SHLV . . .	1

3718	SHLVK	1

3719	SKIRL	1

3720	SKLEG	1

3721	SKLGA	1

3722	SKLKG	1

2191	SKLRM	1

3723	SKLRN	1

3724	SKLRR	1

3725	SLLEE	1

3726	SLLGT	1

3727	SLLNG	1

2138	SQLKV	1

3728	SQLLE	1

3729	SRLMA	1

3730	STLLM	1

3731	STLVG	1

3732	TALRG	1

	TG . . .	1

2469	TGLAR	1

3733	TGLGL	1

3734	TGLLK	1

2157	TGLRV	1

3735	TGLVD	1

3385	THEKP	1

3736	THFRT	1

3737	THIR . . .	1

3738	THLAR	1

2449	THLKQ	1

3739	THLLK	1

3740	THLMK	1

331	THLRP	1

3741	THLVK	1

3742	THMK	1

3743	THVKK	1

3744	TKLKM	1

3745	TKLKR	1

3746	TKLNM	1

3747	TKLRK	1

3748	TKLRP	1

3749	TKLS . . .	1

3750	TKLTI	1

3751	TMLGG	1

3752	TMLKL	1

3753	TMLPG	1

3754	TPLKR	1

3755	TPLRA	1

3756	TQLKK	1

3757	TQLKL	1

1941	TQLKV	1

3758	TR*RL	1

3759	TRLKL	1

110	TRLRE	1

	TS . . .	1

3760	TTLGI	1

3761	TYLKK	1

3762	VELDP	1

3763	VELVN	1

3764	VKLQQ	1

3765	VKLRL	1

3766	VKLRN	1

3767	VKLRV	1

3768	VLLKS	1

3769	VLLQM	1

3770	VMLKD	1

3771	VMLMG	1

3772	VPLAL	1

3773	VPLER	1

3774	VPLNT	1

3775	VPLSS	1

3776	VPLVP	1

	VQ*G . . .	1

3777	VRLEE	1

3778	VRLQA	1

3779	VVTA . . .	1

3780	WHLKK	1

	YG . . .	1

TABLE 20

ZF4 selection on G: C change at nt 10 of core
motif in CBS. Sequences reflect position 2 to 6.

SEQ
ID		Read
NO:	Sequence	#

61	AKLRV	5924

3325	TKLRV	4888

64	TKLKV	3542

2137	SKLKV	3056

3319	AKLKV	2451

65	SKLRV	1583

3375	SKLRT	474

3350	GKLRV	320

63	AKLRI	254

3345	GKLKV	237

3312	AKLKI	164

1986	AGLKR	132

3322	TKLKI	129

1957	GGLKV	78

3326	SKLKI	76

3334	SKLRI	76

3527	ARLGT	64

3781	VALGS	48

3454	GVLQK	46

	TRS . . .	39

60	AHLRK	30

3782	AKLVV	26

3783	TKLRA	24

3784	LGLRG	18

3652	MRLAR	15

3785	TKLKA	14

3722	SKLKG	13

3361	TKLRI	13

3365	AKLRT	12

	NGR . . . .	12

3786	PNLAV	12

3787	GGLEV	10

158	GHLKK	10

3788	PREAI	10

3789	TKLKG	10

3790	TKLIV	9

3791	WILRA	9

3792	AK*RG	8

3414	AKLRA	8

3311	AKLRL	8

3793	EK*KV	8

106	GKLRI	8

3310	HHLTK	8

3385	THEKP . . . .	8

3794	TK*RG	8

3795	TKLRT	8

3315	AKLKL	7

3796	AKLRE	7

3437	AKLTV	7

3353	EKLKV	7

2187	SKLKE	7

3797	TKLRG	7

3509	AKLRG	6

1386	EHLRR	6

3798	EKLRV	6

3799	RALW . . .	6

2438	SKLKA	6

3504	AKLKG	5

3390	AKLRM	5

3400	EVLTM	5

3314	QHLRK	5

3800	SKLVV	5

1851	STLKV	5

3801	TKLKE	5

3802	TKLNV	5

3316	GHLVK	4

3320	GKLRL	4

3803	KDALQYESEC	4

	G . . .

3804	LSLVD	4

3805	QKLKV	4

3806	RELKE . . . .	4

3807	RILGS	4

163	THLKK	4

3309	THLRA	4

3808	TKIRV	4

160	TKLRL	4

3809	TKLRM	4

3810	TKLVV	4

3811	TKVRV	4

3812	TRSHSR . . . .	4

159	AHLKK	3

3436	AKLKA	3

3813	AKLRD	3

1909	ATLKV	3

3532	ATLRV	3

3536	DGLES	3

3814	GGLKG	3

3418	GHLRG	3

162	GHLRK	3

3815	GKLIV	3

3816	GKLKG	3

3317	GKLKI	3

3451	GKLVP	3

3817	KKLHW . . .	3

3408	QALSI	3

3818	RTLS . . . .	3

3819	SKLRA	3

3820	SKVRV	3

3427	SLLAG	3

3821	TK*SV	3

3822	TKLAV	3

3823	TKLRE	3

3824	TKSRV	3

3825	TKVKV	3

3826	VMLMM	3

3430	VPLAG	3

3431	AGLLG	2

3827	AILQV	2

3501	AKIRV	2

3435	AKLIV	2

3503	AKLKE	2

3828	AKLMV	2

3829	AKLSV	2

3830	AKVKV	2

3521	ANLSN	2

2315	DKLRV	2

3831	ETLMH	2

3416	GALMG	2

3444	GGLQK	2

3445	GGLTM	2

3333	GHLKM	2

3832	GKSKV	2

3592	GLLGE	2

3452	GLLGL	2

3453	GNLGM	2

2554	GTLKR	2

3456	HLLEN	2

3457	IGLQR	2

3833	IKLRV	2

3834	KALHT	2

3835	KGLMM	2

3836	MELAE	2

3423	MGLVG	2

3460	MRLGE	2

3656	MTLPL	2

2615	NGLMR	2

3402	NGRS . . .	2

3837	NKLKV	2

3838	PRLLA	2

3465	PRLRH	2

3839	PRLSR	2

3840	QGLEA	2

2434	SELKV	2

3470	SKLKR	2

3841	SKLRE	2

3842	SKLRG	2

	TH*R . . .	2

3843	TKIKV	2

161	TKLKL	2

3476	TKLMV	2

3389	TKLQV	2

3844	TKLRD	2

3845	TKLSV	2

3477	TPLNI	2

3478	TRLQK	2

3024	TSLTR	2

1919	TTLRV	2

	V	2

3481	AALES	1

3846	AELKA	1

3847	AELKV	1

3484	AELLG	1

3486	AGLAA	1

3848	AGLKH	1

2475	AGLRR	1

2498	AGLSR	1

2473	AGLTR	1

1988	AGLVR	1

3490	AHLKA	1

287	AHLKV	1

90	AHLRV	1

3495	AHLSP	1

3849	AKIRE	1

3850	AKLAV	1

3851	AKLGV	1

3852	AKLMI	1

3853	AKLNV	1

3854	AKLRF	1

3855	AKLRN	1

3387	AKLTI	1

3856	AKLWV	1

3857	AKRRV	1

3858	AKSKV	1

3859	AKVRG	1

3860	ALLKV	1

3517	AMLIM	1

3861	AMLKV	1

3440	AMLMQ	1

3519	AMLRG	1

3862	AQLKV	1

3863	AQLRV	1

3525	AQLVD	1

1945	ARLKV	1

3864	ARLRI	1

1993	ARLRM	1

1947	ARLRV	1

3865	ATLQV	1

3866	AVLKV	1

3867	AYPRE	1

3868	CGLHW . . .	1

3869	CKLRV	1

1995	DALDR	1

3535	DELRV	1

1852	DGLKV	1

2005	DGLLR	1

3537	DGLMD	1

3870	DGLTG	1

3538	DGLVG	1

3871	DHLKR	1

206	DHLNV	1

3543	DLLGV	1

3544	DLLLN	1

3545	DNLRE	1

3546	DPLAR	1

3872	DRLTI	1

3873	DVLKG	1

3874	DVLRG	1

3875	EALVH	1

3551	EELMM	1

3267	EGLRG	1

3552	EGLVE	1

201	EHLRV	1

3349	EHLVR	1

3562	EQLGP	1

3876	EQLMT	1

3564	ERLGR	1

3565	ESLMA	1

3566	ETLSH	1

3877	EVLAA	1

3567	EVLGI	1

	G . . .	1

3571	GDLSG	1

3573	GGLDQ	1

3878	GGLKD	1

3879	GGLKI	1

2659	GGLNR	1

3575	GGLPE	1

	GH*R . . .	1

3393	GHLKR	1

3446	GHLLR	1

3580	GHLMV	1

3330	GHLRR	1

3363	GHLRV	1

3419	GHLTL	1

3448	GHLVG	1

3582	GILAG	1

3880	GILRM	1

3881	GK*RG	1

3584	GKLKA	1

3382	GKLKM	1

3882	GKLML	1

3883	GKLQV	1

3588	GKLRA	1

3884	GKLRQ	1

3885	GKLRT	1

3394	GKLTL	1

3593	GLLLD	1

3594	GLLMG	1

3364	GLLPG	1

3595	GLLRG	1

3886	GPLGQ	1

3597	GPLGV	1

3887	GPLMG	1

3888	GQLKA	1

3889	GRLAV	1

3890	GRLNA	1

3601	GSLST	1

3602	GSLVK	1

3603	GVLAG	1

3604	GVLLV	1

3607	HALRT	1

3455	HGLLP	1

3612	HIRS . . .	1

3891	HPLTV	1

3892	HRLTR	1

3614	IELVQ	1

3615	IGLGL	1

3893	IKLKV	1

3894	IMLKS	1

3618	IMLVE	1

3895	IQSGE	1

3896	IQVTLA	1

3897	IRLAL	1

3621	IRLG . . .	1

3338	IRLGV	1

3342	ITLM . . .	1

3624	IVLAA	1

3898	KALRG	1

3628	KKLLE	1

3899	KKLRE	1

3900	KKLVR	1

3629	KMLPP	1

3630	KNLIK	1

3631	KSLMP	1

3458	KTLGV	1

3632	LALGG	1

3633	LGLGA	1

3634	LGLVG	1

	LQ . . .	1

3636	LRLIG	1

3901	LSLDG	1

3637	LTLQR	1

3638	LVLRR	1

	MA . . .	1

3339	MALGL	1

3641	MALTR	1

3902	MELDR	1

3642	MGLDP	1

3643	MGLGE	1

3644	MGLQN	1

3646	MKLEQ	1

3903	MKLQA	1

3904	MKLRV	1

3647	MLLRN	1

3649	MLLVN	1

3905	MPLLA	1

3650	MPLRA	1

3906	MRLARHIRS	1

	HTGERP . . .

3653	MRLMG	1

3655	MSLER	1

3907	MSLVN	1

3657	MTLSD	1

3658	MVLAG	1

3908	MVLQE	1

3909	MVLVG	1

	N . . .	1

3910	NDALEYESEC	1

	GP . . .

3911	NDALQYESV	1

	CVP . . .

2504	NGLQR	1

3912	NGLVV	1

3913	NK*NV	1

3914	NKLRV	1

3660	NLAH	1

3661	NLLPT	1

3663	NRLGG	1

3664	NTLPK	1

	NV . . .	1

3915	NVLGG	1

3462	NVLNK	1

3916	PGLAA	1

3665	PGLHG	1

3669	PKLGL	1

3917	PKLRA	1

3670	PLLKS	1

3464	PLLMP	1

3918	PNLAG	1

3919	PNYW . . .	1

3671	PQLTG	1

3672	PREAM	1

3673	PTLQR	1

3920	PVLDH	1

	Q	1

3921	QALTN	1

3674	QELGR	1

3675	QGLPV	1

3682	QILLH	1

3467	QKLNL	1

3684	QNLHK	1

3685	QPLIK	1

3687	QTLAE	1

3922	QVLRK	1

3689	RALSA	1

3923	RELVR	1

	RGL . . .	1

3924	RGLDM	1

3925	RGLDR	1

3691	RGLTA	1

3926	RGLVA	1

2953	RGLVR	1

3692	RGLVV	1

3694	RHLRE	1

3697	RILPR	1

3698	RKLIV	1

3927	RKLKA	1

3928	RKLKV	1

3929	RKLRE	1

3930	RKLRV	1

3931	RKVRV	1

3700	RLLGA	1

3701	RLLMP	1

3932	RMLQE	1

3703	RMLVP	1

3933	RPLEV	1

3705	RRLVN	1

3706	RTLML	1

3707	RTLTQ	1

	S*G . . .	1

3708	SDLHV	1

2581	SGLGR	1

3710	SGLLV	1

2486	SGLVR	1

1848	SHLKV	1

3331	SHLRL	1

3934	SKFKV	1

3935	SKFRV	1

3936	SKIRT	1

3469	SKLHL	1

3937	SKLKD	1

3358	SKLKL	1

3938	SKLKM	1

3939	SKLQI	1

92	SKLRL	1

3940	SKLSV	1

3941	SKLTV	1

3337	SKLVL	1

3942	SKSRT	1

3943	SKVKV	1

3944	SKVRT	1

3725	SLLEE	1

3726	SLLGT	1

3945	SNLKG	1

3946	SNLTH	1

3728	SQLLE	1

1857	SRLKV	1

3730	STLLM	1

3947	TALIS	1

3732	TALRG	1

3948	TELIG	1

3949	TELKV	1

	TG*S . . .	1

2469	TGLAR	1

3733	TGLGL	1

2157	TGLRV	1

3385	THEKP	1

3737	THIR . . .	1

3738	THLAR	1

3429	THLQK	1

3318	THLRK	1

3344	THLRL	1

3329	THLRT	1

3950	TKLHV	1

3951	TKLKD	1

3744	TKLKM	1

3745	TKLKR	1

3952	TKLKT	1

3953	TKLMA	1

3746	TKLNM	1

3954	TKLQI	1

3955	TKLR . . .	1

3956	TKLTV	1

3957	TKLWV	1

3958	TKSRD	1

3751	TMLGG	1

3959	TMLKV	1

3753	TMLPG	1

3960	TMLRV	1

3754	TPLKR	1

1864	TRLKV	1

110	TRLRE	1

2168	TRLRG	1

1883	TRLRV	1

3961	TRSHS . . .	1

3962	TTIRV	1

3760	TTLGI	1

1849	TTLKV	1

3963	TTLSA	1

3964	TTLVP	1

3965	TVLAP	1

3966	TVLPM	1

3967	VALTK	1

3763	VELVN	1

3479	VGLGQ	1

3968	VGLLR	1

3969	VKLLV	1

3764	VKLQQ	1

3766	VKLRN	1

3767	VKLRV	1

3768	VLLKS	1

3970	VLLMA	1

3971	VLLPS	1

3770	VMLKD	1

3771	VMLMG	1

3972	VNLLE	1

3772	VPLAL	1

3773	VPLER	1

3774	VPLNT	1

3775	VPLSS	1

3776	VPLVP	1

	VQ*G . . .	1

3973	VQLPV	1

3777	VRLEE	1

3778	VRLQA	1

2994	VTLGR	1

3974	YTHMK	1

TABLE 21

ZF4 selection on G: A change at nt 10 of core
motif in CBS. Sequences reflect position 2 to 6.

SEQ
ID
NO:	Sequence	Read #

61	AKLRV	408

3350	GKLRV	294

	TRS	180

64	TKLKV	170

3320	GKLRL	166

3402	NGRS	155

3325	TKLRV	124

3390	AKLRM	109

160	TKLRL	109

3345	GKLKV	107

3312	AKLKI	92

3319	AKLKV	88

186	EKLRI	84

3655	MSLER	68

3975	NGRSPVC	67

3416	GALMG	66

3976	AELIR	63

2581	SGLGR	63

3915	NVLGG	61

3977	RGLT	61

3978	TLLMG	58

3451	GKLVP	57

3430	VPLAG	57

3682	QILLH	55

3979	TLPL	55

3980	*MLTS	54

3981	EMLTS	53

2137	SKLKV	53

3615	IGLGL	52

3322	TKLKI	52

3495	AHLSP	51

3828	AKLMV	51

3982	DALRG	51

3633	LGLGA	51

3805	QKLKV	51

3408	QALSI	50

3983	PLLET	49

3984	PSLM	49

3452	GLLGL	48

3985	TLLVG	48

3766	VKLRN	48

62	GGLGL	47

3419	GHLTL	47

3986	GPLHI	46

3649	MLLVN	46

3987	VELNS	46

3988	AKLIT	45

3394	GKLTL	45

3946	SNLTH	45

3989	AT*RR	44

3544	DLLLN	44

3596	GMLGG	44

3923	RELVR	44

3990	SPLLS	44

3991	DKLRR	43

3570	GALIM	43

3992	GLLG	43

3993	GLMM	42

3994	IHLAD	42

3995	TLTQ	42

3996	TRSHSS	42

3997	ALMQ	41

1947	ARLRV	41

3321	GHLRL	41

3456	HLLEN	41

3998	HTLNM	41

3999	PMLVD	41

3469	SKLHL	41

4000	GK*KL	40

3440	AMLMQ	39

3546	DPLAR	39

3328	GKLKL	39

3914	NKLRV	39

3732	TALRG	39

3827	AILQV	38

3435	AKLIV	38

3311	AKLRL	38

3612	HIRS	38

3382	GKLKM	37

3592	GLLGE	37

3453	GNLGM	37

3582	GILAG	36

4001	GPLAL	36

3908	MVLQE	36

3669	PKLGL	36

4002	ARLGL	35

4003	EELLK	35

3647	MLLRN	35

3685	QPLIK	35

288	AHLAV	34

3400	EVLTM	34

3460	MRLGE	34

3548	EALMA	33

4004	PLLGV	33

3671	PQLTG	33

3877	EVLAA	32

4005	HPLQQ	32

3916	PGLAA	32

3467	QKLNL	32

4006	SKLNN	32

4007	TRLRN	32

3438	AKLVL	31

4008	DLLV	31

462	DSLLA	31

4009	GELRT	31

4010	RLLGV	31

2700	AALTR	30

3444	GGLQK	30

2615	NGLMR	30

4011	NRLQ	30

4012	PALGN	30

4013	PLLGM	30

4014	PPLMQ	30

4015	TQLEE	30

4016	VGLEG	30

3543	DLLGV	29

3572	GGLDL	29

3418	GHLRG	29

4017	KTLRE	29

4018	PRLR	29

4019	PSLGV	29

4020	RR*PS	29

3735	TGLVD	29

3429	THLQK	29

	DGLMDHIRSH

4021	TGERPF	28

3459	LSLLK	28

4022	MVLVP	28

4023	SELTG	28

4024	SGLKH	28

3754	TPLKR	28

4025	VGLG	28

60	AHLRK	27

3506	AKLMN	27

63	AKLRI	27

4026	DRLGP	27

4027	GLLGR	27

3617	IMLRE	27

4028	KQLQP	27

	MA*S	27

	NGR	27

3694	RHLRE	27

4029	RPLLR	27

4030	RSLRL	27

65	SKLRV	27

3427	SLLAG	27

3760	TTLGI	27

3484	AELLG	26

2473	AGLTR	26

3538	DGLVG	26

4031	GALG	26

4032	GDLSP	26

3573	GGLDQ	26

3580	GHLMV	26

3317	GKLKI	26

4033	GKLSL	26

3603	GVLAG	26

4034	LRLNL	26

4035	MTLGN	26

4036	PMLAA	26

3375	SKLRT	26

3746	TKLNM	26

4037	ALIG	25

4038	AQLAN	25

4039	DGLAM	25

3575	GGLPE	25

4040	GLPV	25

3631	KSLMP	25

2601	NGLNR	25

4041	SHMK	25

3477	TPLNI	25

3965	TVLAP	25

4042	VLLME	25

3431	AGLLG	24

4043	GALPR	24

4044	GKLIL	24

3882	GKLML	24

3604	GVLLV	24

4045	KQLTD	24

4046	LKLIG	24

3636	LRLIG	24

4047	LRLMS	24

3663	NRLGG	24

4048	PNYWP	24

4049	RHLVP	24

4050	SRLGA	24

3855	AKLRN	23

4051	DRLAS	23

3547	DSLGE	23

3563	ERLAA	23

106	GKLRI	23

4052	GSLS	23

664	HRLGG	23

4053	MDLLL	23

4054	MTLGA	23

4055	PPLER	23

4056	PVLPG	23

3674	QELGR	23

3818	RTLS	23

4057	SLLQG	23

2157	TGLRV	23

3476	TKLMV	23

3773	VPLER	23

4058	APLGM	22

1386	EHLRR	22

2607	GALVR	22

2659	GGLNR	22

3446	GHLLR	22

4059	GILAK	22

4060	GMLPD	22

3597	GPLGV	22

4061	GSLPM	22

3602	GSLVK	22

3166	GVLAR	22

3634	LGLVG	22

3637	LTLQR	22

4062	NGRSPVET	22

3666	PGLRA	22

4063	PMLRV	22

4064	TLML	22

90	AHLRV	21

3515	ALLMA	21

4065	ASLGQ	21

3870	DGLTG	21

3267	EGLRG	21

223	EHLAV	21

4066	ELILE	21

4067	GH*RS	21

4068	GHLAM	21

3589	GKLRM	21

4069	GLLP	21

4070	GTLAI	21

4071	IRLKK	21

4072	KELRR	21

3627	KILPE	21

4073	LHLPI	21

3423	MGLVG	21

3905	MPLLA	21

4074	NELRG	21

3462	NVLNK	21

4075	PHLNG	21

3464	PLLMP	21

4076	RLLGS	21

4077	RTLIS	21

4078	SC*AS	21

3708	SDLHV	21

92	SKLRL	21

4079	VKLMN	21

4080	VTLIG	21

4081	AGLQE	20

4082	ALHT	20

4083	DPLVD	20

	E	20

4084	EALDA	20

4085	GALAT	20

4052	GSLS	20

4086	GTLLM	20

4087	IKLRP	20

	LQ	20

	NGP	20

3684	QNLHK	20

4088	RRLLD	20

3726	SLLGT	20

3948	TELIG	20

4089	TGLMG	20

4090	TKLLL	20

4091	TTLGA	20

4092	VE*DP	20

3968	VGLLR	20

4093	AGLGI	19

4094	AGLLQ	19

3526	ARLAG	19

4095	AVLSH	19

3535	DELRV	19

4096	DRLAG	19

4097	ERLSN	19

4098	ETLM	19

4099	GELRG	19

3590	GKLVA	19

4100	GRLNR	19

4101	GRLRL	19

4102	IMLAG	19

4103	IVLDP	19

4104	KVLAP	19

4105	LMLGM	19

3641	MALTR	19

4106	MPLRE	19

4107	RLLGP	19

3819	SKLRA	19

4108	SMYRS	19

4109	THLAK	19

3762	VELDP	19

4110	VGLTR	19

3775	VPLSS	19

4111	VQLPT	19

2538	AALRR	18

4112	AGLD	18

3517	AMLIM	18

3519	AMLRG	18

4113	DVLPG	18

3562	EQLGP	18

3393	GHLKR	18

3880	GILRM	18

4114	GLLV	18

4115	GLMN	18

4116	GMLVG	18

4117	GPLTI	18

4118	GRLE	18

4119	GSLQS	18

4120	GVLVS	18

4121	HKLLK	18

3614	IELVQ	18

3619	IPLGD	18

3632	LALGG	18

3648	MLLSH	18

4122	MRLKV	18

4123	MRLRS	18

4124	MSLSP	18

4125	PALGG	18

3665	PGLHG	18

3673	PTLQR	18

4126	QPLAG	18

4127	SK*VV	18

3842	SKLRG	18

4128	TLIN	18

4129	TLLTP	18

4130	DALME	17

4131	EALNK	17

4132	EGLPT	17

4133	ELLKS	17

4134	GELTD	17

3884	GKLRQ	17

3161	GMLRR	17

4135	GPLVS	17

4136	GQLMM	17

4137	GQLVG	17

4138	KGLEG	17

4139	QGLDN	17

4140	RALVS	17

4141	RGLAT	17

3426	SKLMV	17

3800	SKLVV	17

3729	SRLMA	17

4142	TLHE	17

2168	TRLRG	17

3864	ARLRI	16

201	EHLRV	16

4143	GHLKS	16

4144	GLLKH	16

3890	GRLNA	16

4145	GVLSI	16

4146	GVLST	16

3607	HALRT	16

3900	KKLVR	16

3638	LVLRR	16

4147	MPLVP	16

3661	NLLPT	16

4148	PKLQP	16

4149	PVLMG	16

4150	QALIG	16

4151	RGLIT	16

3691	RGLTA	16

3705	RRLVN	16

4152	RVQD	16

3725	SLLEE	16

4153	TELPM	16

	TGL	16

3751	TMLGG	16

3776	VPLVP	16

4154	APLDL	15

4155	ARLGR	15

4156	DALSA	15

4157	EGLAG	15

50	GGLVR	15

4158	GGLVS	15

3363	GHLRV	15

3815	GKLIV	15

3595	GLLRG	15

4159	GMLGT	15

4160	GPLLG	15

4161	HIRSH	15

3457	IGLQR	15

4162	IMLV	15

3897	IRLAL	15

304	KALGT	15

3898	KALRG	15

4163	LHLQG	15

4164	MELMT	15

4165	MPLGG	15

4166	PGLAD	15

4167	PTLEV	15

4168	RQLGM	15

4169	RVLRG	15

2525	SGLLR	15

4170	SVLRV	15

3733	TGLGL	15

4171	TVLAG	15

4172	VGLA	15

4173	VGLRG	15

3770	VMLKD	15

3774	VPLNT	15

2994	VTLGR	15

	WR	15

	A	14

4174	AALHH	14

3490	AHLKA	14

4175	ALLGV	14

3525	AQLVD	14

4176	ARLHA	14

4177	DGLG	14

4178	DHLVG	14

4179	DILRG	14

4180	DQLVE	14

4181	DQLVG	14

4182	EKLMM	14

4183	ELLTP	14

3564	ERLGR	14

4184	GALRS	14

3445	GGLTM	14

3583	GKLHE	14

4185	GKLNI	14

3406	GKLVL	14

4186	GRLLE	14

3628	KKLLE	14

3458	KTLGV	14

4187	MALPE	14

3653	MRLMG	14

4188	NDALQYES	14

3662	NRLES	14

3461	NSLTR	14

4189	PKLRS	14

4190	PRLPP	14

4191	PVLKL	14

4192	QKLAN	14

4193	QKLKL	14

4194	RALPK	14

3697	RILPR	14

4195	THLGR	14

3753	TMLPG	14

4196	VALGT	14

4197	VKLHE	14

4198	VTLG	14

4199	ARLLG	13

4200	ARLTG	13

4201	ASLGA	13

4202	DLLSG	13

3545	DNLRE	13

4203	EALTI	13

3551	EELMM	13

4204	ETLS	13

4205	GALGS	13

3381	GHLRT	13

4206	GPLVL	13

4207	GRLGA	13

4208	GRSYMA	13

4209	GVLGS	13

4210	HPLLV	13

4211	ITLSP	13

3642	MGLDP	13

4212	MLLNG	13

4213	MRLAE	13

4214	NMLSR	13

4215	PGLGG	13

4216	PGLVP	13

3670	PLLKS	13

3468	SHLRV	13

4217	SRLGV	13

2469	TGLAR	13

4218	TLMG	13

4219	TRLMM	13

	TRLREHIRSHT

4220	GERPF	13

4221	VELGP	13

4222	VHLAR	13

4223	VKLVG	13

3486	AGLAA	12

4224	APLRV	12

4225	EALV	12

4226	EVLPE	12

4227	GALMN	12

4228	GLQA	12

4229	GLTG	12

4230	GTLGD	12

4231	HLLGP	12

4232	LKLKL	12

4233	MALRK	12

4234	MVLTG	12

4235	NGLIE	12

4236	NKLVV	12

4237	PALNV	12

4238	PMLRL	12

4239	PQLLG	12

4240	PVLRV	12

4241	QPLKR	12

3924	RGLDM	12

4242	RGLEN	12

3700	RLLGA	12

4243	RRLMV	12

2486	SGLVR	12

4244	SPLSG	12

3728	SQLLE	12

4245	SRLGR	12

4246	TGLVG	12

3403	THLRR	12

3809	TKLRM	12

4247	TKLVM	12

4248	TLLG	12

4249	TMLPR	12

4250	TNLRL	12

4251	TPLGE	12

4252	TPLVG	12

4253	TRLLT	12

4254	VGLGR	12

4255	VKLQ	12

3768	VLLKS	12

4256	AGLML	11

3398	AKLTL	11

3521	ANLSN	11

4257	ARLLT	11

2880	ATLLR	11

4258	EGLGG	11

4259	EGLHL	11

3333	GHLKM	11

3889	GRLAV	11

4260	GVLG	11

4261	LGLEG	11

4262	LNLQP	11

4263	LRLRT	11

4264	MELGD	11

4265	MLLQR	11

4266	MLPP	11

4267	MSLGG	11

4268	PKLII	11

4269	PNLQT	11

4270	PPLLS	11

4271	PTLGM	11

4272	QKLMT	11

3687	QTLAE	11

3701	RLLMP	11

4273	RRLVG	11

4274	SNLIM	11

3730	STLLM	11

3738	THLAR	11

4275	TLTM	11

4276	TRLGG	11

3478	TRLQK	11

4277	VGLLA	11

4278	VKLRM	11

4279	VLLGG	11

4280	VQ*GG	11

3777	VRLEE	11

4281	AGLSG	10

4282	AGLTE	10

4283	AGLVA	10

4284	ALSA	10

4285	ATLMK	10

2468	DGLAR	10

206	DHLNV	10

4286	EALAI	10

4287	EELVE	10

4288	EMLIP	10

4289	EPLAA	10

4290	ERLQE	10

3878	GGLKD	10

3588	GKLRA	10

3591	GKLVV	10

4291	GMLRV	10

4292	GPLME	10

4293	GVLSP	10

4294	IKLMG	10

4295	IPLNR	10

4296	MLLKG	10

4297	MRLPR	10

4298	MSLRE	10

3918	PNLAG	10

4299	PPLMV	10

4300	PTLGV	10

4301	RGLRN	10

3692	RGLVV	10

4302	RSLIV	10

4303	RTLGE	10

4304	SSLGV	10

3947	TALIS	10

4305	TGLGT	10

3344	THLRL	10

3822	TKLAV	10

4306	TKLLG	10

4307	TLIG	10

4308	TNLLR	10

4309	TTLGG	10

4310	VILGA	10

3972	VNLLE	10

3481	AALES	9

4311	AALGL	9

4312	AELMR	9

4313	AGLDG	9

1988	AGLVR	9

3534	DELMR	9

4314	DSLVI	9

4315	EKLKA	9

3798	EKLRV	9

4316	GKLIA	9

4317	GNLVT	9

4318	GRLLI	9

4319	GRLRS	9

3239	GSLIR	9

2554	GTLKR	9

4320	HELMK	9

4321	KMLGG	9

4322	LGLIQ	9

4323	LKLER	9

4324	LPLNG	9

4325	MGLGV	9

3658	MVLAG	9

3909	MVLVG	9

2540	NGLAR	9

3668	PILLQ	9

4326	PMLTV	9

4327	PPLII	9

4328	QRLVE	9

3698	RKLIV	9

4329	RKLKE	9

4330	RRLHE	9

4331	RVLGA	9

2532	SALAR	9

4332	SC*RP	9

4333	SGLDA	9

4334	SQLDR	9

2507	TGLLR	9

3952	TKLKT	9

4335	TSLTE	9

2342	AGLKM	8

4336	AGLRS	8

4337	AHLGQ	8

3493	AHLR	8

4338	ALME	8

2875	ASLRR	8

1995	DALDR	8

4339	DGLHG	8

4340	DGLLQ	8

3550	EELGL	8

4341	EKLRS	8

3876	EQLMT	8

4342	ERLAR	8

3569	GALGR	8

4343	GELKA	8

2295	GGLVV	8

3341	GHLRM	8

4344	GLML	8

4345	GLQN	8

4346	GLTA	8

4347	GMLGE	8

4348	GPLRR	8

4349	GVLDT	8

4350	GVLNT	8

4351	IQLAD	8

4352	KGLTM	8

4353	MELGN	8

4354	MPLMR	8

3657	MTLSD	8

4355	NGLAM	8

4356	NGLQD	8

4357	NTLDV	8

4358	PHLSM	8

4359	PILLG	8

4360	PVLQG	8

4361	QGLGG	8

4362	QKLQI	8

4363	QPLIA	8

3926	RGLVA	8

3727	SLLNG	8

4364	SRLTD	8

4365	TLLGD	8

4366	TRSHSSV	8

3024	TSLTR	8

4367	TTLGD	8

4368	VKLAP	8

3973	VQLPV	8

3367	AALRK	7

159	AHLKK	7

4369	AKLHP	7

4370	AVLEN	7

3571	GDLSG	7

4371	GELGV	7

187	GKLVT	7

3593	GLLLD	7

3594	GLLMG	7

4372	GLMA	7

4373	GLNR	7

4374	GLVV	7

4375	GPLPV	7

4376	GSLTQ	7

4377	GVLRG	7

4378	HPLAV	7

4379	HTLGM	7

4380	IQLGG	7

4381	KLLGD	7

3630	KNLIK	7

4382	MALAR	7

4383	MELEP	7

4384	MGLAN	7

3643	MGLGE	7

4385	MPLDG	7

4386	NVLGR	7

4387	PGLPE	7

4388	PHLQN	7

4389	PRLGS	7

4390	PSLLV	7

4391	PTLAR	7

4392	QMLER	7

4393	RDLGS	7

4394	RGLGN	7

4395	RLLEK	7

3703	RMLVP	7

4396	SVLSG	7

4397	TGLVN	7

4398	TLA*SH	7

4399	TRLHT	7

3967	VALTK	7

3771	VMLMG	7

4400	VVLAG	7

4401	AGLVG	6

3315	AKLKL	6

4402	AR*PS	6

1945	ARLKV	6

2005	DGLLR	6

4403	DKLHR	6

2203	DKLKV	6

4404	ERLPV	6

4405	GDLVE	6

4406	GELGE	6

4407	GGLMQ	6

4408	GLLT	6

4409	GLPG	6

4410	GSLRT	6

4411	GTLQV	6

4412	GVLKS	6

4413	HGLVN	6

4414	IELGR	6

4415	KPLEL	6

4416	MKLE	6

3664	NTLPK	6

4417	PALMR	6

303	PHLVV	6

4418	PPLVV	6

4419	QALVP	6

4420	QELGG	6

3370	QHLRR	6

4421	QTLGV	6

4422	RILEP	6

4423	RLLMN	6

4424	RPLVG	6

4425	RRLEP	6

4426	SGLRA	6

4427	SKLMA	6

3940	SKLSV	6

4428	TMLEP	6

4429	TRSQ	6

4430	VALRK	6

4431	VDLSG	6

4432	VMLLG	6

4433	VPLSE	6

2718	AGLDR	5

4434	ARLPV	5

4435	ARYGC	5

1909	ATLKV	5

2317	DGLRA	5

4436	ERLLQ	5

4437	ETLMG	5

4438	GHLML	5

4439	GHLQG	5

4440	GKLMV	5

4441	GPLG	5

4442	GPLTM	5

4443	GQLV	5

4444	GSLTL	5

4445	GTLRA	5

4446	GTLTG	5

3310	HHLTK	5

4447	IVLVR	5

4448	MALVR	5

4449	MELGK	5

4450	MGLEG	5

4451	MGLMA	5

4452	MPLNR	5

4453	NMLGG	5

4454	NPLEL	5

4455	NSLGG	5

4456	PRLLQ	5

4457	PRLVK	5

2953	RGLVR	5

4458	RHLRS	5

4459	RSLVV	5

	RSPV*ERMWI

4460	LRA	5

4461	RTLNA	5

4462	TELN	5

4463	VKLRA	5

4464	VLLQD	5

4465	VMLG	5

4466	AGLNG	4

4467	AHLRM	4

3414	AKLRA	4

4468	AR*RA	4

4469	ARLPE	4

4470	AVLNK	4

	DALQYESECG

4471	GLNH	4

3030	DTLLR	4

4472	EGLRD	4

4473	ESLMG	4

	G	4

4474	GELV	4

4475	GGLRP	4

158	GHLKK	4

3584	GKLKA	4

4476	GLIG	4

4477	GLIS	4

4478	GLLGN	4

4479	GMLVN	4

4480	GPLED	4

4481	GPLQA	4

4482	GTLTV	4

4483	GVLGI	4

4484	IDLGM	4

4485	IELGG	4

4486	IGLAT	4

4487	KKLMP	4

4488	KLLGE	4

4489	KLLLG	4

3629	KMLPP	4

4490	MGLTL	4

4491	MNLGM	4

4492	MPLMV	4

3650	MPLRA	4

3651	MQLGG	4

2085	MRLRM	4

4493	PALTV	4

4494	PGLAL	4

4495	PGLMG	4

4496	PHLMS	4

4497	PQLSA	4

4498	PRLKA	4

4499	QKLIR	4

4500	RELGV	4

4501	RGLHQ	4

4502	RGLIG	4

4503	RGLMG	4

4504	RTRSH	4

4505	SQLDT	4

4506	TELGG	4

163	THLKK	4

3309	THLRA	4

4507	TKLGV	4

4508	TMLEG	4

4509	VSLGV	4

4510	VSLTA	4

4511	VSLVG	4

1986	AGLKR	3

4512	AGLQN	3

4513	AGLRV	3

3516	ALLRR	3

4514	ARLRT	3

4515	ASLQK	3

4516	ASLR	3

2772	ATLSR	3

4517	DILGE	3

4518	EELRM	3

4519	EGLTG	3

4520	EMLKE	3

4521	ESLLG	3

3565	ESLMA	3

4522	ETLAG	3

4523	EVLVQ	3

2521	GALKR	3

2745	GGLGR	3

162	GHLRK	3

4524	GKLRS	3

4525	GLKT	3

4526	GLLGV	3

4527	GMLLP	3

4528	GMLSG	3

3887	GPLMG	3

4529	GRLAP	3

4530	GSLLR	3

4531	GTLTM	3

	GVI	3

4532	ILLQQ	3

4533	KLLQM	3

4534	LGLPG	3

4535	MELVL	3

4536	MGLAG	3

4537	MGLPV	3

3644	MGLQN	3

4538	MQLAD	3

4539	MSLLR	3

4540	MSLPE	3

4541	NGLKQ	3

2504	NGLQR	3

4542	NGRSPV*E	3

4543	NPLSR	3

4544	NQLVA	3

4545	NTLGL	3

4546	PRLRV	3

4547	PVLLM	3

4548	PVLTG	3

3314	QHLRK	3

4549	QQLL	3

4550	RGLVN	3

4551	RHLVV	3

4552	RLLAE	3

4553	RLLPG	3

4554	RPLIT	3

4555	RVLMN	3

4556	RVLQR	3

2580	SGLER	3

161	TKLKL	3

4557	TLLPG	3

110	TRLRE	3

3249	TSLER	3

4558	VGLPA	3

4559	VPLRP	3

4560	VRLMP	3

4561	VSLGE	3

4562	AALTK	2

4563	AALVK	2

4564	AHLTP	2

4565	AILRT	2

4566	AKLNS	2

3853	AKLNV	2

3509	AKLRG	2

4567	ALLGA	2

4568	ARLLR	2

3528	ARLRA	2

4569	DVLG	2

4570	EELQS	2

3552	EGLVE	2

4571	ELLGP	2

4572	ERMC	2

4573	EVLAG	2

4574	GALGE	2

4575	GDLVP	2

4576	GELRI	2

4577	GGLEL	2

4578	GHLSP	2

4579	GKLEA	2

4580	GKLKR	2

2912	GKLRR	2

4581	GKLVI	2

4582	GLHQ	2

4583	GLLR	2

4584	GLMV	2

4585	GLTL	2

117	GNLVR	2

4586	GPLVG	2

4587	GQLVD	2

4588	GRLSV	2

4589	GVLAV	2

3609	HGLTG	2

4590	HVLEL	2

4591	IELEM	2

4592	IGLQA	2

4593	KGLGN	2

4594	KILPV	2

4595	KPLPG	2

4596	KSLRM	2

4597	KTLGT	2

4598	LGLAA	2

4599	LGLGG	2

4600	LVLQE	2

4601	MGLAS	2

4602	MLLEE	2

771	MLPA	2

3652	MRLAR	2

4603	MSLRQ	2

4604	MTLGT	2

4605	NGLIV	2

4606	NHLRM	2

	NLA	2

4607	PALIM	2

4608	PGLAG	2

4609	PLLRA	2

4610	PPLDG	2

4611	PPLIM	2

4612	PPLLG	2

4613	PQLTE	2

4614	PVLDG	2

4615	QGLTT	2

4616	QRLAV	2

4617	RELGG	2

4618	RGLDG	2

4619	RGLTE	2

4620	RHLGA	2

4621	RSLMI	2

4622	RSLRP	2

3721	SKLGA	2

4623	SKLGE	2

	T*LT	2

2443	TALKV	2

4624	THLR	2

1864	TRLKV	2

4625	TRLPP	2

4626	VELGD	2

3763	VELVN	2

2459	VGLGG	2

4627	VGLKD	2

4628	VKLHV	2

4629	VKLLS	2

4630	VQLTK	2

4631	VRLK	2

4632	VRLPP	2

4633	AALEN	1

4634	AALGP	1

4635	AALGT	1

4636	AALKI	1

4637	AALMN	1

4638	AALMQ	1

2865	AALMR	1

4639	AALRV	1

4640	AALSS	1

4641	AELGP	1

4642	AELRA	1

3485	AELRI	1

4643	AGIAA	1

4644	AGILQ	1

4645	AGLDS	1

4646	AGLG	1

4647	AGLGG	1

4648	AGLGN	1

4649	AGLGP	1

4650	AGLGQ	1

4651	AHFRV	1

4652	AHLRG	1

4653	AHLRP	1

4654	AKFRM	1

4655	AKLE	1

4656	AKLGE	1

4657	AKLGL	1

4658	AKLHA	1

3504	AKLKG	1

4659	AKLLG	1

4660	AKLML	1

4661	AKLQP	1

3854	AKLRF	1

4662	AKLRQ	1

4663	AKLS	1

4664	AKLTN	1

4665	AKLWL	1

4666	ALDA	1

4667	ALIM	1

4668	ALKG	1

4669	ALLGE	1

4670	ALLRS	1

4671	ALTG	1

4672	ALTR	1

4673	AMLPD	1

4674	AMLR	1

4675	APLAG	1

4676	APLGP	1

4677	AQLAD	1

4678	AQLLL	1

4679	AR*RG	1

4680	ARLAA	1

3527	ARLGT	1

4681	ARLMS	1

4682	ARLRS	1

4683	ARLTE	1

4684	ARYGR	1

4685	ASLGP	1

4686	ASLRP	1

4687	AT*RS	1

4688	ATLAK	1

4689	ATLEV	1

4690	ATLKI	1

4691	ATLMG	1

4692	ATLNM	1

4693	ATLNV	1

4694	AVIG	1

4695	CGLGR	1

4696	DALQP	1

1999	DALTV	1

4697	DELM	1

4698	DELMN	1

4699	DELRA	1

4700	DGLE	1

4701	DGLEK	1

3536	DGLES	1

4702	DGLML	1

	DGLTGHIRSHT

4703	GERPF	1

4704	DGVAM	1

4705	DHLVD	1

4706	DILG	1

4707	DILRT	1

2348	DKLKG	1

4708	DKLMM	1

4709	DLLA	1

4710	DLLAR	1

103	DNLRV	1

4711	DRLAA	1

4712	DRLGG	1

4713	DSLPE	1

4714	DSLV	1

3874	DVLRG	1

4715	DYLNV	1

4716	EALA	1

4717	EALKV	1

4718	EALMV	1

4719	EALTN	1

4720	EELAP	1

	EELMMHIRSH

4721	TGERPF	1

	EELVEHIRSHT

4722	GERPF	1

3377	EHLRL	1

3349	EHLVR	1

4723	EKLIV	1

3353	EKLKV	1

4724	ELLAR	1

4725	ELLPS	1

4726	EMLVA	1

4727	EQLGT	1

4728	ERLAV	1

93	ERLRV	1

4729	ETLNS	1

4730	ETSSH	1

4731	EVLAV	1

3567	EVLGI	1

4732	EVLIQ	1

4733	EVLQE	1

4734	GALGL	1

4735	GALGV	1

4736	GALIS	1

4737	GALMQ	1

4738	GALRD	1

4739	GALRG	1

4740	GAVMN	1

4741	GE*GI	1

4742	GELKV	1

4743	GELML	1

4744	GELMR	1

4745	GELRV	1

4746	GELTG	1

4747	GFLAR	1

4748	GGFRD	1

4749	GGLA	1

4750	GGLAE	1

368	GGLGA	1

4751	GGLGE	1

4752	GGLGP	1

4753	GGLHP	1

1957	GGLKV	1

4754	GGLMD	1

4755	GGLMT	1

4756	GGLNI	1

2357	GGLRG	1

4757	GGLRL	1

4758	GGLSG	1

4759	GGLVG	1

4760	GGVGL	1

4761	GHLAI	1

4762	GHLQC	1

3159	GHLQR	1

3330	GHLRR	1

4763	GHLSV	1

3448	GHLVG	1

3316	GHLVK	1

4764	GILAR	1

4765	GILSG	1

4766	GKLAI	1

4767	GKLGG	1

4768	GKLIG	1

4769	GKLII	1

4770	GKLIT	1

	GKLKMHIRSH

4771	TGERPF	1

4772	GKLLK	1

4773	GKLNA	1

4774	GKLPT	1

4775	GKLQA	1

3587	GKLR	1

3588	GKLRA	1

4776	GKLRE	1

4777	GKLT	1

4778	GKLTM	1

4779	GLAA	1

4780	GLIV	1

4781	GLLEK	1

4782	GLLGG	1

4783	GLLMV	1

3364	GLLPG	1

4784	GLLQD	1

4785	GLLTG	1

4786	GLSG	1

4787	GLSGR	1

4788	GLSV	1

4789	GLVN	1

4790	GLVQ	1

4791	GMLAG	1

4792	GNLSN	1

727	GPLA	1

4793	GPLKP	1

4794	GPLRP	1

4795	GPLVP	1

4796	GQLGP	1

4797	GQLLE	1

4798	GR*ML	1

4799	GRLGG	1

4800	GRLLG	1

4801	GRLMP	1

4802	GRLVS	1

4803	GRYGC	1

3279	GSLRV	1

4804	GSLSK	1

4805	GSLSP	1

4806	GTLKL	1

4807	GTLLL	1

2685	GTLLV	1

4808	GTLMT	1

2192	GTLRV	1

4809	GTLTE	1

4810	GVIN	1

	GVL	1

4811	GVLDN	1

4812	GVLE	1

4813	GVLKD	1

3454	GVLQK	1

4814	GVLRL	1

4815	GVLSG	1

2220	GVLTG	1

4816	GVMN	1

4817	GVPV	1

4818	HELMR	1

4819	HLLVP	1

4820	HPLDR	1

4821	HPLLS	1

4822	HPVKE	1

4823	HTLKM	1

4824	HTLLK	1

4825	HTLNI	1

3178	HTLNK	1

4826	HTLRP	1

4827	IALPG	1

4828	IELAL	1

4829	IELG	1

4830	IELHL	1

4831	IGIQR	1

4832	IGLGA	1

4833	IGLRL	1

4834	IHLAG	1

4835	IHLRM	1

4836	IKLTG	1

4837	IMLPR	1

4838	IQLMG	1

4839	IQLRL	1

4840	IRLAA	1

4841	IRLGP	1

3338	IRLGV	1

4842	IRLRR	1

4843	ISLVG	1

4844	ITLMV	1

4845	ITLRG	1

4846	ITLRP	1

4847	ITLVG	1

4848	IVLPG	1

	KG	1

4849	KGLAT	1

4850	KGLDL	1

4851	KGLMR	1

4852	KGRSPVET	1

4853	KIIV	1

4854	KILLA	1

4855	KKLAG	1

4856	KKLGV	1

4857	KKLRI	1

4858	KLLAG	1

4859	KLLRV	1

4860	KPLAA	1

4861	KPLMV	1

4862	KRLEG	1

4863	KSLVG	1

4864	KTLEG	1

4865	KTLRG	1

2404	KTLRV	1

4866	KTLVG	1

4867	KVLPV	1

4868	LAHGT	1

4869	LGLGP	1

4870	LGLGV	1

4871	LKVKL	1

4872	LNLHT	1

4873	LRLIM	1

4874	LRVIG	1

4875	LSLSG	1

4876	LTLQQ	1

4877	LVLRG	1

4878	MALRG	1

4879	MELIG	1

4880	MGLRV	1

4881	MLAA	1

4882	MLLIS	1

4883	MLLLP	1

4884	MLLMV	1

4885	MLLPP	1

4886	MLLPV	1

4887	MLLV	1

4888	MLLVG	1

4889	MLVG	1

4890	MMLDP	1

4891	MPLGA	1

4892	MPLGL	1

4893	MPLLG	1

4894	MRLEE	1

4895	MRLGA	1

4896	MRLGG	1

4897	MRLGR	1

3654	MRLVG	1

4898	MSLHG	1

4899	MSLQQ	1

4900	MTLER	1

	MVL	1

4901	MVLMN	1

4902	MVLNT	1

4903	MVLRG	1

4904	MVLVT	1

4905	MVVAS	1

4906	NDALQYD	1

	NDALQYESEC

4907	GP	1

4908	NELLR	1

4909	NELMR	1

4910	NELRV	1

4911	NGLG	1

	NGLIVHIRSHT

4912	GERPF	1

	NGR	1

4913	NGRPPG*E	1

4914	NGRSPVR	1

4915	NILMG	1

4916	NKLAR	1

4917	NKLRA	1

4918	NKLRG	1

4919	NKLVA	1

4920	NKLVK	1

4921	NMLGV	1

4922	NNLIN	1

1838	NRLRE	1

4923	NRLRI	1

4924	NSLV	1

4925	NSLVA	1

	NVHP*VVGLA

4926	A	1

4927	NVLGE	1

4928	PALAG	1

4929	PALGP	1

4930	PALV	1

4931	PASV	1

4932	PDLRA	1

4933	PGITE	1

4934	PGLAP	1

4935	PGLHE	1

4936	PGVAA	1

4937	PGVVP	1

4938	PHLKR	1

4939	PKLIF	1

4940	PLRG	1

4941	PMLAG	1

4942	PMLTM	1

4943	PNLAS	1

3786	PNLAV	1

3919	PNYW	1

4944	PNYWS	1

4945	PQLVV	1

4946	PQSRG*RG	1

4947	PR*GA	1

4948	PRLRL	1

4949	PSFQ	1

4950	PTLAK	1

4951	PVLKV	1

4952	PVLMT	1

2602	QALKR	1

4953	QALRG	1

4954	QALSP	1

4955	QGLHL	1

3675	QGLPV	1

4956	QILLQ	1

	QILLRHIRSHT

4957	GERPF	1

4958	QILLY	1

4959	QILPE	1

4960	QMLAR	1

4961	QPLAV	1

4962	QPLTM	1

4963	QRLGG	1

4964	QTLAV	1

4965	QTLGG	1

4966	QTLGP	1

4967	REIVR	1

4968	RELRR	1

4969	RGLAA	1

4970	RGLDN	1

4971	RGLNS	1

4972	RGLRS	1

4973	RGLTG	1

4974	RGLVE	1

4975	RGYGT	1

	RHE	1

4976	RHLKM	1

4977	RLLGL	1

4978	RP*SG	1

4979	RPLAG	1

4980	RQLGK	1

4981	RQLLE	1

4982	RRLEA	1

4983	RRLET	1

2126	RRLGD	1

4984	RRLGS	1

4985	RRLSE	1

4986	RRLTP	1

4987	RRVVG	1

	RSH	1

4988	RTLKL	1

4989	RTLVG	1

4990	RVLEP	1

4991	RVLRE	1

	SC**A	1

4992	SCLK	1

4993	SGILV	1

4994	SGLGG	1

4995	SGLGL	1

4996	SGLGT	1

4997	SGLLG	1

4998	SGLNL	1

4999	SGLRL	1

5000	SGLVG	1

3331	SHLRL	1

3425	SKLIL	1

2438	SKLKA	1

3722	SKLKG	1

5001	SKLLG	1

3334	SKLRI	1

2191	SKLRM	1

3337	SKLVL	1

5002	SL*HG	1

5003	SLLRT	1

5004	SNLTY	1

5005	SNYWP	1

5006	SPLIG	1

5007	SPLKI	1

5008	SPLRN	1

2138	SQLKV	1

5009	SQMK	1

	SR*G	1

1857	SRLKV	1

5010	SRLMT	1

5011	SRLVT	1

5012	SSLGA	1

5013	SSLGL	1

5014	STLQK	1

5015	SVLVG	1

5016	SVLVS	1

	T	1

5017	TALEA	1

5018	TALKG	1

5019	TELE	1

5020	TELIR	1

5021	TELPR	1

5022	TELRV	1

5023	TGLAD	1

5024	TGLGA	1

5025	THLAN	1

5026	THLAV	1

3318	THLRK	1

3808	TKIRV	1

3785	TKLKA	1

5027	TKLLR	1

5028	TKLME	1

3802	TKLNV	1

3955	TKLR	1

3783	TKLRA	1

3361	TKLRI	1

5029	TKLRR	1

5030	TKLVL	1

5031	TKSGV	1

5032	TLIS	1

5033	TLLIR	1

5034	TLLM	1

5035	TLLMQ	1

5036	TLNG	1

5037	TLQP	1

5038	TMLDP	1

5039	TMLRE	1

5040	TNLVG	1

5041	TPLIV	1

5042	TPLMQ	1

5043	TPLSD	1

5044	TPLSI	1

5045	TQLED	1

5046	TRLGA	1

5047	TRLMI	1

5048	TRLRL	1

1883	TRLRV	1

5049	TRLTG	1

5050	TSLSE	1

5051	TTLEP	1

5052	TTLGV	1

1849	TTLKV	1

1919	TTLRV	1

5053	TVLGG	1

5054	TVLT	1

	V*KS	1

5055	VALHT	1

5056	VDLLL	1

5057	VELAP	1

5058	VELN	1

5059	VELNN	1

5060	VELRV	1

5061	VGLPV	1

5062	VGLQA	1

2652	VGLQR	1

5063	VGLRN	1

5064	VGLRV	1

5065	VGLSP	1

5066	VGLSQ	1

5067	VHLAL	1

5068	VKLMA	1

5069	VKLQN	1

3765	VKLRL	1

5070	VLLAA	1

5071	VLLIE	1

5072	VLLKI	1

5073	VLLTP	1

5074	VLMV	1

5075	VLQR	1

5076	VMLRG	1

3772	VPLAL	1

5077	VPLVG	1

5078	VQLPM	1

5079	VQLRV	1

5080	VRLEG	1

5081	VRLGG	1

3778	VRLQA	1

5082	VRLVR	1

	VTG	1

5083	VTLER	1

5084	VTLGS	1

	WRN	1

TABLE 22

ZF4 selection on G:A change at
nt 11 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

118	GNLRR	3407

69	ANLRR	1937

117	GNLVR	1794

116	SNLRR	1771

5085	SNLKR	1208

68	TNLRR	862

119	GNLKR	850

138	GNLAR	805

2582	SNLVR	764

2609	GNLQR	562

70	GNLTR	531

121	NNLRR	486

2914	GNLIR	475

2494	ANLVR	455

2706	GNLNR	373

2517	GNLLR	360

2620	ANLKR	326

2524	SNLAR	269

2963	SNLQR	261

139	GNLMR	251

2695	SNLMR	228

2746	GNLHR	220

5086	SNLTR	209

5087	NNLKR	202

5088	SNLIR	199

5089	ANLMR	191

2621	ANLNR	179

74	TMLRR	158

5090	SNLNR	155

5091	ANLTR	136

5092	ANLQR	125

2595	TNLKR	118

73	AMLRR	111

2567	GNLSR	107

2542	ANLAR	102

66	ATLRR	96

2558	HNLRR	90

2538	AALRR	81

2496	SNLLR	77

5093	ANLER	73

2556	SMLRR	62

5094	ANLHR	59

5095	ANLLR	58

3032	SMLKR	51

2544	SNLSR	47

2541	TNLQR	47

2521	GALKR	44

2641	GALRR	44

3347	AHLRR	42

2823	HMLRR	40

2047	HMLKR	36

5096	RNLQR	35

71	AMLKR	31

2722	GMLKR	31

3161	GMLRR	29

2131	SALKR	28

5097	SNLER	26

5098	KNLQR	25

5099	RNLRR	24

2584	GTLRR	21

2978	TMLKR	21

2481	GNLER	20

5100	QNLKR	19

67	RRLDR	19

2638	STLRR	19

2526	TNLNR	17

2575	QNLRR	16

2523	SALRR	16

2714	TNLHR	16

2551	ANLIR	15

1985	AALKR	14

48	ATLKR	14

2875	ASLRR	13

2587	NTLRR	13

2511	TNLVR	13

3330	GHLRR	12

2691	NNLMR	12

2617	TALKR	12

5101	KNLER	11

2518	NNLVR	11

3403	THLRR	11

5102	SMLQR	10

2561	TNLMR	10

2737	TTLRR	10

2475	AGLRR	9

2622	ATLTR	9

3050	HNLKR	9

5103	KNLVR	9

2464	SGLRR	9

2769	VNLRR	9

5104	AMLTR	8

2882	AVLRR	8

3393	GHLKR	8

5105	TNLTR	8

3017	ATLNR	7

2739	ATLVR	7

5106	HNLMR	7

2734	TALRR	7

4308	TNLLR	7

5107	AMLQR	6

52	ANLSR	6

2509	ASLKR	6

2876	ASLTR	6

2801	ATLMR	6

5108	GMLER	6

5109	RLLIN	6

5110	SGLLK	6

2649	TNLAR	6

5111	AHLVR	5

3012	ATLHR	5

2881	ATLQR	5

2599	ENLRR	5

3084	HMLQR	5

72	HMLTR	5

5112	ISLRV	5

2543	NNLAR	5

3205	SNLHR	5

2153	STLKR	5

5113	AHLKR	4

2879	ATLIR	4

2623	DNLRR	4

2592	GALTR	4

5114	GNLRK	4

5115	KKLLR	4

5116	MNLRR	4

5117	MVLLR	4

5118	NNLQR	4

5119	QNLVR	4

5120	RNLAR	4

3396	SHLRR	4

2962	SMLHR	4

2679	TNLER	4

5121	TVLLV	4

2738	AALNR	3

2770	AALVR	3

1986	AGLKR	3

2539	ETLRR	3

3159	GHLQR	3

3449	GHLVR	3

5122	GMLNR	3

5123	GMLTR	3

5124	GMLVR	3

2608	GNLGR	3

5125	GNLRG	3

5126	GNLVK	3

2600	GSLRR	3

2554	GTLKR	3

56	HTLRR	3

3010	HVLRR	3

5127	KNLRR	3

5128	MNLKR	3

3407	NGRSPV...	3

2712	NMLRR	3

2757	PNLIR	3

3370	QHLRR	3

2956	SALNR	3

5129	STLEV	3

2967	STLNR	3

5130	TALRS	3

1305	THLKR	3

5131	TNLIR	3

2700	AALTR	2

5132	AMLNR	2

5133	ANLRL	2

5134	ANLRW	2

2654	ATLAR	2

5135	DALLV	2

2528	GGLIR	2

4764	GILAR	2

3160	GILRR	2

	GN*S...	2

2522	GNLDR	2

5136	GNLNK	2

5137	GNLRP	2

5138	GNLRS	2

5139	GTLIR	2

3081	GTLMR	2

2626	GTLVR	2

5140	HGLET	2

5141	HMLNR	2

2644	HNLVR	2

5142	KNLMR	2

2637	NNLLR	2

2756	NSLRR	2

5143	PGLLG	2

5144	RNLVR	2

5145	SMLNR	2

2677	SMLTR	2

2487	SNLDR	2

2850	STLMR	2

2970	SVLRR	2

2462	TGLRR	2

5146	TMLQR	2

2766	TSLKR	2

2860	TTLKR	2

3075	TVLRR	2

5147	AALRS	1

5148	ADLER	1

3089	ADLVR	1

2798	AGLMR	1

1431	AHLTR	1

2871	AILTR	1

5149	AMLAR	1

5150	AMLHR	1

5151	AMLIR	1

5152	ANFRR	1

5153	ANIQR	1

5154	ANLDR	1

2771	ANLGR	1

5155	ANLVG	1

5156	ANSRR	1

5157	ANVRR	1

5158	APLRR	1

2799	ASLQR	1

2880	ATLLR	1

5159	ATLRS	1

5160	AYFRR	1

5161	CNLAR	1

5162	CNLNR	1

5163	CNLVR	1

2591	DNLKR	1

2506	DNLVR	1

2778	GALNR	1

3035	GDLAR	1

2816	GDLRR	1

2780	GDLTR	1

2027	GGLKR	1

2461	GGLRR	1

2909	GGVRR	1

5164	GHLNR	1

5165	GNFRR	1

5166	GNFVG	1

5167	GNLAG	1

5168	GNLAS	1

5169	GNLHK	1

5170	GNLLS	1

5171	GNLMS	1

5172	GNLNH	1

5173	GNLQS	1

5174	GNLRH	1

5175	GNLS...	1

5176	GNLTK	1

5177	GNLTQ	1

5178	GNLTW	1

5179	GNLVW	1

5180	GNLWR	1

5181	GNSKR	1

5182	GNSQR	1

5183	GNSRR	1

5184	GNVQR	1

5185	GNVTR	1

5186	GQLAL	1

2819	GSLKR	1

2747	GTLNR	1

5187	GY*LR	1

2661	HNLAR	1

2752	HNLQR	1

5188	ITLQR	1

5189	KILGN	1

5190	KNLKR	1

1356	KNLTR	1

5191	KSLRR	1

5192	LNLRR	1

5193	LNLVR	1

2664	NMLKR	1

2690	NNLIR	1

5194	NNLNR	1

2726	NNLTR	1

5195	NNSRR	1

2788	NTLAR	1

2939	NTLIR	1

2628	NTLKR	1

2940	NTLNR	1

5196	PRLRG	1

5197	QHLKR	1

2574	QMLKR	1

2593	QTLRR	1

5198	RLIIN	1

5199	RNLKR	1

3292	SALQR	1

2559	SGLKR	1

5200	SHLKR	1

3202	SILNR	1

5201	SKLTR	1

2647	SMLIR	1

5202	SMLVR	1

5203	SNLFR	1

5204	SNLIH	1

5205	SNLRK	1

5206	SNLRQ	1

5207	SNLSG	1

5208	SNLTS	1

5209	SNLVW	1

5210	SNSRR	1

5211	SNVKR	1

5212	SNVRG	1

2698	STLVR	1

5213	TMFRR	1

3109	TMLNR	1

2680	TNLGR	1

5214	TNLLS	1

5215	TPTRS	1

5216	TQLVL	1

2589	TSLRR	1

5217	VNLTR	1

2997	VTLRR	1

TABLE 23

ZF4 selection on G:C change at
nt 11 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID
NO:	Sequence	Read #

73	AMLRR	3064

74	TMLRR	2212

2556	SMLRR	1556

3161	GMLRR	1320

2722	GMLKR	1160

3032	SMLKR	1049

71	AMLKR	797

2978	TMLKR	515

2823	HMLRR	478

2047	HMLKR	429

66	ATLRR	261

5102	SMLQR	248

5107	AMLQR	212

5132	AMLNR	125

5104	AMLTR	124

5146	TMLQR	123

2712	NMLRR	119

2664	NMLKR	102

2677	SMLTR	98

72	HMLTR	93

5123	GMLTR	88

5150	AMLHR	72

5122	GMLNR	68

2962	SMLHR	63

5145	SMLNR	59

48	ATLKR	58

5124	GMLVR	50

5141	HMLNR	47

3084	HMLQR	47

5149	AMLAR	46

5218	AMLVR	45

3109	TMLNR	38

5219	GMLHR	34

5202	SMLVR	34

2533	SMLAR	29

2638	STLRR	27

2970	SVLRR	27

67	RRLDR	26

118	GNLRR	24

2737	TTLRR	24

2882	AVLRR	23

5151	AMLIR	22

2913	GMLAR	22

5220	GMLQR	22

2584	GTLRR	19

2875	ASLRR	18

5221	HMLAR	17

2587	NTLRR	17

69	ANLRR	16

2713	QMLRR	16

3017	ATLNR	15

2574	QMLKR	15

5222	RRLKN	15

5223	AMLMR	14

2801	ATLMR	14

5224	GMLIR	14

5225	EMLRR	13

117	GNLVR	13

5226	RTLAL	13

5227	SMLSR	13

116	SNLRR	13

2647	SMLIR	12

1986	AGLKR	11

	TRS	11

2739	ATLVR	10

	TRS...	10

2538	AALRR	9

3012	ATLHR	9

2582	SNLVR	9

5228	TMLTR	9

68	TNLRR	9

5229	TMLVR	8

3075	TVLRR	8

2027	GGLKR	7

2914	GNLIR	7

2609	GNLQR	7

3407	NGRSPV...	7

2559	SGLKR	7

5230	TMLMR	7

2860	TTLKR	7

2881	ATLQR	6

2622	ATLTR	6

5231	GMLMR	6

70	GNLTR	6

2554	GTLKR	6

5085	SNLKR	6

2965	SSLKR	6

5232	AMLER	5

5233	AMVRR	5

2494	ANLVR	5

119	GNLKR	5

5086	SNLTR	5

5234	TMLAR	5

3987	VELNS	5

2654	ATLAR	4

2879	ATLIR	4

2606	EMLKR	4

138	GNLAR	4

139	GNLMR	4

5087	NNLKR	4

5235	SMLMR	4

2153	STLKR	4

2462	TGLRR	4

5093	ANLER	3

2620	ANLKR	3

2621	ANLNR	3

5092	ANLQR	3

2509	ASLKR	3

2520	DMLRR	3

2641	GALRR	3

2706	GNLNR	3

5236	HLLRR	3

5237	HMLHR	3

3010	HVLRR	3

5238	KTLRR	3

	LL...	3

121	NNLRR	3

2477	SGLTR	3

5239	SMLKN	3

3203	SMLLR	3

2963	SNLQR	3

2967	STLNR	3

1985	AALKR	2

2738	AALNR	2

3516	ALLRR	2

5240	AMLLR	2

5241	AMLRH	2

5242	AMLRS	2

5243	AMLRW	2

5244	AMLSR	2

5094	ANLHR	2

2802	AVLKR	2

5108	GMLER	2

5245	GMLKN	2

5246	GMLRW	2

5247	GMVRR	2

2600	GSLRR	2

2921	GVLRR	2

3039	HILKR	2

5248	HILRR	2

5249	HMLRS	2

3040	HMLVR	2

2558	HNLRR	2

56	HTLRR	2

5250	MGLST	2

5251	NMLIR	2

2628	NTLKR	2

2593	QTLRR	2

5252	RMLKR	2

5253	RMLQR	2

	RN*P...	2

5254	SMFKR	2

2524	SNLAR	2

2850	STLMR	2

5255	TLLRR	2

5256	TMIRR	2

5257	TMVRR	2

5258	VIKR...	2

5259	AKLQR	1

3062	ALLKR	1

5260	AMFRR	1

5261	AMIRR	1

5262	AMITR	1

5263	AMKTR	1

5264	AMLCR	1

5265	AMLHS	1

5266	AMLPR	1

4674	AMLR...	1

3519	AMLRG	1

5267	AMLRK	1

5268	AMLTM	1

5269	AMLWR	1

5270	AMYT...	1

2542	ANLAR	1

5271	ARLRR	1

4682	ARLRS	1

1947	ARLRV	1

3251	ASLNR	1

2878	ATLER	1

3025	ATLGR	1

5159	ATLRS	1

2772	ATLSR	1

5272	CMLRR	1

2640	DMLKR	1

3078	DMLQR	1

5273	DMVKR	1

5274	EMLNS	1

2539	ETLRR	1

5275	GLLKR	1

5276	GLLQS	1

5277	GLLSR	1

5278	GMIKR	1

5279	GMLKT	1

5280	GMLRM	1

5281	GMLTW	1

2746	GNLHR	1

2517	GNLLR	1

5282	GRLKR	1

5283	GRLKS	1

5284	GRLRV	1

2747	GTLNR	1

2626	GTLVR	1

3001	GVLKR	1

2483	HALRR	1

2531	HLLKR	1

5285	HLLNS...	1

5286	HMLLR	1

5287	HMLMR	1

5288	HMVRR	1

5106	HNLMR	1

2784	HVLKR	1

5189	KILGN	1

5289	KMLKR	1

5290	LMLGK	1

5291	MLRR	1

5292	NLLKR	1

5293	NMLGR	1

5294	NTFRR	1

2939	NTLIR	1

2940	NTLNR	1

5295	PMLMR	1

5296	PVVKR	1

2692	QSLKR	1

5297	RMFRR	1

5298	RMLRR	1

2956	SALNR	1

2523	SALRR	1

2464	SGLRR	1

3004	SILKR	1

3470	SKLKR	1

5201	SKLTR	1

5299	SLLNR	1

5300	SMFRR	1

5301	SMIKR	1

5302	SMLGR	1

5303	SMLKW	1

5304	SMSRR	1

5305	SMVKR	1

2496	SNLLR	1

5090	SNLNR	1

2792	SQLKR	1

1876	SRLKR	1

5306	SRLRR	1

2845	SSLAR	1

2698	STLVR	1

2699	SVLKR	1

5307	TILRR	1

5308	TMLER	1

5309	TMLGR	1

5310	TMLHR	1

5311	TMLLR	1

5312	TMLRH	1

5313	TMLWR	1

2595	TNLKR	1

2856	TNLSR	1

5215	TPTRS	1

5314	VMLKR	1

5315	VSLRK	1

2997	VTLRR	1

5316	WMLKR	1

5317	WMLRR	1

5318	YMLKR	1

5319	YMLRR	1

TABLE 24

ZF4 selection on G:T change at
nt 11 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

66	ATLRR	6399

67	RRLDR	1155

2584	GTLRR	1073

2737	TTLRR	1024

2638	STLRR	970

3017	ATLNR	770

2739	ATLVR	727

48	ATLKR	708

2587	NTLRR	670

2538	AALRR	657

2801	ATLMR	456

2654	ATLAR	418

2554	GTLKR	399

2875	ASLRR	366

2622	ATLTR	363

2593	QTLRR	298

2539	ETLRR	292

2881	ATLQR	291

2879	ATLIR	261

2153	STLKR	252

2628	NTLKR	237

56	HTLRR	227

2882	AVLRR	208

2880	ATLLR	171

1985	AALKR	141

2878	ATLER	134

3012	ATLHR	130

2860	TTLKR	125

2509	ASLKR	95

73	AMLRR	93

3010	HVLRR	81

2523	SALRR	63

5248	HILRR	60

74	TMLRR	59

2967	STLNR	58

2131	SALKR	47

2738	AALNR	46

2483	HALRR	44

2641	GALRR	41

2843	QTLKR	41

2783	HTLKR	39

3032	SMIKR	39

1930	HALKR	36

2970	SVLRR	36

2802	AVLKR	35

2556	SMIRR	34

3161	GMLRR	33

2722	GMLKR	31

2850	STLMR	31

2698	STLVR	31

2626	GTLVR	28

2521	GALKR	27

2747	GTLNR	27

2590	TTLQR	27

2921	GVLRR	25

118	GNLRR	24

116	SNLRR	24

2589	TSLRR	24

69	ANLRR	23

2997	VTLRR	23

2700	AALTR	22

71	AMLKR	22

2697	STLQR	22

5320	ATLRK	21

117	GNLVR	21

2823	HNIRR	20

2772	ATLSR	17

5321	RTLQR	17

2734	TALRR	17

2819	GSLKR	16

3018	STLIR	16

2717	AALQR	15

2800	ASLVR	15

2849	STLHR	15

2489	SSLRR	14

2978	TMLKR	14

3075	TVLRR	14

2876	ASLTR	13

3081	GTLMR	13

2047	HNIKR	13

2966	STLLR	13

2762	STLTR	13

2681	TTLNR	13

70	GNLTR	12

5189	KILGN	12

68	TNLRR	11

3864	ARLRI	10

2502	ETLKR	10

2600	GSLRR	10

2684	GTLAR	10

5322	KTLER	10

5323	QTLMR	10

3028	SILRR	10

5085	SNLKR	10

2617	TALKR	10

2799	ASLQR	9

3001	GVLKR	9

121	NNLRR	9

2877	ATLDR	8

138	GNLAR	8

2914	GNLIR	8

5324	KTLQR	8

5325	RTLRR	8

5102	SMLQR	8

2965	SSLKR	8

1947	ARLRV	7

2607	GALVR	7

5139	GTLIR	7

2784	HVLKR	7

3067	MTLRR	7

5086	SNLTR	7

2582	SNLVR	7

2620	ANLKR	6

119	GNLKR	6

5326	HILNR	6

5327	MTLMR	6

2770	AALVR	5

5107	ANIQR	5

2609	GNLQR	5

2940	NTLNR	5

3027	NTLVR	5

3196	QTLTR	5

5328	RTLKR	5

2666	SALTR	5

2699	SVLKR	5

5104	AMLTR	4

2621	ANLNR	4

2494	ANLVR	4

5158	APLRR	4

3025	ATLGR	4

5329	ATVRR	4

2530	DTLRR	4

3160	GILRR	4

5122	GMLNR	4

3033	GTLLR	4

2707	GTLQR	4

5330	GVLSR	4

5331	HRLKI	4

2830	HTLVR	4

5332	KTLIR	4

5238	KTLRR	4

5087	NNLKR	4

2756	NSLRR	4

2939	NTLIR	4

2677	SMLTR	4

2524	SNLAR	4

2963	SNLQR	4

2550	STLAR	4

5333	TILAR	4

2766	TSLKR	4

2857	TTLAR	4

2618	TTLMR	4

3117	AILRR	3

5089	ANLMR	3

3090	ASLAR	3

5334	ASLHR	3

5335	ATLNK	3

5336	ATLRG	3

2583	EALRR	3

3049	GILKR	3

5123	GMLTR	3

2706	GNLNR	3

4375	GPLPV	3

5337	GPLVR	3

3245	GSLSR	3

72	HMLTR	3

2827	HSLRR	3

5338	HVLNR	3

5339	NSLKR	3

5340	NTLMR	3

5341	NVLRR	3

2950	QTLQR	3

5342	RRLNR	3

2956	SALNR	3

3292	SALQR	3

2733	SVLTR	3

1986	AGLKR	2

2475	AGLRR	2

1988	AGLVR	2

5150	AMLHR	2

5151	AMLIR	2

5343	ARLKI	2

3251	ASLNR	2

3244	ASLSR	2

5344	ATFRR	2

5345	ATLNW	2

5346	ATLRW	2

2634	ESLRR	2

3151	ETLVR	2

2778	GALNR	2

2815	GALQR	2

5124	GMLVR	2

2517	GNLLR	2

3230	HALTR	2

5141	HMLNR	2

2558	HNLRR	2

2586	HTLMR	2

2613	HTLQR	2

5347	IALAG	2

5348	MSLRR	2

5349	MTLLR	2

5350	MTLVR	2

3407	NGRSPV...	2

2664	NMLKR	2

2712	NMLRR	2

3191	PTLRR	2

5351	QRLSV	2

4424	RPLVG	2

5352	RRIDR	2

5353	RRLDS	2

5354	RRVDR	2

5355	RSLIR	2

5356	RTLIR	2

5357	SDLTV	2

2962	SMLHR	2

5358	SRLKI	2

2564	SSLVR	2

5359	STVRR	2

2651	TTLTR	2

2767	TTLVR	2

57	TVLKR	2

2546	AALAR	1

2864	AALLR	1

5360	AALNS	1

3367	AALRK	1

3410	AALRL	1

5147	AALRS	1

5361	AAVRR	1

5259	AKLQR	1

3510	AKLRR	1

3062	ALLKR	1

5149	AMLAR	1

5132	AMLNR	1

5218	AMLVR	1

5094	ANLHR	1

5092	ANLQR	1

5091	ANLTR	1

	AP*C...	1

5362	APLHR	1

5363	APLKR	1

5364	APLMR	1

5365	APLVR	1

5366	APYP...	1

5271	ARLRR	1

2874	ARLTR	1

5367	ARLVG	1

5368	ASFRR	1

5369	ASLER	1

3250	ASLMR	1

	AT*G...	1

5370	ATFKR	1

5371	ATFRT	1

5372	ATFTR	1

5373	ATIRR	1

5374	ATLES	1

5375	ATLFR	1

5376	ATLHW	1

5377	ATLIS	1

5378	ATLNH	1

5379	ATLNS	1

5380	ATLQG	1

5381	ATLQW	1

5382	ATLRI	1

5383	ATLRP	1

5384	ATLWR	1

5385	ATSVR	1

5386	ATVAR	1

5387	AVLGR	1

5388	AVLLR	1

5389	AVLNR	1

3121	AVLTR	1

3991	DKLRR	1

2640	DMLKR	1

5390	DRLRA	1

2656	DTLNR	1

5391	EPLVM	1

3038	ETLAR	1

3043	ETLQR	1

2592	GALTR	1

2816	GDLRR	1

2913	GMLAR	1

139	GNLMR	1

5392	GPFKR	1

5393	GPLGL	1

5394	GPLKR	1

5395	GSLGA	1

2781	GSLQR	1

2660	GSLTR	1

5396	GTFRR	1

3014	GTLDR	1

2917	GTLER	1

2918	GTLGR	1

5397	GTLMW	1

5398	GTLRK	1

2562	GTLTR	1

386	GTLVS	1

5399	GTSNR	1

5400	GTSRR	1

5401	GVLRK	1

5402	GVVRR	1

2749	HALMR	1

3246	HALQR	1

3039	HILKR	1

5403	HILQR	1

2578	HTLAR	1

2689	HTLLR	1

2828	HTLNR	1

3180	HTLRG	1

3181	HTLSR	1

3099	HVLHR	1

5404	KTLLR	1

5405	KTLVR	1

5406	MALRM	1

5407	MPLAR	1

4452	MPLNR	1

5408	MPLVR	1

	MRS	1

2833	MTLKR	1

4923	NRLRI	1

2788	NTLAR	1

2837	NTLHR	1

3015	NTLLR	1

2941	NTLQR	1

5409	NTLRW	1

3006	NTLTR	1

5410	NTLVS	1

5411	NTVRR	1

2942	NVLKR	1

5412	PPLKR	1

5413	PSLKR	1

5414	PTFHR	1

5415	QKLA...	1

2574	QMLKR	1

2692	QSLKR	1

3195	QTLHR	1

5416	QTLIR	1

5417	QTLRQ	1

3248	QTLVR	1

	RN*P...	1

5418	RRLAG	1

5419	RRLAR	1

5420	RRLDG	1

5421	RRLHR	1

5422	RRLVR	1

5423	RRSDR	1

5424	RRVEK	1

5425	RTLER	1

5426	RTLNR	1

5427	RTLRG	1

5428	SAVKR	1

2559	SGLKR	1

5201	SKLTR	1

2647	SMLIR	1

5145	SMLNR	1

5304	SMSRR	1

5088	SNLIR	1

5429	SPLRR	1

5430	SRLRI	1

5431	STLCR	1

2848	STLER	1

5432	STLKS	1

5433	STLRI	1

5434	STSRR	1

5435	SVLRK	1

5436	TALIR	1

5437	TALMR	1

2764	TALTR	1

5146	TMLQR	1

5438	TMLRG	1

5131	TNLIR	1

2595	TNLKR	1

5439	TPIMM	1

5215	TPTRS	1

1883	TRLRV	1

5440	TRSP...	1

2858	TTLGR	1

2859	TTLIR	1

5441	TTLRS	1

5442	TVLNR	1

3308	VSLRR	1

2995	VTLKR	1

5443	VTLQR	1

5444	VVLGN	1

5445	WRLDR	1

5446	WTLRR	1

TABLE 25

ZF3 selection on G:A change at
nt 13 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

81	GQLTV	1094

5447	GQINV	906

78	GELVV	766

5448	AELIV	643

5449	TELIV	552

5450	QELLV	528

5451	GELIV	525

5452	GELTV	505

80	GQLIV	476

5453	QELLT	457

5454	SELIV	416

5455	GQLLV	372

5456	SGLIV	372

5457	GQLII	361

5458	AELLV	311

5459	VELLI	277

5460	AELVV	271

5461	AQLIV	267

76	SQLIV	265

82	TELII	251

83	QGLLV	247

5462	SQLII	243

79	QQLLI	224

5463	AGLIV	221

5464	QELVV	209

5465	GELLV	206

86	GELLT	202

5466	SQLLV	199

5467	GELVI	194

75	QQLIV	179

5468	QELII	177

5469	TQLIV	176

5470	VELII	172

5471	VELLV	160

5472	GELLI	151

85	GQLLT	150

5473	NELLI	149

5474	GQLLI	148

5475	SQLLI	140

5476	AQLLV	136

5477	GQLIT	132

5478	GQLTI	129

5479	TELIT	122

5480	TELLI	118

5481	TELLV	116

5482	QELLI	112

5483	AGLVV	106

5484	GSLLV	104

5485	AQLVV	102

5486	HPPEE	100

5487	SQLVV	100

77	QQLLV	98

5488	QELIV	95

5489	SELII	91

5490	AQLII	90

5491	QQLVV	90

5492	TGLLV	88

5493	NQLII	88

5494	GQLVI	81

5495	AGLLV	80

5496	NQLLV	73

5497	QELGV	69

5498	GALVV	68

5499	SQLTV	67

5500	GELTT	67

5501	GELII	65

3710	SGLLV	63

5502	AELII	60

5503	TQLII	59

5504	QQLII	59

5505	AQLIT	58

5506	SQLIT	58

5507	SSLIV	57

5508	SELTV	57

5509	NELLV	57

5510	TQLLV	56

5511	QGLIV	55

5512	QELVI	55

5513	NELIV	55

5514	TELLT	53

TABLE 26

ZF3 selection on G:T change at
nt 13 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		Read
NO:	Sequence	#

79	QQLLI	1145

5452	GELTV	1108

81	GQLTV	933

5474	GQLLI	748

5447	GQLVV	545

5457	GQLII	518

80	GQLIV	479

78	GELVV	477

5515	GELIT	438

5466	SQLLV	432

5462	SQLII	431

85	GQLLT	404

5516	SQLSM	365

84	QQLLT	349

75	QQLIV	312

5486	HPPEE	308

5453	QELLT	300

5475	SQLLI	282

4773	GKLNA	281

5451	GELIV	263

5455	GQLLV	225

76	SQLIV	219

5517	RALLI	216

5518	ENLLI	201

5476	AQLLV	174

5519	PDLKR	174

86	GELLT	172

5505	AQLIT	164

5520	GQLVT	138

5521	GULLS	116

5450	QELLV	112

5522	GELNP	112

5523	GQLIQ	98

5524	PTLVG	98

5525	LVLAD	95

5526	EALRA	94

5467	GELVI	87

1926	STLKA	87

5494	GQLVI	85

5463	AGLIV	82

5527	GQLTL	82

5528	NVLGT	81

5529	KGLGP	79

5530	MQLRR	79

3026	GDLQR	75

5531	VLLPN	71

5532	MRLGD	69

5533	GQLAQ	67

4074	NELRG	67

5500	GELTT	66

5534	GELVT	64

333	STLVV	63

5535	VDLAV	61

5536	AQLTI	59

5537	DALPA	57

5538	SVLQL	57

5539	GPLGN	56

5540	GHLLL	52

5541	DVLDP	51

5542	SSLSI	50

5543	KMLAD	50

TABLE 27

ZF3 selection on G:C change at
nt 13 of core motif in CBS.
Sequences reflect position
2 to 6.

SEQ
ID		#
NO:	Sequence	Reads

173	RKHD	4641

175	RKAD	1938

174	RRSD	1299

681	RRHD	868

682	RKTD	182

683	NVSM	146

684	RQSD	76

685	RKND	69

686	SENV	69

687	VDHR	60

688	AQIV	58

689	KTPH	56

690	PKIV	51

691	GAEP	42

692	MIVE	40

693	VVGN	40

694	KGPE	36

695	GKVM	33

696	TEPG	33

697	TPHN	32

698	MPGG	31

699	DLEK	28

700	GTDN	27

701	ISRL	25

702	ATGL	21

703	ASNP	19

704	GAPT	17

705	HSPN	17

706	RPVA	16

177	RKDD	6

707	MIVD	4

708	RHRK	3

709	RKHV	3

710	RKQD	3

711	RKSD	3

712	DHHT	2

713	GKHD	2

714	MKAD	2

715	RKAE	2

716	RRAD	2

717	APIG	1

718	AQNR	1

719	DMDA	1

720	EAPM	1

721	EEMM	1

722	EPIR	1

723	GALE	1

724	GENV	1

725	GKAD	1

726	GKVD	1

727	GPLA	1

728	GRIE	1

729	IEKL	1

730	KAAS	1

731	KEEH	1

732	LKVD	1

733	LUVE	1

734	LMTQ	1

735	MASL	1

736	MGIG	1

737	MPGD	1

738	MSLG	1

739	NDMT	1

740	NMHT	1

741	NRIV	1

742	PENA	1

743	QKHD	1

744	QVPD	1

745	RASD	1

746	REHD	1

747	RGHD	1

748	RKHA	1

749	RKHY	1

750	RKLD	1

751	RKPD	1

752	RKVD	1

753	RKYD	1

754	RMSD	1

755	RRLD	1

756	RRND	1

757	RRRD	1

758	RRSG	1

759	RWHD	1

760	SHRL	1

761	SQHV	1

762	SSHD	1

763	TTHV	1

764	VHHV	1

765	WKAD	1

766	WKHD	1

REFERENCES

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Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered CCCTC-binding factor (CTCF) variant comprising at least one amino acid residue in at least one zinc finger that differs in sequence from the amino acid sequence of a wild-type CTCF, wherein the engineered CTCF variant binds to a mutant CTCF binding sequence (CBS) with a higher affinity than wild-type CTCF, the mutant CBS comprising at least one nucleotide base that differs in sequence from the nucleotide sequence of a consensus CBS, wherein the at least one amino acid residue that differs in sequence from the amino acid sequence of the wild-type CTCF is selected from the group consisting of amino acid residues at position(s) −1, +1, +2, +3, +5, and +6 of any of ZF7, ZF6, ZF5, ZF4, and ZF3 of the engineered CTCF variant.

2. The engineered CTCF variant of claim 1, wherein the mutant CBS has a Thymine (T), Adenine (A), or Guanine (G) residue at position 2 of the consensus CBS motif, the engineered CTCF comprising an amino acid residue threonine, asparagine, or histidine at ZF7 position +3.

3. The engineered CTCF variant of claim 1, wherein the mutant CBS has a G residue at position 2 of the consensus CBS motif, the engineered CTCF comprising the amino acid sequence DHLQT, EHLNV, AHLQV, EHLRE, DHLQV, EHLKV, EHLVV, DHLRT, or DHLAT at ZF7 positions +2 to +6.

4. (canceled)

5. The engineered CTCF variant of claim 1, wherein the mutant CBS has a T, or A residue at position 5 of the consensus CBS motif, the engineered CTCF at ZF6 positions +2 to +6 comprising:

the amino acid sequence NAMKR, GNMAR, EGMTR, SNMVR, or NAMRG; wherein the mutant CBS has a T residue at position 5 of the consensus CBS motif; or

the amino acid sequence EHMGR, DHMNR, THMKR, EHMRR, or THMNR, wherein the mutant CBS has a G residue at position 5 of the consensus CBS motif.

6. The engineered CTCF variant of claim 1, wherein the mutant CBS has a T, or C residue at position 6 of the consensus CBS motif, the engineered CTCF at ZF6 positions −1 to +3 comprising:

the amino acid sequence MNES or HRES, wherein the mutant CBS has a T residue at position 6 of the consensus CBS motif; or

the amino acid sequence RPDT, RTDI, or RHDT, wherein the mutant CBS has a G residue at position 6 of the consensus CBS motif.

7. The engineered CTCF variant of claim 1, wherein the mutant CBS has a C, A, or T residue at position 7 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 comprising:

the amino acid sequence HGLKV, HRLKE, HALKV, SRLKE, or DGLRV, wherein the mutant CBS has a T residue at position 7 of the consensus CBS motif;

the amino acid sequence HTLKV, or HGLKV, wherein the mutant CBS has an A residue at position 7 of the consensus CBS motif; or

the amino acid sequence SRLKE, HRLKE or NRLKE, wherein the mutant CBS has a C residue at position 7 of the consensus CBS motif.

8. The engineered CTCF variant of claim 1, wherein the mutant CBS has a C, A, or T residue at position 8 of the consensus CBS motif, the engineered CTCF at ZF5 positions +2 to +6 comprising:

the amino acid sequence ATLKR, QALRR, GGLVR, or HGLIR, wherein the mutant CBS has a T residue at position 8 of the consensus CBS motif;

the amino acid sequence ANLSR, TGLTR, HGLVR, or GGLTR, wherein the mutant CBS has an A residue at position 8 of the consensus CBS motif;

the amino acid sequence HTLRR, TVLKR, ADLKR, or HGLRR, wherein the mutant CBS has a C residue at position 8 of the consensus CBS motif.

9. The engineered CTCF variant of claim 1, wherein the mutant CBS has a T, A, or C residue at position 10 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 comprising:

the amino acid sequence AHLRK, wherein the mutant CBS has a T residue at position 10 of the consensus CBS motif;

the amino acid sequence AKLRV, EKLRI, or AKLRI, wherein the mutant CBS has an A residue at position 10 of the consensus CBS motif; or

the amino acid sequence TKLKV, wherein the mutant CBS has a C residue at position 10 of the consensus CBS motif.

10. The engineered CTCF variant of claim 1, wherein the mutant CBS has a T, A, or C residue at position 11 of the consensus CBS motif, the engineered CTCF at ZF4 positions +2 to +6 comprising:

the amino acid sequence ATLRR or RRLDR, wherein the mutant CBS has a T residue at position 11 of the consensus CBS motif;

the amino acid sequence TNLRR, ANLRR, or GNLTR, wherein the mutant CBS has an A residue at position 11 of the consensus CBS motif; or

the amino acid sequence AMLKR, HMLTR, AMLRR, or TMLRR, wherein the mutant CBS has a C residue at position 11 of the consensus CBS motif.

11. The engineered CTCF variant of claim 1, wherein the mutant CBS has a T, A, or C residue at position 13 of the consensus CBS motif, the engineered CTCF at ZF3 positions +2 to +6 comprising:

the amino acid sequence QQLIV, SQLIV, QQLLV, GELVV, or QQLLI, wherein the mutant CBS has a T residue at position 13 of the consensus CBS motif;

the amino acid sequence GQLIV, GQLTV, GKLVT, TELII or QGLLV, wherein the mutant CBS has an A residue at position 13 of the consensus CBS motif; or

the amino acid sequence QQLLT, GQLLT, GELLT, or QQLLI, wherein the mutant CBS has a C residue at position 13 of the consensus CBS motif.

12. The engineered CTCF variant if claim 1, wherein the mutant CBS has A, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:

(i) the amino acid sequence AKLKK, AKLRK, AHLRV, AKLRV, or SKLRL at ZF4 positions +2 to +6 of the engineered CTCF;

(ii) the amino acid sequence ERLRV, NRLKV, SRLKE, or NRLKV at ZF5 positions +2 to +6 of the engineered CTCF;

(iii) the amino acid sequence RPDT, RTET, or RADV at ZF6 positions −1 to +3 of the engineered CTCF; and

(iv) the amino acid sequence DNLLA, SNLLV, DNLMA, or DNLRV at ZF7 positions +2 to +6 of the engineered CTCF.

13. The engineered CTCF variant of claim 1, wherein the mutant CBS has G, G, T, and T residues at positions 2, 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:

(i) the amino acid sequence GHLKK, AHLRK, or GKLRI at ZF4 positions +2 to +6 of the engineered CTCF;

(ii) the amino acid sequence SRLKE, DALRR, DGLKR, or TRLRE at ZF5 positions +2 to +6 of the engineered CTCF;

(iii) the amino acid sequence at RPDTMKR or RTENMKM at ZF6 positions −1 to +6 of the engineered CTCF; and

(iv) the amino acid sequence EHLKV, DHLLA, or HHLDV at ZF7 positions +2 to +6 of the engineered CTCF.

14. The engineered CTCF variant of claim 1, wherein the mutant CBS has A, and A residues at positions 2, 5, and 11 of the consensus CBS motif, respectively, the engineered CTCF comprising:

(i) the amino acid sequence SNLRR, GNLVR, GNLRR, GNLKR, ANLRR, NNLRR, or TNLRR at ZF4 positions +2 to +6 of the engineered CTCF;

(ii) the amino acid sequence EHMKR, EHMRR, THMKR, EHMNR, or EHMAR at ZF6 positions +2 to +6 of the engineered CTCF; and

(iii) the amino acid sequence DNLLT, DNLLV, DNLQT, DNLLA, DNLAT, DNLQA, DNLMA, or DNLMT at ZF7 positions +2 to +6 of the engineered CTCF.

15. (canceled)

16. The engineered CTCF variant of claim 1, wherein the mutant CBS that has T, and T residues at positions 6, 7, and 10 of the consensus CBS motif, respectively, the engineered CTCF comprising:

(i) the amino acid sequence GHLKK, AHLKK, TKLRL, TKLKL, GHLRK, THLKK, or AHLRK at ZF4 positions +2 to +6 of the engineered CTCF;

(ii) the amino acid sequence TRLKE or SRLKE at ZF5 positions +2 to +6 of the engineered CTCF; and

(iii) the amino acid sequence RADN, RHDT, RRDT, RPDT, RTSS, or RNDT at ZF6 positions −1 to +3 of the engineered CTCF.

17. The engineered CTCF variant of claim 1, wherein the engineered CTCF variant interacts with cohesion to mediate the formation of an enhancer-promoter loop to modulate gene expression.

18. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an engineered CTCF variant according to claim 1.

19. The method of claim 18, wherein the subject has cancer.

20. A method of activating or repressing expression of a gene under the control of a mutant CBS of claim 1, the gene being aberrantly expressed under the control of the mutant CBS, the method comprising contacting the mutant CBS with an engineered CTCF according to any one of claims 1-17, thereby regulating the expression of the gene.

21. The method of claim 20, wherein the engineered CTCF activates or represses expression of the gene by interacting with cohesion to mediate the formation of an enhancer-promoter loop.

22. A pharmaceutical composition comprising an engineered CTCF variant according to claim 1.

23. A gene expression system for regulation of a gene, the system comprising a nucleic acid encoding an engineered CTCF variant according to claim 1.

24. A method of altering the structure of chromatin comprising contacting an engineered CTCF variant according to claim 1 with a mutant CBS to form a binding complex, such that the structure of the chromatin is altered.

25. A method of modulating expression of a gene that is under the control of a CBS bearing one or more mutations, the method comprising contacting the CBS bearing one or more mutations with an engineered CTCF variant according to claim 1.

26. A kit comprising an engineered CTCF variant according to claim 1 and instructions for use in a method described herein.