WO1993004076A1 - Facteur de transcription de yy1 et procede d'isolation dudit facteur - Google Patents

Facteur de transcription de yy1 et procede d'isolation dudit facteur Download PDF

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WO1993004076A1
WO1993004076A1 PCT/US1992/006840 US9206840W WO9304076A1 WO 1993004076 A1 WO1993004076 A1 WO 1993004076A1 US 9206840 W US9206840 W US 9206840W WO 9304076 A1 WO9304076 A1 WO 9304076A1
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transcription factor
transcription
sequence
dna
protein
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PCT/US1992/006840
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Yang Shi
Edward Seto
Thomas Shenk
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Trustees Of Princeton University
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • This application relates to the YY1 transcription factor, which is a eukaryotic protein regulating the expression of various genes.
  • the application also relates to the isolation and cloning of the YY1
  • Transcription factors can be divided into two classes; those that activate and those that repress transcription.
  • a variety of eukaryotic transcriptional activators has been described (reviewed in Johnson and McKnight, 1989; Mitchell and Tjian, 1989).
  • Discrete domains that participate in transcriptional activation have been identified, including the acidic amino acid stretch in GAL4 (Gill and Ptashne, 1987), the glutaminerich sequence in Spl (Courey and Tjian, 1988) and the proline-rich region in CTF/NF-1 (Mermod et al., 1989). Relatively few transcriptional repressors have been described in eukaryotes.
  • the Drosophila Kr ⁇ ppel protein is the only instance in which functional domains capable of mediating repression have been identified.
  • An N- terminal, alanine-rich domain from the Kr ⁇ ppel protein fused to the DNA binding domain of the lac repressor can repress transcription of target genes containing lac operator sequences (Licht et al., 1990).
  • a function contained within or very close to the C-terminal zinc finger domain of the Kr ⁇ ppel protein in addition to its role in DNA binding (Zuo et al., 1991).
  • GCF human transcriptional repressor was cloned, termed GCF, that binds to GC-rich sequences (Kageyama and Pastan, 1989). Domains within the protein that mediate
  • RNA molecules include upstream and downstream promoter-proximal elements, enhancers, repressors, and silencers, which modulate the rate of specific initiation by RNA
  • promoter-proximal region between -45 to +30 contains two highly conserved motifs, the TATA sequence at around -30 and CA at +1 (Bucher et al., 1986).
  • TATA element-binding factor TFIID has been purified and cloned from several organisms and has provided invaluable insight into the process of
  • a sequence located at -50 to -70 of the adeno- associated virus (hereinafter referred to as: AAV) P5 promoter mediates adenovirus EIA-induced transcriptional activation of the promoter (Chang et al., 1989). This same element mediates transcriptional repression in the absence of EIA. Although two distinct cellular proteins were found to interact with the sequence, only one of them, YY1, is involved in transcriptional repression. In addition to the sequence between -50 to -70 (P5-60 site),
  • YY1 binds to the sequence surrounding the transcription initiation region of the promoter (P5+1 site). Both binding sites are capable of repressing transcription directed by heterologous promoters. EIA not only relieves repression exerted by YY1 but stimulates transcription through the YY1 binding site. Thus, EIA can activate transcription through the same cis element that mediates repression.
  • YY1 was expressed as a GAL4-YY1 chimera.
  • the GAL4-YY1 hybrid protein was able to direct both
  • YY1 repressed transcription directed by a TATA element plus initiator sequence both within transfected cells ( Figure 6) and in cell-free transcription extracts (Figure 7).
  • YY1 also repressed activity of the SV40 enhancer/promoter when its binding site was inserted into the 5' side of the enhancer element (Table 1), indicating that repression is not limited to a specific promoter or sequence context.
  • a single copy of the YY1 binding site reduced transcription 10 to 75-fold ( Figure 6 and
  • methylation interference experiments indicated that the proteins from the two sources contact the same bases within the YY1 binding site.
  • oligonucleotide affinity chromatography The 68kD factor might well be modified or associated with different accessory factors in the presence of EIA, but there is no evidence for a second protein with YY1 DNA-binding specificity in infected as compared to uninfected cells.
  • RNA blot analysis was performed using the cDNA to probe HeLa cell mRNA, and a single 2.6 kb band was identified.
  • a consensus sequence for initiation of translation (Kozak, 1984a; Kozak, 1984b) is present near the 5' end of the cDNA (position 241-243), although the reading frame remains open to its 5' side. It seems likely that this AUG serves as the normal translation start site since it is used at good efficiency in a reticulocyte lysate to produce a protein that migrates in SDS polyacrylamide gels as a polypeptide close to 68 kD in size.
  • transcription factor YY1 denotes a mammalian transcription factor that can both enhance and repress transcription and which is native to mammalian tissue.
  • the term also refers to any bioactive portions of the YY1 factor that have either the repressor or enhancer functions of YY1, or other activities characteristic of YY1.
  • Natural allelic variations of YY1 may exist in nature and may be distinguished by amino acid differences in the overall sequence or by deletions, substitutions, insertions, inversions or additions of one (or more) amino- acids in the sequence. In addition, the location of and degree of post translational modification might depend on the nature of the host cellular
  • transcription factor YY1 All such allelic variations and modifications resulting in derivatives of transcription factor YY1 are included within the scope of this invention, as long as it maintains either YY1's characteristic enhancement or repression of transcription activity. Alternatively, such derivatives may maintain the ability to bind to the characteristic YY1 binding sites.
  • YY1 contains four C 2 H 2 -type zinc fingers (underlined in Figure 11) that exhibit sequence similarity (73% identity) to those of the REX-1 protein (Hosier et al., 1989).
  • REX-1 is a zinc finger protein whose expression is rapidly reduced by retinoic acid treatment of F9 teratocarcinoma cells. Its DNA recognition site, as well as its functions remain unclear.
  • the YY1 zinc finger motifs, as well as those of REX-1, are related to those of the GLI-Kr ⁇ ppel family of genes (Ruppert et al., 1988).
  • Three of the four fingers in YY1 belong to the GLI subgroup with the consensus amino acid sequence:
  • YY1 can either repress or activate depending on the intracellular milieu.
  • the Drosophila Kr ⁇ ppe ' l protein can also repress or activate transcription, depending on the context of its binding site (Frasch and Levine, 1987; Licht et al., 1990;
  • YY1 as identified by SEQ ID NO:l does not contain an alanine-rich sequence, but it does contain a glycine-rich (42%) segment between amino acids 157 to 201 ( Figure 11); which, given the similarity of glycine and alanine residues might serve the same function.
  • Kr ⁇ ppel protein contains an activation domain or performs this function by interacting with an adaptor protein; however we have identified an acidic domain between amino acid 12 to 53 of YY1 shown in SEQ ID NO:1 ( Figure 11) that can activate transcription when removed from the context of the YY1 protein and fused to a heterologous DNA-binding domain. It is of note that YY1 contains a stretch of 11 consecutive histidine residues between amino acids 70-80. The basic histidine stretch might conceivably neutralize the putative activating function of the nearby acidic domain under repressing conditions.
  • the YY1 protein can bind to its recognition site with equal efficiency whether present in uninfected or adenovirus-infected, EIA-containing
  • adeno-associated virus In the absence of a helper virus, adeno-associated virus normally integrates into the genome of its host cell and remains transcriptionally silent. YY1 binding within the P5 promoter presumably helps maintain the viral chromosome in its latent state. A domain that represses transcription within the Epstein-Barr virus BZLFl promoter includes a YY1 binding site.
  • YY1 helps to regulate expression of the BZLFl gene, whose product, in turn, mediates the switch from latent to lytic infection (Rooney et al., 1988). Thus it is possible that YY1 plays a role in the maintenance of latency in several different virus systems.
  • oncogenes jun and fos are positive acting transcription factors which together form the activity known as AP-1.
  • Recessive oncogene, pRB appears to repress the activity of the EZF transcription factor. Since YY1 influences transcription, it is possible that YY1 might exhibit oncogenic properties if it were mutated, over expressed, or not expressed at all within a cell. If YY1 exhibits oncogenic properties, then antisense nucleic acids, antibodies and chemotherapeutic agents that specifically target the protein could prove valuable in cancer
  • FIG. 1 Identification of YY1 binding activity in HeLa cells. Band shift assay of YY1 binding activity in HeLa cells. 32 P-labeled P5-60 oligonucleotide, corresponding to the sequence from -49 to -71 of the AAV P5 promoter was used as substrate for binding with crude HeLa nuclear extracts. P5ML is an oligonucleotide containing an MLTF (major late transcription factor) binding site (Chang et al., 1989). Numbers above the lanes indicate the molar excess of unlabeled, competitor DNAs. Bands
  • YY1 protein used. Complexes corresponding to YY1 binding at the P5 +1 site only (BI), +1 and-60 (BII), as well as free probe DNA, are labeled.
  • FIG. 4 Methylation interference analysis of YY1 binding sites in the P5 promoter. The methylation interference pattern on the non-coding strand of the P5 promoter is shown. The left-most lane represents
  • Lanes 2 and 3 correspond to BI and BII in ( Figure 3) after chemical cleavage. Interference at the +1 and the -60 positions are indicated at the right. Strong methylation interference bases are indicated by solid dots. Weak interference sites are indicated by solid rectangles.
  • YY1 is shown as a stippled rod; factor 2 is represented by an open oval; MLTF is shown as a left-ward striped rectangle and TFIID is a right-ward striped rectangle. Wild-type and various mutant derivatives of P5-60 and P5+1 YY1 binding
  • Figure 7 Representative autoradiogram of in vitro transcription from synthetic promoters. Results are presented as means + SD of three independent reactions using at least two separate preparations of template DNA and nuclear extracts.
  • oligonucleotides were used as substrates for binding with partially purified YY1.
  • P5ML is an oligonucleotide containing an MLTF binding site. Numbers above the lanes indicate the molar excess of unlabeled, competitor DNAs. Bands corresponding to YY1 complex and free DNA are labeled. Refer to Figure 5 for the exact sequences of both wild-type and mutant P5-60 and P5+1 YY1 binding sites. Figures 9-10. Visualization of purified YY1 protein and determination of its binding specificity.
  • FIG. 9 Silver staining of YY1 protein at different purification stages.
  • the left-most lane contains
  • Lane 1 crude HeLa nuclear extract; lane 2, flow through from HIC chromatography; lane 3, HIC column fraction containing no YY1 activity; lane 4, peak HIC fraction containing YY1; lane 5, eluate from the first round of affinity column chromatography; lanes 6 to 10, flow through and sequential washes from the first round affinity column chromatography; lanes 11 to 13, purified
  • YY1 protein eluted from second round affinity column purification.
  • residues (aa 43-52) and eleven histidine residues (aa 70- 80) is underlined as are the four zinc-finger sequences near the C-terminus of the protein and potential polyadenylation signals within the 3' untranslated region.
  • FIG. 12 Bacterially synthesized YY1 protein binds specifically to its cognate sites. Ten ng of purified, renatured YY1-HIS fusion protein was incubated with the 32 P-labeled P5-60 oligonucleotide (YY1 site at -60).
  • Oligonucleotide P5+1 contains YY1 binding sequence at the cap site of the P5 promoter.
  • AP-1 represents an
  • oligonucleotide containing an AP-1 binding site whose sequence is taken from the promoter of the human
  • pGAL4-TKCAT contains five GAL4 binding sites placed upstream of the TK TATA box in plasmid pBL2CAT.
  • pGAL4-YY1 contains the entire coding region of YY1 cDNA with GAL4 (1-147) fused to its N-terminus.
  • pGAL4-YY1 ⁇ is a derivative of pGAL4-YY1, lacking 83 amino acids at the C-terminus.
  • FIG. 14-15 Assays were performed by transfecting either HeLa (Fig. 14) or NXE 3T3 (Fig. 15) cells. CAT assay results were plotted with mean ⁇ SD from three independent transfections.
  • the line connected by solid dots represents data collected from co-transfeetion with GAL4 (1-147) only.
  • the line connected by solid squares represents data collected from co-transfection with GAL4 YY1.
  • the line connected by solid triangles represents data collected from co-transfection with pGAL4-YY1 ⁇ .
  • the line connected by open squares represents co-transfectio of pGAL4 YY1 with TKCAT.
  • Each co-transfection assay contained lO ⁇ g of target plasmids.
  • Figures 16-17 Models for YY1-mediated transcriptional repression and activation.
  • Figure 16. YY1 protein as a repressor is represented by a shaded rectangle.
  • YY1 as an activator is shown as a shaded oval.
  • EIA protein is represented by a dark square. In the absence of EIA, YY1 represses
  • YY1 is converted from a repressor to an activator by either modifying it or by physically associating with it.
  • YY1 protein exists as a transcriptional activator with the activation domain being blocked by another cellular protein, represented by a dark circle.
  • EIA dissociates the cellular protein and unmasks the transcriptional activation domain of YY1.
  • P5+1 element can function as a
  • FIG. 1 In vitro transcription of the F5 promoter.
  • Wildtype and mutant templates illustrated at the top of each panel, were transcribed in HeLa nuclear extracts, and product RNAs were assayed by reverse transcription.
  • the expected size of the reverse transcripts are shown on the left side of the autoradiogram.
  • Molecular weight markers are derived from 32 P-labelled Msp I-digested pBR322 fragments. Figure 19. Specific transcription with the P5+1 element.
  • Wildtype and mutant templates illustrated at the top of each panel, were transcribed in HeLa nuclear extracts, and product RNAs were assayed by reverse transcription. The expected size of the reverse transcripts are shown on the left side of the autoradiogram.
  • Molecular weight markers are derived from 32 P-labelled Msp I-digested pBR322 fragments. Figures 20-23. Transcription directed by the F5+1 element in the absence and presence of YY1 activities.
  • FIG. 20 HeLa nuclear extracts were depleted for YY1 activities by two sequential passages through a YY1- specific DNA affinity column. ⁇ lectrophoretic mobility shift assay (EMSA) was used to monitor YY1 activity.
  • ESA ⁇ lectrophoretic mobility shift assay
  • FIG. 21 HeLa nuclear extracts depleted for YY1.were used to transcribe template P5+1. Anti-YY1 or preimmune antibodies were added where indicated. ,Primer extension products specific to the template are indicated by arrow.
  • FIG. 22 Drosophila embryo extracts devoid of YY1 were used to transcribe template pP5+l. Anti-YY1 or preimmune antibodies were added where indicated. Primer extension products specific to the template are indicated by arrow.
  • FIG. 23 Western blot analysis of YY1.
  • the arrow indicates the expected size of YY1 protein from Hela extract. Prestained high-molecular-weight markers (BRL) were used as standards.
  • FIG. 25 In vitro transcription of the LeIF-J+1 sequence elements. Reverse transcription products of the RNA are indicated by arrow.
  • FIG. 26 EMSA of YY1-related factor binding to the Tdt and LeIF-J Inr. Formation of complex I is specifically inhibited by addition of excess Tdt Inr and the P5 Inr oligonucleotides but not by the addition of an API oligonucleotide. Complex II and III are nonspecific.
  • Complex IV is specifically competed by addition of excess LeIF-J Inr and the P5 Inr oligonucleotides but not by the addition of an API oligonucleotide.
  • Complex V is specifically competed by addition of excess LeIF-J Inr and the P5 Inr oligonucleotides but not by the addition of an API oligonucleotide.
  • sequence-specific DNA protein complex was present but was masked by a co-migrating complex formed by a non-specifi DNA binding activity in HeLa cells (complex II, Figure 1). This protein (termed factor 2) was detected after removal of the non-specific DNA-binding activity by chromatography.
  • DNase I footprinting experiments were performed with DNA affinity purified YY1 protein.
  • the substrate used was the P5 transcriptional control region (-96 to +24) taken from plasmid P5-CAT190 (Chang et al., 1989).
  • a footprint covering the P5-60 sequence element was readily visible ( Figure 2, lane 6).
  • a second footprint was also detected at the transcription initiation region (P5+1 sequence) of the promoter
  • Figure 4 shows the methylation interference patterns observed on the non-coding strand of the P5 promoter.
  • the DNA- protein complex BI in Figure 3 corresponds to YY1 binding at P5+1 site only ( Figure 4, lane 2).
  • Complex BII represents YY1 binding at both P5+1 and at P5-60 sites of the promoter. Additional complexes migrating between BI and free DNA ( Figure 3) were also analyzed. No
  • the P5-60 element capable of mediating EIA-induced transcriptional activation contains partially overlapping binding sites for two cellular proteins, YY1 and factor 2.
  • YY1 and factor 2 Two cellular proteins
  • pTI contains a minimal synthetic promoter similar to that described by Smale and Baltimore (1989). It was constructed by inserting oligonucleotides containing the Tdt initiator and the TATA sequence from the adenovirus major late promoter into BamHI/SacI and EcoRI/SacI sites of pSP72, respectively. Oligonucleotides containing the P5-60 or P5+1 sequence, and various mutant derivatives thereof were inserted into the EcoRV site of pTI (-50 from initiation of transcription). This same set of promoter constructs was used for in vitro transcription analysis or in vivo transfection experiments with the CAT reporter gene at +52, downstream of the transcription initiation site.
  • Oligonucleotides containing the P5-60 or P5+1 sequence, as well as various mutant versions of the two sequences (as shown in Fig. 5) were placed directly upstream of the TATA box in construct pTI.
  • the chloramphenicol acetyltransferase (CAT) reporter gene was introduced downstream of the initiator sequence in all the constructs described above.
  • the YY1 binding site can mediate repression when inserted upstream of th SV40 promoter/enhancer domain.
  • Each cell free transcription reaction (25 ⁇ l) contained 100 ng template DNA, 12 mM HEPES (pH 7.9), 12% (v/v) glycerol, 60 mM KCl, 0.12 mM EDTA, 0.3 mM PMSF, 0.3 mM DTT, 10 mM MgCl 2 , 500 ng poly [dG-dC], 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM DTP, 1 mM creatine phosphate, and approximately 72-96 ⁇ g of HeLa cell nuclear extracts.
  • Reactions were incubated for 1 hr at 30°C, and terminate by the addition of 225 ⁇ l of stop buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, 1% SDS, 20 ⁇ g/ml yeast tRNA).
  • RNA products were analyzed by primer extension assay (McKnight and Kingsbury, 1982).
  • Reaction products were assayed by primer extension and the expected product of 79 nucleotides is denoted by an arrow at the right as shown in Figure 7. Reaction products were quantified relative to those produced by pTI, and results are presented as mean ⁇ SD of three independent reactions using at least two separate preparations of template DNA and nuclear extracts.
  • binding of YY1 to its cognate sites causes transcriptional repression.
  • the transcriptional repression can be relieved by adenovirus EIA protein.
  • YY1 responds to EIA by activating transcription since a functional YY1 binding site, in the presence of EIA, stimulates transcription to a greater extent than observed in constructs carrying mutant sites unable to bind the factor. This implies that YY1 is a
  • transcriptional repressor and can be converted into a transcriptional activator by EIA.
  • CAT plasmids carrying either the P5-60 or the P5+1 or mutant sequence upstream of the entire SV40 enhancer/promoter domain (pSVECAT) (see Figure 5 for the sequences of the wild- type and mutant YY1 binding sites).
  • the pSV40ECAT recombinants carrying wild-type and mutant P5-60 or P5+1 sequences were constructed by inserting double stranded oligonucleotides into pSV40ECAT at the BamHI site (5' to the SV40 enhancer). Two ⁇ g of each plasmid was
  • pP5-60SV40ECAT contained multiple copies of the wild-type P5-60 sequence, identified as SEQ ID N0:3; pP5- 60(mt2)SVECAT, identified as SEQ ID NO:5, contained multiple base-pair substitutions in the P5-60 sequence and showed substantially diminished binding for both YY1 and factor 2 as determined by band shift assays.
  • the wild-type P5-60 sequence caused at least a 15-fold reduction of CAT activity compared to pSV40ECAT with no insert (Table 1, compare pP5-60SVECAT and pSVECAT).
  • SEQ ID NO:5 failed to repress CAT activity, suggesting that either YY1, factor 2 or both contributed to repression.
  • P5+1 sequence that only binds YY1 was tested.
  • YY1 activity was purified by hydrophobic interaction column chromatography followed by two rounds of
  • Nuclear extract was prepared from HeLa cells grown in spinner culture supplemented with 10% calf serum as described (Dignam et al., 1983).
  • YY1 activity was partially purified from HeLa nuclear extract by chromatography on a TSK phenyl- 5PW matrix in an LKB UltroPac HPLC column (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.). Peak fractions containing YY1 activity were pooled and dialyzed to a final concentration of 0.1 M KCl.
  • a linker containing a BamHI cleavage site was added to the ends of an oligonucleotide
  • the most highly purified material contained a major polypeptide with an apparent molecular weight of 68 kD ( Figure 9).
  • the 68 kD protein band was excised from the gel, protein was recovered by elution, denatured in 6 M guanidine and renatured in DNA binding buffer (Wang, et al., 1987).
  • YY1 protein was visualized by silver staining (Oakley et al., 1980). The band was then excised out of the gel, minced and incubated with 2 ml of elution buffer
  • Affinity-purified YY1 proteins were pooled and concentrated by precipitation with acetone at -80°C overnight.
  • the protein pellet was resuspended in sample buffer, boiled and subjected to electrophoresis on a 12.5% SDS polyacrylamide gel.
  • the protein was
  • Two degenerate oligonucleotides were designed based on the amino acid sequence corresponding to the ends of the sequence: a 17- mer [5'GA(TC)AT(ATC)GA(TC)CA(TC)GA(AG)AC3'], identified as SEQ ID NO: 10, corresponding to the coding strand sequence and a 27-mer corresponding to the opposite strand at the other end of the sequence with inosine replacing nucleotides at the wobble position (5' GTA ATC IGG IGG GTT GTT ITC ICC IAT 3') identified as SEQ ID NO: 11. These two oligonucleotides resulted in a combined degeneracy of greater than 16,000 fold.
  • TGTT CTTCAACCACTGT-3' identified as SEQ ID NO:12 was synthesized based on the internal sequence of the 63 bp DNA fragment and was used as a probe to screen a HeLa ⁇ gtll cDNA library (D98/AH-2, a generous gift of T.
  • nested deletion constructs were made from both directions using the Erase-a-Base kit (Promega Corp., Madison, WI) with minor modifications. Briefly, the recombinant plasmid was linearized by two restriction enzymes by cleavage at two restriction sites adjacent to each other in the polylinker region to generate both 3' and 5' overhangs. After exonuclease IIl digestion, the DNA was subjected to electrophoresis on a 1% low melting agarose gel. The shortened DNA fragments were excised from the gel and ligation was performed after the low melting agarose was dissolved and diluted. Overlapping deletion constructs were sequenced by the dideoxy method (Sanger et al., 1977).
  • Figure 11 shows the YY1 cDNA as part of the sequenc identified as SEQ ID NO:l and its amino acid sequence (identified as SEQ ID:2).
  • the cDNA encodes a 414 amino acid zinc finger protein, and it includes the 21 amino acids determined by peptide sequencing of purified YY1.
  • YY1 cDNA was verified by expressing it as a fusion protein in E. coli, purifying it and testing its ability to bind to YY1 recognition sites. Expression and Purification of a HIS-YYl Fusion Protein Which Binds to YYl Recognition Sites
  • a DNA fragment containing the entire YY1 coding region beginning from the putative translation initiation AUG was isolated by digestion of clone 14-1 with Styl/Hindlll. The ends of this fragment were blunted by Klenow polymerase and cloned into the pDS56-6xHIS vector (Abate et al., 1990; a generous gift of R. Gentz, Hoffman-LaRoche & Co., Basel, Switzerland) in frame at its BamHI site (also blunt ended).
  • the resulting fusion protein contains 12 additional amino acids, MRGSHHHHHHGS, at the N-terminus of the YY1 protein and the six histidine residues allow the protein to be purified by chromatography on a nickel chelate matrix (Hochuli et al., 1987; Abate et al., 1990).
  • the fusion plasmid was introduced into a bacterial strain RR (Maniatis et al., 1982). Usually a liter of bacterial culture was grown to optical density of 0.7 to 0.8 units. Synthesis of the fusion protein was induced by 1 mM IPTG. Bacteria were allowed to grow in the presence of IPTG for 2 hrs. They were pelleted by centrifugation and lysed in 12 ml guanidine-HCl (pH 7.8) by incubating overnight at 4°C. The insoluble debris were removed by centrifugation at 18 krpm in a SS34 rotor.
  • the nickel chelate column was processed as follows: a 1 ml (bed volume) column was first washed with 10 ml distilled H 2 O, followed by 8 ml of 0.1 M NiSO 4 and another 2 ml of distilled H 2 O the column was then equilibrated with 10 ml of 6 M guanidine-HCl (pH 8.0). Bacterial lysate in the same buffer was loaded and the column washed first with 10 ml of 6 M guanidine-HCl (pH 8.0) and then with 10 ml of 6 M guanidine-HCl (pH 6.0). The fusion protein was eluted with 7 ml of 6 M guanidine-HCl (pH 5.0) and quantified by BioRad protein analysis
  • Figure 12 displays a band shift assay in which a 32 Plabeled oligonucleotide containing the P5-60 sequence was used as probe.
  • the YY1 cDNA was cloned in frame into the histidine fusion protein expression vector pDS56-6xHIS with the putative translation initiation AUG encoding the first amino acid of YY1 in the fusion protein.
  • fusion protein starts from the AUG in the vector and adds 12 additional amino acids to the Nterminus of the YY1 protein.
  • the fusion protein bound to the labeled probe, and the binding could be competed by addition of excess unlabeled P5-60 or P5+1
  • the cDNA encodes a protein that binds to YY1 recognition sites.
  • the YY1 cDNA insert was excised from subclone 14-1 by Apal/Clal and Apal/Hindlll digestion. Both Apal and Clal sites are located in the polylinker regions of the vector flanking the cDNA insert, while the Hindlll site is present in the YY1 cDNA at nucleotide position 1235 ( Figure 11).
  • the Apal/Clal and the Apal/Hindlll DN fragments after their ends were blunted by treatment with the Klenow polymerase, were cloned into the EcoRI site (also blunted with Klenow polymerase) of GAL4 (1- 147) expression plasmid. Due to the presence of the polylinker sequence, the fusion proteins encoded by the chimeric genes carry an additional 14 amino acids that connect the C-terminus of the GAL4 sequence to the N- terminus of the YY1 sequence.
  • Plasmids containing adenovirus 12S or 13S EIA cDNAs expressed under control of the cytomegalovirus immediate early promoter were provided by J. Nevins (Duke University).
  • YY1 were each cloned into the GAL4 fusion vector, pG4 (Ma and Ptashne, 1987).
  • the fusion proteins (GAL4-YY1 and GAL4 YY1 ⁇ ) contain the GAL4 DNA binding domain (amino acids 1-147) at their N-terminus ( Figure 13), directing the fusion protein to promoters containing GAL4 binding sites.
  • HeLa cells and NIH3T3 cells were grown on 10 cm dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Fortyeight hours after transfection, cells were lysed by repeated freeze/thaw cycles and extracts assayed for CAT activity (Gorman et al., 1982).
  • truncated fusion protein failed to repress because it was grossly unstable and failed to accumulate within
  • the target GAL4-TKCAT gene was co-transfected with the parent plasmid pG4 which only expresses the truncated GAL4 protein. No repression was detected in HeLa cells ( Figure 14) and a modest activation was observed in
  • adenovirus EIA proteins The ability of adenovirus EIA proteins to relieve repression and activate through the fusion protein was examined by cotransfecting HeLa cells with the plasmid encoding the GAL4-YY1 protein, the plasmid carrying the GAL4-responsive reporter construct, and plasmids encoding either the 12S or 13S EIA protein. 10 ⁇ g of target plasmids ( ⁇ GAL4-TKCAT or pTKCAT) were transfected into HeLa cells with plasmids encoding either the EIA 12S or the EIA 13S mRNAs. The results are expressed as mean ⁇ SD of three independent transfections. (Table 2).
  • the relative CAT activity reported as 1 represents an average CAT conversion of 15%.
  • the GAL4-YY1 protein inhibited CAT expression by a factor of about 12.
  • the 12S EIA protein relieved the repression, and the 13S EIA protein relieved the repression and further activated CAT expression by a factor of about 3.
  • YY1 protein when fused to the GAL4 DNA binding domain, can repress transcription.
  • the repression can be
  • EIA protein relieved by either EIA protein, and the 13S EIA protein can activate to a modest extent through the fusion protein.
  • the cloned YY1 protein mimics the behavior of the endogenous activity in all respects.
  • YY1 is an Initiator Sequence-Binding
  • adeno-associated virus type 2 P5 promoter +1 region (P5+1 element) sequence is necessary and sufficient for accurate basal transcription. Further, partially purified YY1 can restore basal level transcription from a P5+1 element in a HeLa extract depleted for YY1 or a Drosophila embryo extract devoid of
  • YY1 interacts with DNA elements centered at -60 and at the transcription initiation region of the adenoassociated virus P5 promoter. Since association of YY1 at the +1 region may point to novel means of
  • nucleotide -50 or -39 to +24 both of which include only the TATA and P5+1 elements, were actively transcribed (Fig. 18, lanes 2, 3).
  • Plasmid p ⁇ -20/+24 With the exception of p ⁇ -20/+24, plasmids shown in Figure 18 were previously described (Chang et al., 1989). Plasmid p ⁇ -20/+24 was created by deleting the DNA sequences between the Ava I and Kpn I sites in plasmid p- 50. Plasmids pMLTATA and pSpl (referred to as plasmids II and III, respectively in Smale, Schmidt, Berk and Baltimore, 1990) were provided by Stephen Smale
  • RNA in Figure 18 utilized a synthetic oligonucleotide primer complementary to the CAT gene sequence that was present downstream of promoter elements, 5'-GCCATTGGGATATATCAACGGTGG-3'.
  • RNA synthesized in Figure 19 used a synthetic oligonucleotide complementary to the SP6 promoter sequence in the vector (Promega). All experiments were carried out independently a minimum of three times with at least two separate plasmid and nuclear extract preparations to ensure data reproducibility.
  • Tdt terminal deoxynucleotidyltransferase
  • plasmid template pP5+1 produced a transcript of the expected length as detected by primer extension analysis.
  • plasmid template with a mutation in the P5+1 sequence so that it no longer binds YY1 did not produce detectable specific product (Fig. 19, lane 3).
  • the adenovirus type 2 major late promoter TATA box or the SV40 Spl binding sites alone promoted a low level of transcription initiation (Fig. 19, lanes 7,10), but the promoter strength and fidelity increased in the presence of an Inr element (Fig. 19, lanes 8, 11).
  • YY1 also diminished transcription to its original level s (Fig. 19, lanes 9, 12).
  • the P5 promoter TATA which is identical to the SV40 promoter TATA sequence, directed a detectable level of transcription only in the presence of the P5+1 element (Fig. 19, lanes 4, 5).
  • oligonucleotides SEQ ID NO: 14 (5'- CGGGAGGGTCTCCATTTTGAAG-3' and SEQ ID NO: 15 5'- CCCTCCCGCTTCAAAATGGAGA-3') were annealed, ligated, then coupled to sepharose columns.
  • Nuclear extracts were prepared from 36 liters of HeLa cells grown in spinner culture in SMEM medium supplemented with 5% calf serum and antibiotics. After loading the crude fraction (input), the column was washed and the bound protein eluted as described (Kadonaga et al., 1986).
  • EMSA was used to track YY1 activity. Flow-through from the first passage was reapplied to a fresh column to further deplete YY1 activity.
  • Eluate fractions containing high levels of YY1 activity were pooled and diluted with 6 volumes of buffer Z (20mM HEPES pH 7.8, 12.5 mM MgCl 2 , ImM DTT, 20% glycerol, 0.1% NP40) (Kadonaga et al., 1986); and reapplied to a second column to further purify YY1.
  • buffer Z (20mM HEPES pH 7.8, 12.5 mM MgCl 2 , ImM DTT, 20% glycerol, 0.1% NP40)
  • EMSA using the P5+1 oligonucleotides were performed as previously described (Chang et al., 1989) with the exception that the binding reactions contained 12 mM HEPES pH 7.9, 10% glycerol, 5 mM MgCl 2 , 60 mM KCl, 1 mM DTT, 50 ⁇ g/ml BSA, 0.5 mM EDTA, and 0.05% NP40.
  • Eluate fractions were diluted 1:48 before use for EMSA.
  • HeLa nuclear extracts were prepared as described (Dignam et al., 1983), Drosophila embryo extracts were obtained from Stratagene.
  • Polyclonal antibody against YY1 was prepared by immunizing rabbits with gel-purified YY1 fusion protein (HIS-YY1) produced in E. coli as previously described. Preimmune and anti-YY1 antibodies were purified using Econo-Pac Protein A columns (Bio-Rad) before use.
  • Proteins (30 ⁇ g each, Figure 23 , lanes 1 , 2 ; 0.625 ⁇ g, Figure 23, lane 3) were prepared in SDS denaturation buffer before being separated on a 12.5% SDS- polyacrylamide gel and electroblotted onto a
  • a YY1-specific antibody was employed to be certain that the key factor removed by the DNA affinity chromatography was, indeed, YY1.
  • the antibody was made by immunization of a rabbit with a YY1 fusion protein produced in E.coli, and it can specifically block
  • this antibody detects a single polypeptide migrating at relative molecular mass of
  • triosephosphate isomerase (Boyer et al., 1990), and the human immunodeficiency virus type (Jones et al., 1988) and (Okamoto et al., 1990) promoter +1 regions bear no obvious homology to the P5+1 sequence and unlike the P5+1 element, these sequences are insufficient for basal transcription in the absence of upstream or downstream elements.
  • Tdt Inr deoxynucleotidyltransferase gene (Tdt Inr) (Smale and Baltimore, 1989) and the initiation site of the human leukocyte interferon gene (LeIF-J) (Ullrich et al., 1982) revealed striking similarity to the P5+1 element
  • Oligonucleotides used were: LeIF-J+1, 5'- GATCCCTAGGTTTTCTGGAGACTGAGCT-3' and its complement to produce Bam HI and Sac I ends.
  • EMSA was carried out identical to Fig. 20, and probes used for the assay were 32 P-5' end-labelled double stranded oligonucleotides whose sequences are given in Fig. 24. Molar excess of
  • Non-specific API competitor consisted of oligonucleotides 5'- GGATGTTATAAAGCATGAGTCAGACACCTCTGGCT-3' and its
  • the LeIF-J+1 sequence functions in initiation of transcription as do the Tdt and the P5 Inr.
  • an oligonucleotide consisting of the P5+1 sequence can effectively inhibit the binding of a nuclear factor to the Tdt (Fig. 26, lanes 6-8, complex I) as well as the LeIF-J Inr sequences (Fig. 26, lanes 17-19, complex IV). Since a variety of
  • YY1 can direct the general transcription machinery to initiate RNA synthesis at its binding site.
  • P5+1 element when placed upstream of either a synthetic promoter or the SV40 early promoter/enhancer can repress transcription.
  • this same element can activate transcription when present alone or downstream from the TATA or Spl sites.
  • YY1 when altered, either in its amino acid sequence or in the levels at which it is expressed, may have prognostic value, by predicting how a tumor might respond to various treatments.
  • YY1 and YY1 from tumor cells or other altered states may be used to screen for natural biological products or organic chemical reagents that reverse or alleviate the oncogenic effects of the quantitatively or qualitatively abnormal YY1 produced in tumor cells.
  • YY1 may regulate the replication of viruses (e.g. Epstein-Barr viruses) to whose DNA it binds.
  • viruses e.g. Epstein-Barr viruses
  • agents that influence or modify the activity of YY1 may also alter the behavior of the viral pathogens.
  • Adenovirus EIA proteins can dissociate heteromeric complexes involving the E2F transcription factor: A novel mechanism for EIA trans-activation. Cell 62, 659-669.
  • Adenoassociated virus P5 promoter contains an adenovirus ElA-inducible element and a binding site for the major late transcription factor. J. Virol. 63, 3479-3488.
  • RNA Polymerase II Accurate transcription initiation of RNA Polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475-1489.
  • the thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements.
  • the adenovirus type 5 EIA transcriptional control region contains a duplicated enhancer element Cell 33, 695-703. Heiermann, R. and Pongs, O., Nucleic Acids Res. 13, 2709-
  • initiator codon affect the efficiency of translatio of rat preproinsulin in vivo. Nature 308, 241-246. Kozak, M. (1984b). Compilation and analysis of sequences upstream from the translational start site in
  • Drosophila Kr ⁇ ppel protein is a transcriptional repressor. Nature 346, 76-79. Lillie, J. W., and Green. M. R. (1989). Transcription activation by the adenovirus Ela protein. Nature 338, 39-44.
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Abstract

Facteur de transcription de YY1 à la fois réprimant et amplifiant la transcription dans des types 2 de virus adéno-associés, des virus de l'herpès et d'Epstein-Barr, ainsi que des oncogènes. On a isolé YY1, on l'a cloné en vecteurs d'expression appropriés et on a déterminé sa séquence.
PCT/US1992/006840 1991-08-16 1992-08-14 Facteur de transcription de yy1 et procede d'isolation dudit facteur WO1993004076A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1386639A1 (fr) * 2002-07-31 2004-02-04 Kylix B.V. Identification de 78 gènes impliqués dans le développement de tumeurs et leur utilisation pour le développement de drogues anticancéreuses et le diagnostic du cancer
EP1393776A1 (fr) * 2002-08-14 2004-03-03 Erasmus University Medical Center Rotterdam Identification de gènes impliqués dans le développement de tumeurs et leur utilisation pour le développement de drogues anticancéreuses et le diagnostic du cancer
WO2004056857A2 (fr) * 2002-12-20 2004-07-08 Ernst-Moritz-Arndt-Universität Greifswald Utilisation du facteur de transcription multifonctionnel yin-yang-1 et ses variants pour traiter des maladies, en particulier le diabete de type 1
JP2006508645A (ja) * 2002-08-14 2006-03-16 エラスムス ユニバーシティ メディカル センター ロッテルダム 抗癌剤の開発および癌の診断のための、腫瘍の発生に関与することが同定されたマウスゲノム領域の使用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 51, issued 04 December 1987, D.S. SHORE et al., "Purification and Cloning of a DNA Binding Protein from Yeast that Binds to Both Silencer and Activator Elements", pages 721-732. *
CELL, Volume 54, issued 29 July 1988, C.K. GLASS et al., "The Thyroid Hormone Receptor Binds with Opposite Transcriptional Effects to a Common Sequence Motif in Thyroid Hormone and Estrogen Response Elements", pages 313-323. *
JOURNAL OF VIROLOGY, Volume 63, No. 8, issued August 1989, L.S. CHANG et al., "Adeno-Associated Virus P5 Promoter Contains an Adenovirus E1A-Inducible Element and a Binding Site for the Major Late Transcription Factor", pages 3479-3488. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1386639A1 (fr) * 2002-07-31 2004-02-04 Kylix B.V. Identification de 78 gènes impliqués dans le développement de tumeurs et leur utilisation pour le développement de drogues anticancéreuses et le diagnostic du cancer
EP1393776A1 (fr) * 2002-08-14 2004-03-03 Erasmus University Medical Center Rotterdam Identification de gènes impliqués dans le développement de tumeurs et leur utilisation pour le développement de drogues anticancéreuses et le diagnostic du cancer
JP2006508645A (ja) * 2002-08-14 2006-03-16 エラスムス ユニバーシティ メディカル センター ロッテルダム 抗癌剤の開発および癌の診断のための、腫瘍の発生に関与することが同定されたマウスゲノム領域の使用
WO2004056857A2 (fr) * 2002-12-20 2004-07-08 Ernst-Moritz-Arndt-Universität Greifswald Utilisation du facteur de transcription multifonctionnel yin-yang-1 et ses variants pour traiter des maladies, en particulier le diabete de type 1
WO2004056857A3 (fr) * 2002-12-20 2004-10-28 Univ Ernst Moritz Arndt Utilisation du facteur de transcription multifonctionnel yin-yang-1 et ses variants pour traiter des maladies, en particulier le diabete de type 1

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