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Definitions

• TFs transcription factors
• RNA polymerase II machinery that initiates transcription from promoters
• diverse chromatin-associated factors and complexes that modulate chromatin structure and act as bridges between TFs and RNA pol II (Cramer, 2019).
• the human genome encodes thousands of proteins that arenvolved in various stages of transcriptional regulation, and the ready availability of methods such as ChIP-seq has revealed the genomic binding sites of hundreds of factors in diverse conditions (The ENCODE Project Consortium et al., 2020).
• the defined context alleviates the challenges posed by endogenous gene regulation, where multiple factors bind regulatory elements in concert, hindering causal inference.
• Artificial recruitment has been traditionally used to identify transcriptional activators or transactivation domains (TADs) in individual transcriptional regulators (Ptashne and Gann, 1997).
• TADs transcriptional activators or transactivation domains
• recent studies have characterized the transcriptional effects of large collections of regulators in fruit flies or yeast by individually tethering them to reporter genes (Keung et al., 2014; Stampfel et al., 2015). Due to the limited scalability of the arrayed format, these studies focused on known regulators rather than potentially novel factors.
• An aspect includes a heterologous transcriptional activator comprising: a DNA targeting domain, optionally an enzymatically inactive CRISPR-CAS protein, a zinc finger DNA binding domain, a tet-repressor or transcriptional activator-like effector (TALE) DNA binding domain; and an effector domain comprising: at least one transactivation domain (TAD) selected from the TADs listed in any one of Tables 1 to 6, optionally Table 2 or Table 6, or a functional variant thereof, or at least two TADs selected from the TADs listed in any one of Tables 1- 6, optionally Table 1 or Table 3, or functional variants of any thereof, preferably at least one TAD selected from the TADs listed in Table 4 or Table 5 or Table 6, or functional variants thereof, wherein the DNA targeting domain and effector domain are operably linked.
• TAD transactivation domain
• An aspect includes an isolated nucleic acid encoding an effector domain described herein [0013] An aspect includes an isolated nucleic acid encoding a heterologous transcriptional activator described herein. [0014] An aspect includes an expression construct comprising a nucleic acid described herein operably linked to one or more promoters and one or more transcription termination sites. [0015] An aspect includes a vector comprising a nucleic acid or expression construct described herein, optionally wherein the vector is an adenoviral or lentiviral vector. [0016] An aspect includes a cell comprising a transcriptional activator, nucleic acid, expression construct, or vector described herein.
• An aspect includes a transcriptional activation system comprising: a heterologous transcriptional activator described herein, wherein the DNA targeting domain comprises a CRISPR-Cas protein and at least one gRNA.
• An aspect includes a method of activating transcription of a target gene in a cell, the method comprising: a) introducing into the cell a transcriptional activator, nucleic acid, expression construct, or vector described herein; and b) culturing the cell under suitable conditions such that the effector domain activates transcription of the target gene.
• An aspect includes a screening method, the method comprising: a) introducing into a plurality of cells a transcriptional activator, one or more nucleic acids, one or more expression constructs, or one or more vectors described herein, wherein the DNA targeting domain comprises a CRISPR-Cas protein; and a plurality of gRNAs; or introducing a plurality of gRNAs into a population of cells described herein wherein the DNA targeting domain comprises a CRISPR-Cas protein; b) culturing the plurality of cells such that the one or more gRNAs associate with the CRISPR-Cas protein and guides the transcriptional activator to a CRISPR target site such that the effector domain activates transcription of a target gene; c) optionally treating with an amount of a test drug or toxin; d) optionally culturing the plurality of cells for a period of time to allow for gRNA dropout or enrichment; and e) collecting the plurality of cells, or
• An aspect includes a composition comprising a transcriptional activator, nucleic acid, expression construct, vector, or cell described herein.
• An aspect includes a kit comprising a vial and a heterologous transcriptional activator, nucleic acid, expression construct, vector, cell, or composition described herein and optionally one or more of: an inducing agent, a gRNA or a gRNA expression construct.
• A shows a schematic of the chemically-induced dimerization system to characterize transcriptional activators in human cells.
• B shows the percent of high GFP cells when the indicated constructs were transfected into HEK293T reporter cells and treated with abscisic acid or DMSO for 48 hours.
• C Outline of the pooled ORFeome screen for transcriptional activators.
• D Enrichment of high GFP cells in the pooled ORFeome screen after 48 hour ABA treatment.
• E Enrichment of ORFs in the high GFP pool compared to the unsorted ORFeome.
• F Enrichment of Gene Ontology categories among positive screen hits.
• G Enrichment of InterPro domains among positive screen hits.
• Fig. 2 shows transcriptional activity of transcription factor families. Transcriptional regulators were individually tested for activation of the reporter in an arrayed manner. DNA-binding specificity (shown as sequence logos) is from CisBP (Weirauch et al., 2014). Asterisks indicate statistically significant activators (FDR ⁇ 0.05).
• A Homeobox family proteins
• B Forkhead box proteins
• C Kruppel-like factors
• D SRY-related HMG-box (SOX) proteins
• E Polycomb-group RING Finger (PCGF) proteins. Composition of canonical (cPRC1) and non-canonical (ncPRC1) complexes is shown on the right.
• FIG. 3 shows systematic discovery of transactivation domains in human proteins with TAD-seq.
• A Outline of the TAD-seq pooled assay.
• B Examples of known transactivation domains. Domain organization is shown on top.
• TAD-seq plot shows the fold enrichment of RNAseq reads in the high GFP population or the medium GFP population.
• Each circle shows the mid-point (30th amino acid) of the 60-aa tile. Filled circles indicate statistically significant hits. Grey boxes indicate previously described transactivation domains. C, Examples of novel transactivation domains. Labeling is as in panel B. D, Location of the transactivation domain of HOXA2. E, Sequences of the activating fragments identified in HOXA2. Activating fragments are in bold. The region common to all three fragments that were enriched in the medium GFP population is indicated as overlap of activating fragments. The location of the antennapedia-like hexapeptide sequence is indicated as hexapeptide. F, Location of the transactivation domain in YAF2.
• G Crystal structures of RING1B RAWUL domain bound to the YAF2_RYBP domain of RYBP (PDB 3IXS) and the CBX_C domain of CBX7 (PDB 3GS2).
• H YAF2_RYBP domains and CBX_C domains from indicated proteins were individually tested for transcriptional activity. Asterisks indicate statistically significant activators (FDR ⁇ 0.05). Statistical significance was calculated with unpaired two-tailed t-test assuming equal variance, and corrected for multiple hypotheses with False Discovery Rate (FDR) approach of Benjamini, Krieger and Yekutieli (Benjamini et al., 2006).
• Fig. 4. shows co-factor specificity of transcriptional activators.
• A Proximity partners of indicated transcriptional regulators were identified with BioID2. Enrichment of selected co-factor complexes is shown as a heat map. Average spectral counts (n+1) were normalized to background spectral counts (n+1) of EGFP and Nanoluc baits.
• B Interaction patterns of activating Forkhead transcription factors based on the AP-MS study of (Li et al., 2015). Spectral counts were normalized as in panel A.
• C Left, effect of p300/CBP inhibition by A-485 on the activity of 83 transcriptional regulators.
• Known p300 interactors are shown as solid dark circles, known NuA4 interactors as outlined circles. Right, p300 interactors are significantly more affected than other transcriptional regulators by A-485 treatment. Statistical significance was calculated with one-way ANOVA using Dunnett’s multiple testing correction.
• D Left, effect of BET bromodomain protein inhibition by JQ1 on the activity of 83 transcriptional regulators.
• Known p300 interactors are shown as solid dark circles, known NuA4 interactors as outlined circles. Right, p300 interactors are significantly more affected than other transcriptional regulators by A-485 treatment.
• Fig. 5 shows SRF-C3orf62 fusion generates a potent p300-dependent transcriptional activator that promotes expression of SRF/MRTF target genes.
• A Schematic of the JAZF1-SUZ12 fusion found in low-grade endometrial stromal sarcomas. The transactivation domain identified by TAD-seq is indicated.
• Indicated PYL1 fusions were individually tested for activation of the genomically integrated reporter.
• F SRF-C3orf62 activates expression of serum response element reporter in the absence of cofactors.
• Indicated constructs C-terminally tagged with 3xFLAG-V5 were co-transfected into NIH3T3 cells with an SRE-Firefly luciferase reporter and a constitutive Nanoluc reporter. Relative luciferase activities were measured to assess the activity of each construct.
• NIH3T3 cells stably expressing doxycycline-inducible SRF-C3orf62-GFP or Nanoluc-GFP were treated with doxycycline for 46 hours, of which the last 22 hours in low-serum conditions (0.5% FCS).
• Gene expression patterns were analyzed by RNA-seq. Significantly upregulated (dark circles, top) and downregulated (medium circles, bottom) genes (absolute log2 fold change > 1, FDR ⁇ 0.05).
• Well characterized targets of SRF/MRTF (top, labeled circles) and SRF/TCF (bottom, labeled circles) are indicated.
• Fig.6 shows ORFeome screen for transcriptional activators.
• A Distribution of sequencing reads across the ORFeome in pooled plasmid DNA and in infected cells.
• B Distribution of ORF sizes in the plasmid pool and in infected cells.
• C Transcriptional activity depends on abscisic acid treatment. ORFeome-PYL1 infected cells were treated with 100 ⁇ M ABA and the fraction of high GFP cells measured by flow cytometer over time.
• D The effect of ABA concentration on transcriptional activity.
• Reporter cells transfected with the indicated constructs were treated with increasing amounts of ABA for 48 hours.
• E No high GFP population is observed in ABA treated cells not expressing the ORFeome-PYL1 library.
• F Enrichment of interaction hubs among the hits of the activation screen.
• G Enrichment of yeast two-hybrid autoactivators among the hits of the activation screen.
• H Individual validation of transcriptional activators identified in the activation screen. Indicated constructs were transfected into the reporter cell line and high GFP cell fraction was measured by flow cytometry after 48-hour treatment with ABA. Asterisks indicate statistically significant ABA- dependent increase in high GFP population (FDR ⁇ 5%).
• Fig. 7 shows transcriptional activity of transcription factor and chromatin- associated protein families. Transcriptional regulators were individually tested for activation of the reporter in an arrayed manner. DNA-binding specificity (shown as sequence logos) is from CisBP (Weirauch et al., 2014). Asterisks indicate statistically significant activators (FDR ⁇ 5%).
• Fig. 8 shows differential activity of transcription factors is not explained by expression levels.
• A Schematic of the transcriptional activation assay measuring both reporter gene expression and effector protein expression.
• B Transactivation of Kruppel-!ike factors (left) compared to the expression level of each factor as measured by RFP fluorescence (right). Background RFP intensity is shown as a dashed line.
• C Forkhead TF activity.
• D Homeodomain TF activity,
• E SOX TF activity.
• Fig. 9 shows TAD-seq identifies transactivation domains.
• A High and medium
• GFP population was assessed by flow cytometry after recruiting 60aa fragments to the reporter with ABA for 48 hours.
• High GFP and medium GFP cells were sorted by FACS and ORFs enriched in the pools were identified by next-generationsequencing.
• B Enrichmentand depletion of amino acids among the identified transactivator fragments compared to inactive fragments in the library. Amino acids shown in bold were statistically significantly enriched or depleted.
• C Enrichment of predicted transactivation domains among the active fragments. 9aaTADs were predicted with 9aaTAD prediction tool (https://www.med.muni.cz/9aaTAD/ ' ) using "Moderately Stringent Pattern”. Only 100% confident matches were considered in the analysis.
• ADpred algorithm was described in (Erijman et al. , 2020). Statistical significance was calculated with Fisher’s exact test. D, Predicted TADs among active fragments are iongerthan those in inactive fragments. Statistical significance was calculated with two-tailed t-test assuming equal variance. E, Enrichment of fragments in the high GFP poo! versus the medium GFP pool. Significant hits are shown as outlined circles. F, Individual validation of transactivating fragments. Indicated TADs were fused to PYL1 and transfected into the reporter cells in an arrayed format. Asterisks indicate statistically significant activators (FDR ⁇ 5%).
• Fig. 10 shows systematic discovery of transactivation domains in human proteins with TAD-seq.
• A Examples of known transactivation domains. Domain organization is shown on top.
• TAD-seq plot shows the fold enrichment of RNAseq reads in the high GFP population (dark circles) or the medium GFP population (light circles). Each circle shows the mid-point (30th amino acid) of the 60-aa tile. Filled circles indicate statistically significant hits. Grey boxes indicate previously described transactivation domains.
• B Examples of novel transactivation domains. Labeling is as in panel A.
• Fig. 11 shows transactivation domains of SPDYE4 and YAF2.
• A Alignment of five Spy1/RINGO family proteins; SPDYE4 (SEQ ID HO: 142); SPDYE1 (SEQ ID NO: 143); SPDYE7P (SEQ ID NO: 144); SPDYE2 (SEQ ID NO: 145); and SPDYC (SEQ ID NO: 146).
• SPDYE4 SEQ ID NO: 142
• SPDYE1 SEQ ID NO: 143
• SPDYE7P SEQ ID NO: 144
• SPDYE2 SEQ ID NO: 145
• SPDYC SEQ ID NO: 146
• Dashed box indicates the inferred minimal activating domain. Note that the region of the minimal activating domain is not conserved in SPDYC, which was the only Spy1/RINGO family member that did not activate transcription.
• B Alignment of YAF2_RYBP domains of YAF2 (SEQ ID NO: 147) and RYBP (SEQ ID NO: 148), and CBX_C domains of CBX family proteins (SEQ ID NOs: 149-153). The location of the two beta sheets is indicated with arrows.
• Fig. 12 shows interaction networks of transcriptional activators.
• A BioID2 network of transcriptional activators. Bait proteins (e.g.
• FAM90A1, SPDYE4, SS18L2, SOX7, etc. are shown as light grey rectangles. BAF complex members, p300/CBP, NuA4 complex members, Mediator components, and TFIID components are indicated. The width of the edges indicates average spectral counts of two replicates. For clarity, two highly connected prey proteins (ZNF518A and ZNF518B) were removed from this visualization B, AP-MS network of transcriptional activators. Labeling as in panel A. For clarity, nine highly connected prey proteins (APEH, ACTC1, ALDH1L1, PSDM4, LSS, FLII, PACSIN2, RPS3A, QPCTL) were removed from this visualization. [0036] Fig.
• FIG. 13 shows the effect of small molecule inhibitors on the transcriptional activity of 83 transcriptional regulators.
• A Effect of CDK9 inhibition by flavopiridol.
• Known p300 interactors are shown as solid dark circles, known NuA4 interactors as outlined circles.
• B Effect of Casein kinase 2 inhibition by CX4945.
• Known p300 interactors are shown as solid dark circles, known NuA4 interactors as outlined circles.
• C Effect of DYRK1A/DYRK1B inhibition by AZ191.
• Known p300 interactors are shown as solid dark circles, known NuA4 interactors as outlined circles.
• DYRK1A/DYRK1B interactor DCAF7, and DCAF7 interactor NCKISPD are highlighted.
• D Effect of p300 inhibition by A-485 on transcriptional regulators characterized by AP-MS and BioID. Asterisks indicate statistically significant activators (FDR ⁇ 5%). Statistical significance was calculated with unpaired two-tailed t-test assuming equal variance, and corrected for multiple hypotheses with False Discovery Rate (FDR) approach of Benjamini, Krieger and Yekutieli (Benjamini et al., 2006).
• FDR False Discovery Rate
• Fig. 14 shows transcriptional activation by SRF-C3orf62.
• B Analysis of differentially expressed genes in NIH3T3 cells expressing SRF-GFP, C3orf62-GFP, or SRF-C3orf62-GFP compared to cells expressing Nanoluc-GFP.
• C Gene Ontology enrichment analysis of significantly upregulated and downregulated genes in NIH3T3 cells expressing SRF-C3orf62-GFP.
• D Overlap of genes significantly upregulatedy by SRF-C3orf62-GFP and target genes of SRF/MRTF or SRF/TCF, published previously (Esnault et al., 2014; Gualdrini et al., 2016). Statistical significance was calculated with a hypergeometric distribution test.
• Fig.15 shows the effect of the minimal activating sequence for each individual component of SPDYE4-CITED1-P65-HSF1 fusion on transcriptional activity of an a EGFP reporter. All components were fused to PYL1 dimerization domain and recruited to the reporter with addition of 1 ⁇ M abscisic acid (ABA) for 48 hours. Sequence of each fusion is shown in SEQ ID NOs: 121, 123, 127, 129, 131, 133, and 135. Sequences of each of the individual domains tested are provided in SEQ ID NOs: 47, 90, 101-104, 116, and 118. [0039] Fig.16 shows examples of when fusing two or more transactivation domains and targeting them to the same reporter.
• ABA abscisic acid
• P300core is the catalytic domain EP300 (amino acids 1048-1664). Reporter cells were treated with 1 ⁇ M abscisic acid (ABA) or same volume of DMSO for 48 hours before collection for flow cytometry. Error bars represent S.D. from four replicates.
• Fig. 17 shows activity of tethering different combinations and orientations of activation domains from human SPDYE4, CITED1, p65, and HSF1 proteins to an EGFP reporter (left) or promoter of CD133 gene in HEK293T cells. Recruitment was induced by addition of 1 ⁇ M abscisic acid (ABA) for either 24 or 48 hours before cells were collected.
• Fig. 18 shows the effect of replacing each part of the multi-component SPDYE4-CITED1-P65-HSF1 (SCPH) activator with different activation domains. SPDYE4 activation domain appeared the most indispensable as replacing it with other individually stronger activation domains disrupted activity of the SPCH activator.
• Fig.19 shows the effect of 117 different effector domains comprising different combinations of transactivation domains, or fragments thereof, when used in combination with rTetR or dCas9 based recruitment systems.
• A Transcriptional activation using the 117 effector domains in combination with a rTetR DNA targeting domain.
• nucleic acid means two or more covalently linked nucleotides.
• the term generally includes, but is not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which may be single-stranded (ss) or double stranded (ds).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions.
• nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• oligonucleotide as used herein generally refers to nucleic acids up to 200 base pairs in length and may be single-stranded or double-stranded.
• sequences provided herein may be DNA sequences or RNA sequences, however it is to be understood that the provided sequences encompass both DNA and RNA, as well as the complementary RNA and DNA sequences, unless the context clearly indicates otherwise.
• sequence 5’-GAATCC-3’ is understood to include 5’-GAAUCC-3’, 5’-GGATTC-3’, and 5’GGAUUC-3’.
• functional variant includes modifications of the polypeptide sequences disclosed herein that perform substantially the same function as the polypeptide molecules disclosed herein in substantially the same way.
• functional variants may include active fragments of the polypeptides described herein, for example an N- and/or C-terminal truncation which retains transcriptional activation activity and/or co- activator interaction.
• Functional variants may include variants having one or more substituted amino acids and/or which retain at least a minimal sequence identity to the unmodified or non- variant sequence.
• the functional variant may comprise substitutions of up to 1, 2, 3, or more amino acids for every ten amino acids.
• the functional variant may comprise sequences having at least 80%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein.
• the functional variant may also comprise conservatively substituted amino acid sequences of the sequences disclosed herein.
• Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
• An example of substitutional amino acid variants are conservative amino acid substitutions.
• Functional variants such as active fragments including minimal fragments which can for example be identified as described herein which retain transcriptional activation activity and/or co-activator interaction can be identified for example using the methods described herein.
• a "conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another.
• conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
• the phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.
• heterologous transcriptional activator or “transcriptional activator described herein” as used herein means an engineered fusion protein or engineered dimer comprising: an effector domain comprising at least one transactivation domain (TAD) selected from the TADs listed in Table 2 and functional variants thereof, or at least two TADS selected from the TADs listed in Table 1, Table 2, Table 3, Table 4, Table 5, and/or Table 6 and functional variants of any one thereof, operably linked to a DNA targeting domain.
• TAD transactivation domain
• a first polypeptide may be operably linked to a second polypeptide by covalent linkage (e.g. as a fusion protein), or through one or more interaction components.
• a reporter gene is operably linked to a promoter
• the promoter actuates expression of the reporter gene.
• the transcriptional activator may further comprise one or more interaction components of an interaction system, which provides a functional interaction between the effector domain and/or DNA targeting domain and/or target DNA.
• interaction component is used herein to encompass one or more components of an interaction system, which together provide said functional interaction.
• interaction system as used herein is intended to encompass interaction components that permit covalent or non-covalent interactions, and/or constitutive or inducible interactions.
• Such interaction systems may include for example a peptide linker, optionally a protease-sensitive peptide linker; one or more dimer, trimer, or higher order multimerization components such as an interaction domain, optionally inducible dimer, trimer, or multimerization components, optionally an inducible interaction domain; and/or one or more components which can modulate subcellular localization of the transcriptional activator.
• the interaction system can comprise two or more components.
• the DNA targeting domain and the effector domain may be covalently linked, for example as domains of a single polypeptide (e.g. fusion protein), or may be linked by an interaction component such as an interaction domain for example, that interact under certain conditions (e.g. as a dimer).
• the heterologous transcriptional activator may comprise a single polypeptide, or may comprise a first polypeptide comprising a DNA targeting domain and a first interaction component such as a dimer interaction domain, and a second polypeptide comprising an effector domain and a second interaction component such as a dimer interaction domain, wherein the first and second dimer interaction domain can interact, for example under certain conditions.
• Higher-order multimerization systems such as the SunTag system (Tenenbaum et al., 2014), are also contemplated herein.
• the interaction between the effector domain and/or DNA targeting domain and/or target DNA can be controlled using a variety of inducible interaction systems.
• the effector domain and DNA targeting domain may be linked by a protease- sensitive linker such as a self-cleaving NS3 protease domain, which is stabilized in the presence of an NS3 inhibitor such as grazoprevir.
• localization of the DNA targeting domain and/or effector domain to the nucleus can be controlled by an interaction component such as a localization domain, for example tamoxifen-regulated nuclear localization using estrogen receptor ligand binding domain variants.
• the DNA targeting domain can be linked to a first interaction component such as a first interaction domain and the effector domain can be linked to a second interaction component such as a second interaction domain, such that the first and second interaction domain interact.
• interaction domain means a sequence motif in a first polypeptide (e.g. first dimer interaction domain), that is capable of interacting with a binding partner comprising a sequence motif in a second polypeptide (e.g. second dimer interaction domain) to operably link the first polypeptide and second polypeptide.
• first dimer interaction domain e.g. first dimer interaction domain
• second dimer interaction domain e.g. second dimer interaction domain
• the term is intended to encompass a first or second interaction dimer domain which together form a heterodimer pair that dimerizes for example under suitable inducing conditions.
• Other interaction domains are specifically contemplated and can be identified by the skilled person depending on the desired characteristics.
• Suitable inducible interaction domain pairs include, without limitation: FKBP/FRB (FK506 binding protein/FKBP rapamycin binding), which can be induced with e.g. rapamycin or AP21967; PYL/ABI which can be induced e.g. with abscisic acid; GID1/GAI, which can be induced with e.g. gibberellin or gibberellic acid; and pMag/nMag, which can be induced by e.g. blue light and/or temperature.
• FKBP/FRB FK506 binding protein/FKBP rapamycin binding
• PYL/ABI which can be induced e.g. with abscisic acid
• GID1/GAI which can be induced with e.g. gibberellin or gibberellic acid
• pMag/nMag which can be induced by e.g. blue light and/or temperature.
• DNA targeting domain refers to a polypeptide domain which
• the DNA targeting domain can be any suitable DNA binding domain, for example an enzymatically inactive sequence-specific DNA targeting protein such as a CRISPR-Cas protein, (e.g. dCas9, dCas12, or other Cas-family proteins), a zinc-finger DNA binding domain, a transcriptional activator-like effector (TALE) DNA binding domain, bromodomains, chromodomains, Vietnamese domains, WD40 domains, PHD domains, PWWP domains, or other DNA-binding domains (DBDs) from eukaryotes or prokaryotes (e.g.
• a CRISPR-Cas protein e.g. dCas9, dCas12, or other Cas-family proteins
• TALE transcriptional activator-like effector
• the DNA targeting domain may bind DNA in a sequence specific manner (e.g. Cas-family proteins, zinc- finger DNA binding domains, TALE DNA binding domains) or may bind to specific chromatin modifications (e.g. bromodomains (for acetylated histones) or chromodomains, Vietnamese domains, WD40 domains, PHD domains, PWWP domains etc. (for methylated histones).
• the DNA targeting domain may be a natural (e.g.
• non-engineered DNA binding domain such as for example a DNA binding domain found in a naturally occurring (e.g. endogenous) transcription factor, or the DNA targeting domain may be engineered for example to provide custom sequence specificity (e.g. sequence specificity that differs from the non-engineered DNA binding domain) or altered DNA binding affinity.
• custom sequence specificity e.g. sequence specificity that differs from the non-engineered DNA binding domain
• Methods of engineering for example zinc finger DNA binding domains and TALE DNA binding domains to provide custom DNA binding specificity are known in the art, for example in Maeder et al.2008 and Sanjana et al.2012.
• Enzymatically active Cas9 can also be used when it would lead to repression, for example when the guide is a truncated guide (see for example [24]).
• the DNA targeting domain may have inherent target sequence specificity, for example in the case of zinc-finger DNA binding domains and TALE DNA binding domains, or target sequence specificity may be mediated by additional sequence-specific factors such as e.g. a guide RNA in the case of CRISPR-Cas proteins.
• additional sequence-specific factors such as e.g. a guide RNA in the case of CRISPR-Cas proteins.
• Suitable DNA binding conditions depend on the DNA targeting domain and may include for example the presence of additional factors, such as for example tetracycline in the case in the case of tet-repressors, or a guide RNA in the case of Cas-family proteins.
• effector domain refers to a polypeptide domain comprising at least one transactivation domain (TAD) described herein, for example the TADs listed in Tables 1-5 and functional variants thereof such as active fragments thereof.
• TAD transactivation domain
• the effector domain may comprise two or more, for example two, three, four, or more transactivation domains described herein.
• the active fragment can be about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, or any number between 15 and 70 amino acids, or more than 70 amino acids.
• the active fragment may comprise GFSVDTSALLDLFSP (SEQ ID NO: 104) which corresponds to amino acids 406 to 420 of HSF1.
• the active fragment of HSF1 may comprise amino acids 401 to 427 of HSF1.
• Active fragments of other TADs can be identified by any suitable methods, for example using the methods described herein.
• the heterologous transcriptional activator can be an effector N-terminal or a C terminal fusion, for example the order of the fusion can be effector domain – DNA targeting domain or DNA targeting domain – effector domain (see for example [25], [26], [27] and [28]).
• the effector domain can be fused to the DNA targeting domain by way of a linker.
• two or more TADs may be fused together by way of one or more linkers.
• glycine and glycine serine linkers can be used.
• Transcriptional activators described in the Examples used a variety of glycine serine linkers for example SGGSGGS (SEQ ID NO: 6), GGS, SGGS (SEQ ID NO: 7), and/or GSGSGS (SEQ ID NO: 8).
• Other linkers can also be used for example INSRSSGS (SEQ ID NO: 9).
• CRISPR-Cas refer to a CRISPR Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) protein that binds RNA and is targeted to a specific DNA sequence by the RNA to which it is bound.
• the CRISPR-Cas is a class II monomeric Cas protein for example a type II Cas such as Cas9.
• the Cas9 protein may be Cas9 from Streptococcus pyogenes, Francisella novicida, A. Naesulndii, Staphylococcus aureus or Neisseria meningitidis.
• the Cas9 is from S.
• the Cas protein can also be Cas12a (e.g. dCas12a) for example from Acidaminococcus sp., Lachnospiraceae bacterium, or Francisella tularensis (these have been shown to work as dCas variants), Cas ⁇ (Cas12j) and CasX (Cas12e) may also be used.
• Cas12a e.g. dCas12a
• Cas ⁇ Cas12j
• CasX Cas12e
• dCas9 refers to an enzymatically inactive (or dead) Cas9, which lacks DNA endonuclease activity but retains target DNA binding activity.
• the dCAS9 comprises the sequence of CAS9 and D10A/H840A mutations in the RuvC1 and HNH nuclease domains.
• the dCas9 is a protein comprising an amino acid sequence with at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence identity to a protein encoded by SEQ ID NO: 1 and comprising D10A/H840A mutations and retaining Cas9 target DNA binding activity (e.g. binding the gRNA and the target site).
• dCas12a refers to an enzymatically inactive Cas12a.
• guide RNA refers to an RNA molecule that hybridizes with a specific DNA sequence and minimally comprises a spacer sequence.
• the guide RNA may further comprise a protein binding segment that binds a CRISPR-Cas protein.
• the portion of the guide RNA that hybridizes with a specific DNA sequence is referred to herein as the nucleic acid-targeting sequence, or spacer sequence.
• the protein binding segment of the guide may comprise for example a tracrRNA and/or a direct repeat.
• guide or guide RNA may refer to a spacer sequence alone, or an RNA molecule comprising a spacer sequence and a protein binding segment, according to the context.
• the guide RNA can be represented by the corresponding DNA sequence.
• the guide can be a truncated guide, for example comprising 15 or fewer nucleotides of complementarity to a target site as described in [24] when the enzyme is Cas9.
• Cas9 interacts with a truncated guide, Cas9’s DNA binding capability remains intact while its nucleolytic activity is eliminated. Any length of guide that maintains Cas binding capability can be used.
• spacer or “spacer sequence” as used herein refers to the portion of the guide that forms, or is capable of forming, an RNA-DNA duplex with the target sequence or a portion thereof.
• the spacer sequence may be complementary or correspond to a specific CRISPR target sequence.
• the nucleotide sequence of the spacer sequence may determine the CRISPR target sequence and may be designed or configured to target a desired CRISPR target site.
• the term “tracrRNA” as used herein refers to a “trans-encoded crRNA” which may, for example, interact with a CRISPR-Cas protein such as Cas9 and may be connected to, or form part of, a guide RNA.
• the tracrRNA may be a tracrRNA from for example S. pyogenes.
• a tracrRNA may have for example the sequence of 5’- gtttcagagctatgctggaaacagcatagcaagttgaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtg c-3’ (SEQ ID NO: 2).
• Other tracrRNAs may also be used.
• Suitable tracrRNAs can be identified by a person skilled in the art, including for example 5’- GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC-3’ (SEQ ID NO: 3) or 5’- GTTTCAGAGCTACAGCAGAAATGCTGTAGCAAGTTGAAAT-3’ (SEQ ID NO: 4).
• CRISPR target site or “CRISPR-Cas target site” as used herein mean a nucleic acid to which an activated CRISPR-Cas protein (e.g.
• a CRISPR target site comprises a protospacer-adjacent motif (PAM) and a CRISPR target sequence (i.e. corresponding to the spacer sequence of the guide to which the activated CRISPR-Cas protein is bound).
• PAM protospacer-adjacent motif
• CRISPR target sequence i.e. corresponding to the spacer sequence of the guide to which the activated CRISPR-Cas protein is bound.
• the sequence and relative position of the PAM with respect to the CRISPR target sequence will depend on the type of CRISPR-Cas protein.
• the CRISPR target site of Cas9 or dCas9 may comprise, from 5’ to 3’, a 15 to 25, 16 to 24, 17 to 23, 18 to 22, or 19 to 21 nucleotide, optionally a 20 nucleotide target sequence followed by a 3 nucleotide PAM having the sequence NGG.
• a Cas9 target site may have the sequence 5’- N 1 NGG-3’, where N 1 is 15 to 25, 16 to 24, 17 to 23, 18 to 22, or 19 to 21 nucleotides in length, optionally 20 nucleotides in length.
• the CRISPR target site can be in any suitable genomic locus.
• the CRISPR target site can be in a promoter, enhancer, 3’UTR, or other regulatory element, in a gene, optionally an intron or exon, in a locus corresponding to a non-coding RNA, or in an intergenic region.
• the CRISPR target site is in a promoter or an enhancer.
• Target DNA located in the nucleus of a cell requires a transcriptional activator that can enter the nucleus.
• the transcriptional activator may be nuclear-localized and/or may comprise for example one or more nuclear localization signals (NLS), optionally one or more SV40 NLSs.
• the transcriptional activator comprises two or more NLSs.
• the transcriptional activator may comprise one or more N-terminal NLSs, one or more C-terminal NLSs, one or more internal NLSs, or one or more N-terminal, one or more C- terminal NLSs, and/or one or more internal NLSs.
• the NLS is an SV40 NLS having the sequence PKKKRKV (SEQ ID NO: 22).
• the NLS further comprises an N- and/or C-terminal linker such as INSRSSGS (SEQ ID NO: 9), and optionally has the sequence INSRSSGSPKKKRKVGS (SEQ ID NO: 141).
• the transcriptional activator can also be labelled with a tag.
• suitable tags include but are not limited to Myc, FLAG, HA, V5, ALFA, T7, 6xHis, VSV-G, S- tag, AviTag, StrepTag II, CBP, GFP, mCherry.
• the label can be fused at the N-terminus, the C-terminus or between two components of the heterologous transcriptional activator such as between the DNA targeting domain and the effector domain.
• the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
• the phrase “one or more” in reference to a group of elements includes at least one member of the stated group but not necessarily including one of each of the members of the stated group.
• the element may comprise A; B; C; A and B; A and C; B and C; or A, B, and C.
• Additional members not specifically listed in the group may also be present, for example with reference to the example above, the element may additionally comprise unlisted member D, and accordingly may comprise A and D; B and D; A, C and D; etc.
• heterologous transcriptional activators comprising one or more transactivation domains (TADs), and combinations thereof (“effector domains”) that can be operably linked to a DNA targeting domain to generate a heterologous transcriptional activator, and which can be used to activate gene expression of a desired gene, including an endogenous gene, for example for therapeutic purposes.
• TADs transactivation domains
• effector domains effector domains
• effector domains are operably linked to a DNA- targeting domain that can direct binding of the fusion construct to any locus in the genome.
• heterologous transcriptional activators comprising dCas9 or rTetR functionally associated with an effector domain comprising any one of the TADs listed in Table 1, active fragments thereof, for example those of SEQ ID NOs: 100-104, 107-115, 118-119, 156-160, 162, 164, and 166-185, and combinations of two or more of the TADs or active fragments thereof, for example as listed in Table 3, can be used to activate transcription of a target gene.
• the TAD or active fragment thereof may additionally comprise a linker and/or additional natural sequence e.g.1, 2, 3, 4, 5, 6, 7 or more, for example up to 5, up to 10, up to 20, up to 30, or up to 40 (or any number in between), N- and/or C-terminal amino acids on the end of the TAD or active fragment.
• the TAD labeled “ZXDC short” in Table 3 (SEQ ID NO: 107) comprises a 40 amino acid fragment found in both ZXDC-12 and 13 and comprises an additional 6 N-terminal amino acids of ZXDC-12 natural sequence and an additional 5 C-terminal amino acids of ZXDC-13 natural sequence.
• the active fragment may be shorter than a fragment identified.
• the active fragment may be 20 amino acids, or 25 amino acids of the 40 amino acid fragment found in for example both ZXDC-12 and ZXDC-13, optionally for example an internal portion of SEQ ID NO: 107.
• Shorter active fragments are exemplified for example by SEQ ID NO: 119 which comprises a 20 amino acid fragment found in both HSF1-20 and HSF1-21. Active fragments can be identified as described elsewhere herein.
• the minimal fragment can be identified by comparing active fragments, for example for ATF6, the overlapping fragment shown in Table 5 is HRLDEDWDSALFAELGYFTDTDELQLEAANETYENNFDNL and for KLF7 the overlapping fragment is YFSALPSLEETWQQTCLELERYLQTEPRRISETFGEDLDC.
• one aspect of the disclosure includes a heterologous transcriptional activator comprising a DNA targeting domain, and an effector domain comprising at least one TAD selected from the group consisting of any one of the TADs listed in any one of Tables 1-6, optionally Table 1 or optionally Table 2 or Table 6, active fragments thereof, and combinations thereof for example at least two TADs selected from the TADs listed in Table 1 or Table 3, or functional variants thereof, preferably at least one TAD selected from the TADs listed in Table 4, or Table 5 or Table 6, and/or functional variants thereof, wherein the DNA targeting domain and effector domain are operably linked.
• the at least one TAD is selected from Table 1.
• the at least one TAD is selected from Table 2. [0081] In an embodiment, the at least one TAD is selected from Table 3. [0082] In an embodiment, the at least one TAD is selected from Table 4. [0083] In an embodiment, the at least one TAD is selected from Table 5. [0084] In an embodiment, the at least one TAD is selected from Table 6. [0085] It is understood that the at least two TADs or functional variants thereof can be selected from any Table, for example one from each of Table 3 and Table 4, two or more, for example 3 or 4, from Table 3 etc. It is also contemplated that the grouping can include any sub-combination of the TADS described in any of Tables 1 – 6, for example one or more TADs may be excluded.
• the DNA targeting domain and the effector domain may be operably linked by covalent linkage, for example as domains of a single polypeptide, and/or may be operably linked via one or more interaction components such as interaction domains and/or interact under certain conditions.
• the heterologous transcriptional activator is a single polypeptide.
• the heterologous transcriptional activator further comprises a pair of (i.e. a first and a second) interaction domains, optionally dimer interaction domains, optionally a pair of inducible dimer interaction domains that dimerize under suitable conditions.
• the heterologous transcriptional activator may comprise a first polypeptide comprising a DNA targeting domain and a first dimer interaction domain, optionally an inducible dimerization domain, and a second polypeptide comprising an effector domain and a second dimer interaction domain, optionally an inducible dimerization domain, wherein the first dimer interaction domain and second dimer interaction domain interact, optionally the first inducible dimerization domain and second inducible dimerization domain interact in the presence of one or more inducing agents.
• the dimerization of a heterologous transcriptional activator comprising ABI1 and PYL1 may be induced with the addition of abscisic acid.
• the transcriptional activator comprises a first and second inducible dimerization domain that provide for inducible transcriptional activation in the presence of an inducing agent.
• suitable inducible dimerization domains may be used together. Any suitable inducible dimerization domains may be used, for example the dimerization of ABI1 and PYL1 may be induced with the addition of abscisic acid.
• Other inducible systems include those based on induction with rapamycin, gibberellic acid/gibberellin, and split dCas9-based systems.
• dimerization of GID1 and GAI can be induced by gibberellin
• dimerization of FKBP and FRB can be induced with rapamycin or its analogs, e.g., rapalogs.
• Higher-order multimerization systems such as the SunTag system (Tenenbaum et al., 2014) are also contemplated herein.
• Interaction between the DNA targeting domain and effector domain can also be controlled using other inducible systems.
• Other systems include grazoprevir-induced stabilization (Tague et al. 2018) or tamoxifen- regulated nuclear localization using estrogen receptor ligand binding domain variants.
• the DNA targeting domain can be selected from a variety of DNA binding domains, for example a zinc finger DNA binding domain, transcriptional activator-like effector (TALE) DNA binding domain, dCas9, dCas12 or other Cas-family proteins, or other DNA- binding domains (DBDs) from eukaryotes or prokaryotes (e.g.
• the DNA targeting domain may be a natural (e.g. non-engineered) DNA binding domain, such as for example a DNA binding domain found in a naturally occurring (e.g. endogenous) transcription factor, or the DNA targeting domain may be engineered for example to provide custom sequence specificity (e.g. sequence specificity that differs from the non-engineered DNA binding domain) or altered DNA binding affinity.
• a natural (e.g. non-engineered) DNA binding domain such as for example a DNA binding domain found in a naturally occurring (e.g. endogenous) transcription factor
• the DNA targeting domain may be engineered for example to provide custom sequence specificity (e.g. sequence specificity that differs from the non-engineered DNA binding domain) or altered DNA binding affinity.
• a heterologous transcriptional activator described herein comprises a DNA targeting domain comprising a natural DNA-binding domain
• the effector domain would be targeted to all loci that the transcription factor endogenously binds to, thereby augmenting/replacing the function of the endogenous transcription factor.
• Oct4 transactivation domain increases the efficiency of reprogramming fibroblasts to iPS cells.
• a heterologous transcriptional activator comprising a natural DNA binding domain operably linked to an effector domain could promote e.g., wound healing, transdifferentiation, or tissue regeneration by activating transcription of target genes that are regulated by the endogenous transcription factor.
• an effector domain could be brought to one or more specific loci, or optionally a single locus in the genome in a controlled manner.
• the DNA targeting domain comprises a CRISPR-Cas protein such as dCas9. Enzymatically inactive CRISPR-Cas proteins which retain gRNA and target DNA binding activity can be used.
• the CRISPR-Cas protein is dCas9 having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1 and comprises D10A/H840A and which retains gRNA and target DNA binding activity.
• Other enzymatically inactive CRISPR-Cas proteins are also contemplated can be identified by the skilled person.
• the DNA targeting domain comprises a zinc finger DNA binding domain.
• the zinc finger DNA binding domain is an engineered zinc finger DNA binding domain which has been engineered to bind a specific DNA sequence.
• the effector domain comprises at least one transactivation domain (TAD) described herein, or an active fragment thereof.
• TAD transactivation domain
• various full-length ORFs and TADs identified herein can be used, alone or in combination, to activate transcription of a GFP reporter construct or an endogenous gene such as CD133.
• the effector domain can comprise at least one TAD domain shown in Table 1, and/or an active fragment thereof, such as for example as shown in Table 3.
• the effector domain comprises at least one TAD shown in Table 2, Table 4, and/or Table 5 and/or Table 6 and/or a functional variant of any one thereof, or two or more TADs shown in Table 1, Table 2, Table 3, Table 4, Table 5, and/or Table 6 and functional variants of any one thereof.
• the TAD comprises a polypeptide having a sequence with at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to any one of the TAD domains in Table 1, Table 2, Table 3, Table 4, Table 5, and/or Table 6 and functional variants of any one thereof, and which retains (e.g.
• the effector domain comprises two or more tandem TADs, optionally two TADs, three TADs, four TADs, or more than four TADs, for example 5 TADs, 10 TADs, 15 TADs, 20 TADs, 25 TADs, 30 TADs, or any number of TADs between 5 TADs and 30 TADs, or more than 30 TADs.
• the effector domain comprises two or more TADs or functional variants thereof selected from those listed in Table 1, Table 2, Table 3, Table 4, Table 5, and/or Table 6 and functional variants of any one thereof. In an embodiment, the effector domain comprises three or four TADs selected from those listed in Table 3. In an embodiment, the effector domain comprises one or more of SEQ ID NO: 185, SEQ ID NO: 103, SEQ ID NO: 167, SEQ ID NO: 105, SEQ ID NO: 106, and/or SEQ ID NO: 104. In an embodiment, the effector domain comprises SEQ ID NO: 185, optionally SEQ ID NO: 90, 91, 102, or 157.
• the effector domain comprises SEQ ID NO: 103, optionally SEQ ID NO: 46, 47, or 162. In an embodiment, the effector domain comprises SEQ ID NO: 167, optionally SEQ ID NO: 101, 110, 166, or 172. In an embodiment, the effector domain comprises SEQ ID NO: 105, optionally SEQ ID NO: 116, 117, or 165. In an embodiment, the effector domain comprises SEQ ID NO: 106, optionally SEQ ID NO: 116 or 117. In an embodiment, the effector domain comprises SEQ ID NO: 104, optionally SEQ ID NO: 118, 119, or 159.
• the effector domain comprises SPDYE4-CITED1-RELA-HSF1 (SEQ ID NO: 121); SPDYE4- CITED1-RELA (SEQ ID NO: 123); HSF1-RELA-SPDYE4-CITED1 (SEQ ID NO: 125); SPDYE4-CITED1-p65-miniHSF1 (SEQ ID NO: 127); miniSPDYE4-CITED1-p65-HSF1 (SEQ ID NO: 129); SPDYE4-miniCITED1-p65-HSF1 (SEQ ID NO: 131); SPDYE4-CITED1- minip65(C)-HSF1 (SEQ ID NO: 133); or SPDYE4-CITED1-minip65(N)-HSF1 (SEQ ID NO: 135).
• the effector domain comprises SPDYE4-CITED1-SERTAD2-HSF1; SPDYE4-CITED1-KLF6-HSF1; SPDYE4-CITED1-ZXDC-HSF1; SPDYE4-CITED1-ATF6- HSF1; SPDYE4-CITED1-FOXO1-HSF1; SPDYE4-CITED1-ATMIN-HSF1; SPDYE4-CITED1- p65-SERTAD2; SPDYE4-CITED1-p65-KLF6; SPDYE4-CITED1-p65-ZXDC; SPDYE4- CITED1-p65-ATF6; SPDYE4-CITED1-p65-FOXM1; SPDYE4-CITED1-p65-ATMIN; SPDYE4- C3orf62-p65-HSF1; SPDYE4-DDIT3-p65-HSF1; SPDYE4-FOXO1-p
• the effector domain comprises SPDYE4-C3orf62.2-P_AD- HSF1 (SEQ ID NO: 174); SPDYE4-C3orf62.3-P_AD-HSF1 (SEQ ID NO: 176); SPDYE4- C3orf62_MT-P_AD-HSF1 (SEQ ID NO: 178); SPDYE4-DDIT3_MT-P_AD-HSF1 (SEQ ID NO: 180); SPDYE4-CITED1-P_AD-HSF1_MT (SEQ ID NO: 182); or 3 x ZNF473_KRAB (SEQ ID NO: 184).
• Other combinations are specifically contemplated herein.
• the effector domain may comprise two or more TADs with different transcriptional co-activator preferences.
• the effector domain may comprise a TAD which interacts with CBP/p300 components for example a FOXO TAD, and a TAD which interacts with BET components for example a SPDYE4 TAD.
• the effector domain may comprise two or more TADs with similar transcriptional co-activator preferences.
• the effector domain may comprise two TADs which interact with CBP/p300 components, for example the effector domain may comprise a FOXO1 TAD and a CITED1 TAD.
• Other combinations are specifically contemplated herein.
• “as effective” as used herein means the functional variant retains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100%, or more than 100% transcriptional activation activity and/or co-activator interaction compared to the unmodified or non-variant TAD (e.g. wild-type or full-length TAD).
• Transcriptional activation activity and/or co-activator interaction of variants such as truncations can be determined for example using the methods described herein. For example transcriptional activation activity can be determined using the GFP reporter system described in the Examples.
• Variants can be tethered to the same reporter or endogenous context while controlling for expression levels of each DNA targeting domain (e.g. dCas9). Any differences detected in induced expression of the reporter or target genes when compared to the parental TAD can be contributed to the effect of the variant. Co-activator interaction can be determined for example by AP-MS and/or BioID e.g. as shown in the Examples. [0096] Exemplary TAD and effector domain nucleic acids and polypeptides are provided in Tables 1-6 and SEQ ID NOs: 120-135 and 173-184.
• the effector domain may comprise an amino acid sequence encoded by said nucleic acids, or an amino acid sequence with at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence encoded by the TAD of SEQ ID NOs: 120-135 and 173-184.
• the activity of the encoded polypeptides (fusion or when expressed and activated) of such polypeptides is as effective (e.g. provides at least 80% as effective transcriptional activation) as for example SEQ ID NOs: 120-135 and 173-184.
• the effector domain is fused to the DNA targeting domain by way of a linker.
• two or more TADs are fused together by way of one or more linkers.
• linkers For example, glycine and glycine serine linkers can be used. Transcriptional activators described in the Examples used a variety of glycine serine linkers for example SGGSGGS (SEQ ID NO: 6), GGS, SGGS (SEQ ID NO: 7), and/or GSGSGS (SEQ ID NO: 8). Other linkers can also be used for example INSRSSGS (SEQ ID NO: 9).
• the transcriptional activator comprises one or more nuclear localization signals (NLS). Any suitable NLS can be used. Optionally the NLS is an SV40 NLS.
• the one or more NLS can be one or more N-terminal NLS, one or more C-terminal NLS, one or more internal NLS, and/or combinations thereof.
• the NLS may comprise an NLS of SEQ ID NO: 22.
• the NLS further comprises an N- and/or C-terminal linker such as INSRSSGS (SEQ ID NO: 9), and optionally has the sequence INSRSSGSPKKKRKVGS (SEQ ID NO: 141).
• the transcriptional activator or effector domain may be encoded by a nucleic acid and/or expressed from an expression construct. Accordingly, one aspect of the disclosure is a nucleic acid encoding a transcriptional activator described herein.
• nucleic acid encoding an effector domain of a transcriptional activator described herein.
• the nucleic acid may encode a TAD as provided in any one of Tables 1 to 6, optionally Tables 2, 4, 5 and/or 6, optionally Table 2 or Table 4 or Table 6, or two or more TADs as provided in Tables 1-6.
• the nucleic acid may comprise a nucleic acid of any one of SEQ ID NOs: 120, 122, 124, 126, 128, 130, 132, 134, 173, 175, 177, 179, and 180, or a sequence with at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NOs: 120, 122, 124, 126, 128, 130, 132, 134, 173, 175, 177, 179, and 180, wherein the heterologous transcriptional activator, for example activates transcription about as effectively as the effector domains encoded by SEQ ID NO: 120, 122, 124, 126, 128, 130, 132, 134, 173, 175, 177, 179, and 180, for example at least 80% as effectively, at least 85% as effectively, or at least 90% as effectively, at least 95% as effectively, at least 96% as effectively, at least 97% as effectively,
• the sequence identity is for example relative to the full effector domain sequence or relative to one or more TADs or TAD fragments encoded therein. Other portions, linkers, NLS etc. can be completely different.
• the nucleic acid encoding the effector domain may be suitable for generating a nucleic acid encoding a transcriptional activator described herein.
• the nucleic acid encoding the effector domain may be flanked by suitable cloning sites, or an expression construct or vector comprising said nucleic acid may comprise a cloning site to facilitate insertion of a DNA targeting domain to operably link the effector domain and DNA targeting domain.
• cloning site refers to a portion of a nucleic acid molecule into which a nucleic acid molecule of interest may be inserted, or to which a nucleic acid molecule of interest may be joined, using recombinant DNA technology (cloning).
• the cloning site may be located between the promoter and the polyadenylation signal, such that a nucleic acid molecule of interest may be cloned into the expression cassette in operable linkage with the promoter and the polyadenylation site.
• the cloning site will include the necessary characteristics (such as restriction endonuclease site(s), recombinase recognition site(s), or blunt or overhanging end(s)) to allow insertion of the nucleic acid molecule of interest at the cloning site.
• the cloning site may, for example, be a multiple cloning site (MCS) or polylinker region comprising a plurality of unique restriction enzyme recognition sites to allow a nucleic acid molecule of interest to be inserted.
• the cloning site may include one or more recombinase recognition sites to allow DNA insertion by recombinational cloning; employing site-specific recombinase(s), such as Integrase or Cre Recombinase, to catalyze DNA insertion.
• site-specific recombinase(s) such as Integrase or Cre Recombinase
• recombinational cloning systems include Gateway® (Integrase), CreatorTM (Cre Recombinase), and Echo CloningTM (Cre Recombinase).
• an expression cassette or vector may be provided as a linear molecule, allowing blunt or overhanging ends of a nucleic acid molecule of interest to be joined to blunt or overhanging ends of the expression cassette or vector, for example by ligation or polymerase activity, thus forming a circular molecule.
• the blunt or overhanging ends of the expression cassette or vector may together be viewed as the cloning site.
• a related aspect is an expression construct comprising the nucleic acid encoding the transcriptional activator operably linked to a promoter and a transcription termination site. Any suitable promoter may be used.
• Suitable promoters can be identified by a person skilled in the art, and may include for example CMV, EF1A, or PGK.
• the promoter and/or enhancer sequences of e.g. SEQ ID NOs: 25, 26, 27, and/or 28 can be used in an expression construct. Inducible promoters may also be used.
• the construct is a vector. Any suitable vector may be used. Suitable vectors can be identified by a person skilled in the art, and may include a viral vector, optionally a lentiviral vector or an adenoviral vector.
• Suitable vectors may comprise for example a promoter for expressing effector construct, polyA tail, 3’UTR elements like WPRE to increase stability of expression, insulator sequences, lentiviral packaging signals, a fluorescent protein, and/or an antibiotic resistance marker. Additional suitable components can be identified by a person skilled in the art.
• the transcriptional activator, nucleic acid, construct, or vector is in a cell. Any suitable cell may be used and can be determined by the skilled person on the basis of the desired application.
• the cell may be from any organism.
• the cell is a mammalian cell such as a human cell or a mouse cell.
• the cell is a cell line.
• the cell line may be any suitable cell line.
• the transcriptional activator, nucleic acid, construct, or vector may be introduced into the cell in any suitable manner, for example by transfection. Suitable transfection reagents and methods are routinely practiced in the art and can be identified by the skilled person.
• the construct is a viral vector, optionally a lentiviral vector, and is introduced into the cell by transduction. Suitable transduction methods are routinely practiced in the art and can be identified by the skilled person.
• the cell is stably expressing the heterologous transcriptional activator, optionally the cell is stably transduced, for example prepared using a virus comprising a nucleic acid encoding the heterologous transcriptional activator.
• transcriptional activation system comprising the transcriptional activator described herein, a nucleic acid encoding the transcriptional activator, or construct or vector comprising said nucleic acid or a cell expressing the transcriptional activator.
• the system comprises at least one gRNA.
• the system optionally comprises at least one inducing agent.
• a composition comprising a heterologous transcriptional activator described herein, a nucleic acid described herein, a construct described herein, a vector described herein, a cell described herein and/or a transcriptional activation system described herein.
• the composition can comprise a carrier, such as BSA, or a diluent suitable according to the composition components, optionally water or buffered saline.
• the composition can comprise multiple components such as transcriptional activators, nucleic acids, constructs, vectors or cells comprising the same or different elements.
• a kit for example for activating transcription of a target gene or performing a method described herein the kit comprising a transcriptional activator described herein, a nucleic acid, expression construct, or vector encoding a transcriptional activator described herein, or a cell expressing the transcriptional activator described herein, and optionally a vial housing the transcriptional activator, nucleic acid, expression construct, vector, cell or composition.
• the kit can comprise multiple of one or more of the aforementioned components.
• the kit comprises a gRNA expression construct, an inducing agent, and/or instructions for carrying out the methods described herein.
• Also described herein are methods of activating transcription of a target gene in a cell.
• a transcriptional activator of the disclosure can be targeted to a genomic locus such as a promoter to activate transcription of a target gene in a cell.
• the transcriptional effectors identified herein may be full-length proteins, fragments thereof (transactivation domains), functional variants thereof or combinations of transactivation domains or functional variants thereof. They cover multiple different transcriptional activation strengths from very powerful to moderate to weak.
• Activation strength can be tuned by selecting different TADs or functional variants (e.g. active fragments) for inclusion in the effector domain.
• the activation strength of an effector domain can be determined by the skilled person for example using the MFI or percent GFP positive cells in the recruitment assays shown in the Examples described herein.
• the relative strength of an effector domain can be determined by comparing the MFI or percent GFP positive cells of the specific effector domain in combination with a specific DNA targeting domain and specific DNA target relative to a control such as Renilla in combination with the same DNA targeting domain and specific DNA target.
• High activation can be considered to be for example at least or above 50X control, at least or above 75X control, at least or above 100X control, or at least or above 150X control.
• Medium activation can be considered to be for example at least or above 10X, at least or above 20X, at least or above 30X, or at least or above 40X control, and up to 50X, up to 75X, up to 100X, or up to 150X control.
• Low activation can be considered to be for example at least 2X, at least 2.5X, at least 3X, at least 4X, or at least 5X control and up to 10X, up to 20X, up to 30X, or up to 40X control.
• high activation may be considered to be >50-fold, medium may be 20X to 50X, and low may be at least 3X up to 20X relative to the Renilla control. Suitable activation levels may be selected depending on the desired application.
• another level of control for transcriptional regulation can be added with chemically induced dimerization with e.g. rapalogs or abscisic acid.
• one half e.g. a DNA targeting domain
• the other half e.g. the effector domain
• Treatment with rapalog or abscisic acid would induce the interaction between FKBP and FRB or PYL1 and ABI1, respectively, leading to temporally regulated gene expression.
• the dimerization of a heterologous transcriptional activator comprising ABI1 and PYL1 may be induced with the addition of abscisic acid.
• suitable inducible dimerization domains and inducing agents Any suitable inducible combination of protein dimerization domains and inducing agents may be used, for example the dimerization of ABI1 and PYL1 may be induced with the addition of abscisic acid.
• inducible systems include those based on induction with rapamycin, gibberellic acid/gibberellin, and split dCas9-based systems.
• dimerization of GID1 and GAI can be induced by gibberellin
• dimerization of FKBP and FRB can be induced with rapamycin or its analogs, e.g. rapalogs.
• Higher-order multimerization systems such as the SunTag system (Tenenbaum et al., 2014) are also contemplated herein.
• Interaction between the DNA targeting domain and effector domain can also be controlled using other inducible systems.
• Other systems include grazoprevir-induced stabilization (Tague et al.
• one aspect of the disclosure is a method of activating expression of a target gene in a cell, the method comprising introducing into the cell a transcriptional activator described herein, and culturing the cell under suitable conditions such that the DNA targeting domain guides the transcriptional activator to the target site and the effector domain activates transcription of the target gene.
• the target gene is an endogenous gene.
• the transcriptional activator comprises CRISPR-Cas
• the method further comprises introducing into the cell at least one gRNA that targets a desired genomic locus in the cell, and culturing the cell under suitable conditions such that the at least one gRNA associates with the CRISPR-Cas protein and guides the CRISPR-Cas protein to guide the transcriptional activator to a CRISPR target site such that the effector domain activates transcription of the target gene.
• the method further comprises introducing into the cell at least one inducing agent and culturing the cell under suitable conditions that the first and second inducible dimerization domains associate such that the at least one effector domain activates transcription of the target gene.
• the methods described herein can be used to modulate gene expression of a target gene for example to induce expression of an endogenous gene or modulate chromatin opening in defined regions of the genome.
• some TADs could promote chromatin opening in intergenic regions (i.e. not promoters or enhancers), which could lead to chromatin opening and rearrangement of chromosome folding.
• the methods described herein can be used to identify or screen for one or more genomic loci that are important for cell viability or a phenotype of interest.
• the methods described herein can be used to screen for genes or regulatory elements thereof that are important for resistance or sensitivity to a toxin of interest such as diphtheria toxin.
• the methods described herein can be used to identify regulatory elements that are important for expression of a protein of interest such as CD81.
• the methods described herein can be used in high-throughput screening methods to identify essential or non-essential genes in a cell type by screening for gRNAs that are over- or under- represented in a cell population under certain conditions e.g. drug treatment over time.
• CID chemically-induced dimerization
• HEK293T cell line that contains a stably integrated construct with a 7xTetO array and a basal CMV promoter driving the expression of EGFP was used (Gao et al., 2016).
• Example 7 Methods are as described in Example 7.
• Example 2. ORFeome-wide screen identifies known and novel transcriptional activators.
• 248 putative transcriptional activators were identified, using a cut-off of 5% false discovery rate and at least 4-fold change in read counts between top 1% GFP positive cells and unsorted cells (Figure 1E).
• Gene ontology (GO) analysis revealed significant enrichment for multiple functional categories related to transcriptional activation (Figure 1F).
• the hits were also highly enriched in protein domains found in many transcriptional regulators (Figure 1G), subunits of chromatin-associated protein complexes (Figure 1H), and in interactors of central hubs of transcription, such as RNA polymerase II and histone acetyltransferases CBP and p300 ( Figure 6F). Moreover, the hits were significantly overlapping with human proteins that function in yeast two-hybrid assays as autoactivators (11% in the proteome vs. 29% among the hits; p ⁇ 0.0001, Fisher’s exact test; Figure 6G), which are proteins that activate reporter gene expression in yeast when ectopically recruited to the promoter (Luck et al., 2020).
• sequence-specific transcription factor hits were known activators RELB and MYCL (Barrett et al., 1992; Ryseck et al., 1992) in addition to several master TFs regulating stress response, such as HSF1, ATF6, and DDIT3/CHOP (Vihervaara et al., 2018).
• Co-activators that do not bind DNA themselves but associate with TFs included STAT2, CITED1, and SERTAD1 (Bousoik and Montazeri Aliabadi, 2018; Hsu et al., 2001; Yahata et al., 2001).
• TFIIE TFIIE
• GTF2E1 and GTF2E2 a general transcription factor regulating the assembly of the pre-initiation complex
• CDK9 proteins promoting RNA polymerase II release
• transcriptional elongation e.g., ELL3 and MLLT1(Chen et al., 2018; Cramer, 2019).
• TFs that belong to the same family often have highly similar or even identical sequence specificities (Jolma et al., 2013; Lambert et al., 2018; Weirauch et al., 2014). Nevertheless, even highly related TFs can have distinct effects on transcription and chromatin due to their unique auxiliary domains. In line with this, only some members of transcription factor families were identified as hits in the pooled screen.
• NEUROG1, NEUROG2, NEUROD1, and NEUROD2 can induce neuronal differentiation of iPSCs whereas NEUROD6 cannot (Goparaju et al., 2017). This pattern is concordant with their ability to activate the reporter gene (Figure 7B).
• HOXA1, HOXA2, HOXB1, HOXB2 can activate the b1-ARE autoregulatory element located in the Hoxb1 locus (Di Rocco et al., 1997), and three of these were characterized as activators in the assay (HOXA2 and HOXB2 in the pooled screen and the arrayed assay, HOXA1 in the screen) ( Figure 2A).
• HOXA2 and HOXB2 in the pooled screen and the arrayed assay, HOXA1 in the screen Figure 2A.
• recent work revealed a striking collinearity between the repressive potential, expression pattern, and genomic location of HOX genes (Tycko et al., 2020), with HOX transcription factors in the 5’ end of homeobox clusters being repressive.
• the HOX family activators in the assay are encoded by the most or the next-to-last 3’ genes in their HOX gene clusters.
• a particularly interesting case was that of PCGF family proteins, which are mutually exclusive components of canonical and non-canonical Polycomb Repressive Complexes 1 (PRC1) (Gahan et al., 2020; Gao et al., 2012)( Figure 2E).
• PRC1 canonical and non-canonical Polycomb Repressive Complexes 1
• Figure 2E Although generally thought to act in chromatin compaction and gene silencing in the context of PRC1, PCGF3 was identified as an activator in the original screen. PCGF3 robustly activated the reporter when tested individually, as did PCGF5 (which was not present in the original pooled screen) ( Figure 2E).
• PCGF5 has been previously shown to regulate transcriptional activation (Gao et al., 2014), but no such role has been described for PCGF3.
• PCGF3 and PCGF5 form a distinct group that arose early during animal evolution (Gahan et al., 2020), suggesting that their transcriptional activation function is of ancient origin.
• the primary activation screen identified two proteins (SPDYE4 and SPDYE7P) that belong to the Spy1/RINGO (Rapid INducer of G2/M progression in Oocytes) family of cell cycle regulators.
• Spy1/RINGO proteins bind to and activate Cdk1 and Cdk2 in a cyclin- independent manner, thereby promoting cell cycle progression (Gonzalez and Nebreda, 2020).
• they have not been previously implicated in transcriptional regulation.
• the human genome contains at least 19 Spy1/RINGO family genes and multiple pseudogenes.
• TAD-seq reveals novel human transactivation domains.
• Transcription factors generally activate transcription through transactivation domains (TADs) that interact with co-activators, such as the Mediator, CBP/p300 acetyltransferases, or TFIID. Most TADs are short, unstructured sequences rich in acidic and hydrophobic residues (Sigler, 1988).
• the TAD region is conserved in all family members except SPDYC, the only Spy1/RINGO family protein that was inactive in the recruitment assay ( Figure 2F and 11A).
• some of the uncovered TADs did not have characteristics of typical transactivation domains.
• the three overlapping fragments in HOXA2 that activated transcription spanned a polyalanine stretch between the homeobox DNA-binding domain and the antennapedia-like hexapeptide motif ( Figure 3D and 3E), which interacts with the PBX1 co-activator (Piper et al., 1999).
• This region contained the YAF2_RYBP domain, which folds into an antiparallel beta sheet that binds RING1B, a core PRC1 subunit (Wang et al., 2010) ( Figure 3G).
• the YAF2_RYBP domain of YAF2 and its close homolog RYBP shares sequence and structural homology with the CBX-C domain present in the CBX family Polycomb proteins (Wang et al., 2010)( Figure 11B).
• CBX-C domain containing proteins and YAF2/RYBP interact in a mutually exclusive manner with RING1A and RING1B to form canonical (cPRC1) and non-canonical (ncPRC1) Polycomb complexes ( Figure 3G).
• the YAF2_RYBP domains of YAF2 (SEQ ID NO: 96) and RYBP (SEQ ID NO: 140) and the CBX-C domains of CBX2 (SEQ ID NO: 136), CBX4, CBX6 (SEQ ID NO: 138), CBX7, and CBX8 (SEQ ID NO: 139) were cloned and assayed for activity with the reporter system.
• the YAF2_RYBP motif from both YAF2 and RYBP robustly activated the reporter, consistent with the TADseq results for YAF2 ( Figure 3H).
• CBX-C domains were also activators: CBX-C from CBX2 was the most potent activator, whereas those from CBX6 and CBX8 activated the reporter weakly (Figure 3H). In contrast, CBX4 and CBX7 CBX-C domains had no effect on the reporter. This pattern reflected the evolutionary ancestry of the CBX-C domain proteins ( Figure 3H).
• HEK293 cell lines were established, expressing nine poorly characterized screen hits (C3orf62, C11orf74/IFTAP, NCKIPSD, DCAF7, SS18L2, SPDYE4, FAM90A1, FAM22F/NUTM2F, JAZF1), five known transcriptional regulator hits (SOX7, KLF6, KLF15, CTBP1, HOXA2, and HOXB2), two synthetic transactivators (VP64 and VPR), and negative controls (EGFP and Nanoluc) fused to biotin ligase BirA from Aquifex aeolicus and FLAG epitope tag.
• JAZF1 in contrast, associated with multiple subunits the NuA4 histone acetyltransferase complex in BioID, suggesting that it is a novel subunit of this highly conserved complex.
• SS18L2 in turn, interacted with the BAF chromatin remodeling complex, including core BAF members SMARCA2 and SMARCA4 as well as subunits specific to the canonical BAF (cBAF) and non- canonical BAF (ncBAF) ( Figure 4A and 12)(Centore et al., 2020).
• NCKIPSD also known as SPIN90, interacted with the survival of motor neurons (SMN) complex, which regulates the assembly of ribonucleoprotein complexes but has also been linked to transcriptional activation (Pellizzoni et al., 2001; Singh et al., 2017; Strasswimmer et al., 1999) ( Figure 12).
• NCKIPSD also interacted with DCAF7 ( Figure 12B).
• VP64 which consists of four tandem copies of the viral VP16 transactivation motif, associated with CBP/p300 and mediator subunits MED14 and MED15, consistent with previous reports (Kundu et al., 2000; Yang et al., 2004).
• VPR which is a fusion of VP64, human p65/RELA transactivation domain, and Epstein-Barr virus R transactivator (Chavez et al., 2015), associated with even more co-factors, including CBP/p300 and multiple subunits of the Mediator and TFIID ( Figures 4A and 12A).
• NuA4 interactors were significantly less affected by JQ1 treatment that CBP/p300 interactors or other transactivators (Figure 4D). Although the mechanism by which NuA4 interactors respond to JQ1 treatment in a unique manner requires further research, these results demonstrate how transcriptional activators promote transcription via different co-activator complexes in the context of a single promoter. Indeed, hierarchical clustering of the activators based on their sensitivity to the five compounds revealed multiple distinct groups (Figure 4E). Many paralogous factors (such as CITED1 and CITED2, or PCGF3 and PCGF5) clustered next to each other, indicating that clustering produced functionally relevant groups.
• CITED1 and CITED2, or PCGF3 and PCGF5 clustered next to each other, indicating that clustering produced functionally relevant groups.
• CBP/p300 interactors were primarily in two distinct clusters, as were NuA4 interactors ( Figure 4E). It is likely that other members of these clusters similarly use CBP/p300 or NuA4 as co-activators, such as YAF2 or MYOG for p300, or NOM1 for NuA4.
• Methods are as described in Example 7.
• Example 6. SRF-C3orf62 fusion interacts with CBP/p300 and promotes SRF/MRTF transcriptional program.
• Fusion proteins involving transcriptional regulators are common hallmarks of certain cancers, such as leukemias and sarcomas.
• BTBD18 and NCKIPSD were identified as KMT2A/MLL fusion partners leukemia (Alonso et al., 2010; Sano et al., 2000).
• the fragment of BTBD18 that is fused to MLL contains the transactivation domain identified by TAD-seq ( Figure 10B), implicating the TAD in the oncogenic potential of the fusion product.
• Most MLL fusions involve genes regulating transcriptional elongation, such as the super elongator complex (Winters and Bernt, 2017).
• BTBD18 has also been shown to promote transcriptional elongation (Zhou et al., 2017).
• JAZF1-SUZ12 and SRF-C3orf62 were selected for further characterization.
• the JAZF1-SUZ12 fusion is a hallmark of low-grade endometrial stromal sarcoma (LG-ESS)(Hrzenjak, 2016), bringing together the Polycomb protein SUZ12 and JAZF1 ( Figure 5A)(Piunti et al., 2019).
• SRF- C3orf62 was recently described in a pediatric case of myofibroma/myopericytoma (Antonescu et al., 2017).
• the DNA-binding domain of Serum Response Factor is fused to the C-terminus of C3orf62 ( Figure 5B).
• SRF Serum Response Factor
• the transactivation domain that identified by TAD-seq ( Figure 3C and 10B) is retained in the fusion constructs ( Figure 5A and 5B)
• SUZ12, SRF, JAZF1-SUZ12, SRF-C3orf62, and the C-terminal fragment of C3orf62 fused to SRF (C3orf62-Cterm) were tagged with BirA-FLAG and their interactomes were analyzed with BioID and AP-MS, to complement the data we obtained for JAZF1 and C3orf62.
• SUZ12 proximity partners included other components of the PRC2 complex, such as EZH2, MTF2/PCL2 and C10orf12 (Alekseyenko et al., 2014)( Figure 5C). Strikingly, the JAZF1-SUZ12 fusion protein had both PRC2 and NuA4 subunits as proximity partners ( Figure 5C), indicating that this fusion assembles into a supercomplex of two chromatin-associated complexes that are normally associated with opposing transcriptional activities.
• EPC1-PHF1 fusion that is also associated with LG-ESS similarly assembles the PRC2-NuA4 supercomplex, which leads to aberrant expression of Polycomb targets (Sudarshan et al., 2021).
• JAZF1-SUZ12 and EPC1-PHF1 were recently shown to be strong transcriptional activators (Sudarshan et al., 2021).
• NuA4 integration into the oncogenic supercomplex can override the normally repressive function of PRC2 complexes.
• SRF proximity partners included multiple transcription factors such as ELK1, which forms a ternary complex with SRF on serum response elements (SREs)(Buchwalter et al., 2004)(Figure 5D). However, it did not associate with any transcriptional co-activators.
• C3orf62-Cterm construct and the SRF-C3orf62 fusion robustly identified both CBP and p300 as proximity partners ( Figure 5D).
• AP-MS similarly identified CBP and p300 as prominent SRF-C3orf62 interactors ( Figure 14A). Consistent with these results, both C3orf62-Cterm and SRF-C3orf62 strongly activated the GFP reporter when tethered to the reporter ( Figure 5E).
• SRF-C3orf62 This activity was highly sensitive to CBP/p300 inhibition with A-485 (Figure 5F), in line with the interaction patterns of SRF-C3orf62.
• SRF functions together with either ternary complex factors (TCFs; e.g. ELK1) or myocardin-related transcription factors (MRTFs; e.g. MAL) to regulate target gene expression (Buchwalter et al., 2004; Olson and Nordheim, 2010).
• TCFs ternary complex factors
• MRTFs myocardin-related transcription factors
• SRF-C3orf62 can activate SRF target genes without such cofactors.
• the SRF/TCF pathway which is regulated by MAP kinase signaling, regulates the expression of immediate-early genes (Gualdrini et al., 2016), whereas the actin-Rho signaling dependent SRF/MRTF pathway targets genes involved in cell motility and adhesion (Miralles et al., 2003).
• SRF-C3orf62 can regulate target genes of both pathways, stable doxycycline-inducible NIH3T3 cell lines stably expressing SRF, C3orf62, SRF-C3orf62 and Nanoluc fused to GFP were generated.
• Upregulated genes were also significantly enriched in targets of the E2F transcription factor, a key regulator of cell proliferation (Figure 5I). These signatures are consistent with the oncogenic nature of the SRF-C3orf62 fusion. In addition, many of the most upregulated genes were involved in actin dynamics and myogenesis. For example, one of the most upregulated genes was smooth muscle actin (ACTA2), a known target of SRF/MTRF and a hallmark of SRF fusion positive myofibromas/myopericytomas (Antonescu et al., 2017)(Figure 5H). Consistent with this, another significant signature of the upregulated genes was myogenesis (Figure 5I).
• Example 7 Methods are as described in Example 7.
• FBS fetal bovine serum
• NIH-3T3 cells were obtained from Dr. Sachdev Sidhu’s lab (University of Toronto) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% bovine calf serum (BCS).
• Lentivirus production Lentiviral particles containing the pooled ORFeome and transactivation libraries were produced by transfecting 293T cells with pLX301- ORFs/TADs-PYL1, psPAX2 (Addgene #12260) and pVSV-G (Addgene #8454) at a ratio of 8:6:1. Transfection was performed using XtremeGENE 9 (Roche) on 15-cm dishes according to the manufacturer’s protocol.
• the medium was changed 6-8 hours post-transfection to harvest medium (DMEM + 1.1 g per 100 mL BSA). 72 hours after transfection, supernatant was filtered (0.45 ⁇ M), pooled and collected. A similar protocol was followed for small scale virus production when establishing individual stable cell lines with transfection being performed on 6-well plates using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) reagent.
• Cell line generation A clonal line of the EGFP reporter line was generated expressing ABI-dCas9 (blastacidin, 6 ⁇ g/mL) and gRNA (SEQ ID NO: 10) (co-expressing EBFP2) targeting the pTRE3G promoter.
• NIH3T3 cells Single cells were sorted (FACS Aria IIIu, BD) and expanded and a clone showing induction by a strong transcriptional activator was selected for subsequent experiments.
• entry clones were picked from the hORFeome collection and subcloned into the Gateway compatible pSTV6-TetO-ccdB-EGFP lentiviral plasmid (a kind gift from Payman Samavarchi-Tehrani).
• NIH-3T3 cells were infected in the presence of 8 ⁇ g/mL polybrene and selected with 2 ⁇ g/mL puromycin 24 hours post infection.
• Activation domain tiling library generation A tiling library was generated from 75 proteins identified as activators in the ORFeome screen.
• Oligonucleotides containing a 5’ adapter GGAAGTCAGGGTAGCGGAAGTATG (SEQ ID NO: 23) and a 3’ adapter GGAGGTAGTGTTGAACGCGAAGGC (SEQ ID NO: 24) to generate a 5’ adapter (GSQGSGSM) (SEQ ID NO: 11) and a 3’ adapter (GGSVEREG) (SEQ ID NO: 12) were synthesized as pooled libraries (Twist Biosciences).6x50 ⁇ l PCR reactions were set up using NEBNext Ultra II Q5 master mix (New England Biolabs) with 5 nM oligos as template. PCR conditions were optimized to find the lowest cycle with a clean visible product at the expected 300 bp length.
• thermocycling condition was an initial 30 s at 98°C, then 2 cycles of 98°C for 10 s, 63°C for 20 s, and 72°C for 15 s, followed by 10 more cycles of 98°C for 10 s and 72°C for 30 s with a final extension at 72°C for 5 min.
• Primers were designed to have Gateway compatible flanking sequences.
• the resulting libraries were gel extracted by QIAgen gel extraction kit after loading on a 2% TAE gel for 2 hrs at 60V and subsequently cloned into pDONR221 using 20 separate BP reactions in total of 5 ⁇ l reactions.
• the entry plasmid pool was transformed after an overnight reaction into DH5alpha competent E.
• a clonal EGFP reporter cell line stably co-expressing ABI-dCas9 and a gRNA targeting the promoter were transduced at low multiplicity of infection (MOI) with approximately 30% cell survival after puromycin (1 ⁇ g/mL) selection. Untransduced cells under the same condition were fully eliminated. Sufficient cells were transduced to maintain >500 fold coverage of the libraries. Recruitment was induced by treating cells with 100 ⁇ M abscisic acid (ABA, Sigma) for 48 hours. In parallel, a control batch of cells were treated with equal total volume of DMSO.
• MOI multiplicity of infection
• ABA abscisic acid
• the target ORFeome region was amplified from genomic DNA using primers targeting the T7 promoter and PYL1.
• the product of this reaction was pooled for each sample and further amplified by primers targeting outside the Gateway attB sites for an additional 10 cycles. Amplicons were subsequently separated on 1% agarose gel and any visible PCR product excluding primer dimers were gel purified.
• the final pool was quantified using NEBNext Library Quant Kit for Illumina (New England Biolabs, E7630L) and paired-end sequenced on an Illumina MiSeq.
• TAD sequencing Performed nested PCR on the purified genomic DNA using primers targeting T7 promoter and PYL1 of the backbone vector in the first step creating a ⁇ 470 bp product. Products of the first reaction were then pooled and amplified for an additional 10 steps using primers targeting outside the Gateway sites creating a ⁇ 300 bp product. Libraries were quantified on Qubit dsDNA Broad Range kit and paired-end sequenced on an Illumina MIseq with a custom PAGE-purified R1 sequencing primer.
• cloning adapter sequences were first removed from both ends using cutadapt with CCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGTTGGCGGAAGTC AGGGTAGCGGAAGT (SEQ ID NO: 20) for 5’ and CCGCCACTGTGCTGGATATCAACCACTTTGTACAAGAAAGTTGGGTAGCCTTCGCGTTC AACACTACCTCC (SEQ ID NO: 21) for 3’ adapters.
• Bowtie reference was generated, and reads were mapped using Bowtie v1.2.3 allowing 0 mismatches.
• the edgeR package (Robinson et al., 2010) was used to calculate log2 fold change, p-value, and false discovery rate (FDR) for each ORF by comparing changes in counts from sorted samples to unsorted cells.
• FDR false discovery rate
• SRF reporter assay 30,000 NIH-3T3 cells on 24-well plates were transfected with 8ng SRF reporter (p3DA.luc), 20 ng reference reporter (pcDNA3.1-Nanoluc-3xFLAG-V5) and 50 ng 3xFLAG tagged constructs (Addgene #87063). Transfection was carried out using Lipofectamine 3000 reagent (Thermo Fisher Scientific, L3000001) according to the manufacturer's protocols. Luciferase constructs were a kind gift from Dr. Maria Vartiainen (University of Helsinki). Cells were maintained in low-serum media (0.5% BCS) for 18 hours and stimulated for 7 hours (15% BCS), after which luciferase activity was measured.
• RNA sequencing and analysis NIH-3T3 cells with stable integrations of SRF, C3orf62, SRF-C3orf62 or Nanoluc tagged at the C-terminus with EGFP were induced with 1 ⁇ g/mL doxycycline for 24 hours. RNA was extracted from cells maintained in low-serum conditions (0.5% calf serum) for 22 hours using RNeasy purification kit (Qiagen) and treated with DNase on column. Samples were induced and collected in technical duplicates from 6- well plates.
• Clones were transferred into pDEST-pcDNA5 vector carrying a C-terminal BioID2-FLAG tag (Kim et al., 2016) using Gateway recombinase. Stable HEK293 Flp-In T-REx cell lines were generated as previously reported (Piette et al., 2021). [00171] For AP-MS, cells were grown to 70% confluence on 150 mm dishes before inducing bait expression with 1 ⁇ g/mL tetracycline for 24 hours. Cells were then washed once with 1xPBS, scraped, pelleted, flash-frozen, and stored at -80°C until processing. AP-MS was performed as previously described (Lambert et al., 2015).
• cells were resuspended in cold lysis buffer (50 mM HEPES-NaOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM PMSF, 1 mM DTT, 15 nM Calyculin A and protease inhibitor cocktail (Sigma- Aldrich P8340)) using a 1:4 pellet weight:volume ratio.
• Cells were lysed by one round of freeze-thaw, and lysates sonicated at 4°C using three 10-second bursts at 35% amplitude with 2 s pauses.
• Sonicated lysate was treated with 100U benzonase for 30 minutes at 4°C prior to clearing by centrifugation at 20,000g for 20 minutes at 4°C.
• An equal amount of supernatant from all samples processed within a batch was transferred to a tube containing 25 ⁇ L of pre- washed anti-FLAG magnetic bead 50% slurry (Sigma, M8823) and incubated for two hours at 4°C. Beads were recovered by magnetization and the supernatant discarded.
• Beads were washed once in lysis buffer, and once in 20 mM Tris-HCl pH8.0 with 2 mM CaCl2 and digested on beads with trypsin in two stages (1 ⁇ g trypsin for 4 hours followed by the addition of 0.5 ⁇ g trypsin to the supernatant and overnight incubation at 37°C), as previously described (Taipale et al., 2014). Finally, samples were acidified with 5% formic acid (final concentration) and stored at -80°C.
• BioID For BioID, cells were grown to 70% confluence in 150 mm dishes before inducing gene expression with 1 ⁇ g/mL tetracycline for 18 hours.50 ⁇ M biotin was then added to each plate for 6 hours. Cell pellets were collected as for AP-MS, and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Igepal CA-630, 1mM EDTA, 1 mM MgCl2, protease inhibitor cocktail (Sigma-Aldrich P8340, 1:500), and 0.5% sodium deoxycholate) using a 1:10 pellet weight:volume ratio.
• lysis buffer 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Igepal CA-630, 1mM EDTA, 1 mM MgCl2, protease inhibitor cocktail (Sigma-Aldrich P8340, 1:500
• each sample was treated with 250U Turbonuclease (BioVision 9207-50KU) and 1 ⁇ L RNase A solution (Sigma- Aldrich R6148) and incubated for 30 minutes at 4°C. SDS was then added to a final concentration of 0.25% and after mixing the samples were incubated for another 10 minutes at 4°C followed by centrifugation at 20,000g for 20 minutes. The supernatant was transferred to a tube containing 30 ⁇ l of pre-washed packed streptavidin beads (GE Healthcare, 17-5113- 01). Streptavidin pulldown was done for 3 hours at 4°C.
• Beads were washed once in 1 ml of SDS buffer (2% SDS/50 mM Tris-HCl pH7.5), once in 1 ml lysis buffer, and once in TAP buffer (50 mM HEPES-KOH pH 8.0, 100 mM KCl, 10% glycerol, 2 mM EDTA, 0.1% Igepal CA-630), followed by three 1 ml washes with 50 mM ammonium bicarbonate pH 8.0. After the washes, beads were resuspended in ABC buffer containing 1 ⁇ g trypsin and incubated overnight at 37°C.
• SDS buffer 2% SDS/50 mM Tris-HCl pH7.5
• TAP buffer 50 mM HEPES-KOH pH 8.0, 100 mM KCl, 10% glycerol, 2 mM EDTA, 0.1% Igepal CA-630
• TAD fragments were used: CITED1-8 (SEQ ID NO: 47), CITED2-12 (SEQ ID NO: 49), C3orf62-12 (SEQ ID NO: 45), BRD8-25 (SEQ ID NO: 40), ZXDC-12 (SEQ ID NO: 97), KLF7-1 (SEQ ID NO: 72), ATXN7L3-1 (SEQ ID NO: 35), FAM90A1-20 (SEQ ID NO: 60), SPDYE4-3 (SEQ ID NO: 90), YAF2-7 (SEQ ID NO: 96).
• the SPDYE4-CITED1 TAD fusion protein resulted in a marked increase in transcriptional activation compared to either TAD alone.
• miniTAD sequences were selected as follows: [00180] miniSPDYE4 (SEQ ID NO: 102): Sequence was selected based on the overlap between the two enriched fragments from our tiling screen of the SPDYE4 full length protein. The minimal sequence was designed to include either the acidic rich region or the following beta hairpin. [00181] miniHSF1: The 150 amino acid C-terminal region of HSF1 which contains its activation domain comprises of two known TADs.
• mini-HSF1 (401-420) or “H(mini)”; SEQ ID NO: 119
• miniP65 RelA or p65 is known to have two distinct transactivation domains within its C-terminus.
• the first TAD (“P(mini N-term)”; SEQ ID NO: 105) comprises amino acids 428-520 and the second (“P(mini C-term)”; SEQ ID NO: 106) is the proceeding 521-551 residues. (Schmitz and Baeuerle, 1991; 10.1002/j.1460-2075.1991.tb04950.x) [00183] miniCITED1 (“C(mini)”; SEQ ID NO: 103) was selected to include the C- terminal acidic-rich region within the overlapping region between CITED1-7 and CITED1-8. [00184] Methods: Cells were seeded on 48-well plates.
• each construct was either directly fused TagRFP either directly fused to each component or being co-expressed from the same plasmid was used as a measure of transfection.
• the same gate for RFP+ cells were used to control for the effect of each construct’s expression levels on activity.
• EGFP reporter cells were expanded from a clonal line to control for level of ABI-dCas9 and gRNA targeting the TetO7 sites upstream of the promoter.
• a 293T cell line stably expressing ABI-dCas9 and a pool of 5 gRNAs targeting the promoter were used for all activators.
• CD133 antibody conjugated to APC (Miltenyi Biotec, 130-113- 668) was used. All activator constructs were fused to PYL1 at their C-terminus and were recruited to their targets by treating the cells with 1 ⁇ M abscisic acid for either 24 or 48 hours.
• VPR was much less active as an rTetR fusion than as a dCas9 fusion.
• SCPH and its variants were still the most active constructs in the system.
• a construct containing a miniaturized activation domain of C3orf62 instead of that from CITED1 (“C” in the SCPH construct) and a shorter version of p65 activation domain (“P”) was more potent than the original SCPH construct despite being significantly smaller (1059 bp vs 1611 bp) ( Figure 19A-B).
• Table 2 Subset of fragments identified as TADs by TADseq from Table 1.
• Table 3 Exemplary sequences of TADs and functional variants and active fragments thereof. “ ”
• Table 4 Subset of fragments identified as TADs by TADseq from Table 1.
• Table 5 Subset of active fragments identified as TADs by TADseq.
• Table 6 Subset of fragments identified as TADs.
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• Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts.
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• CREB-binding protein/p300 are transcriptional coactivators of p65. Proc. Natl. Acad. Sci. U. S. A.94, 2927–2932. Gonzalez, L., and Nebreda, A.R. (2020). RINGO/Speedy proteins, a family of non-canonical activators of CDK1 and CDK2. Semin. Cell Dev. Biol.107, 21–27. Goparaju, S.K., Kohda, K., Ibata, K., Soma, A., Nakatake, Y., Akiyama, T., Wakabayashi, S., Matsushita, M., Sakota, M., Kimura, H., et al. (2017).
• TRIP-Br a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1.
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• Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.
• Lambert, J.-P. Picaud, S., Fujisawa, T., Hou, H., Savitsky, P., Uusküla-Reimand, L., Gupta, G.D., Abdouni, H., Lin, Z.-Y., Tucholska, M., et al. (2019).
• HAN11 binds mDia1 and controls GLI1 transcriptional activity. J. Dermatol. Sci.44, 11–20. Muhar, M., Ebert, A., Neumann, T., Umledgeer, C., Jude, J., Wieshofer, C., Rescheneder, P., Lipp, J.J., Herzog, V.A., Reichholf, B., et al. (2016). SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science 360, 800–805.
• DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells. Proc. Natl. Acad. Sci.97, 10412–10417.
• the ORFeome Collaboration a genome-scale human ORF-clone resource. Nat. Methods 13, 191–192. Pellizzoni, L., Charroux, B., Rappsilber, J., Mann, M., and Dreyfuss, G. (2001). A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II. J. Cell Biol.152, 75–86. Piette, B.L., Alerasool, N., Lin, Z.-Y., Lacoste, J., Lam, M.H.Y., Qian, W.W., Tran, S., Larsen, B., Campos, E., Peng, J., et al. (2021).
• Genomics 89, 756–768 Piunti, A., Smith, E.R., Morgan, M.A.J., Ugarenko, M., Khaltyan, N., Helmin, K.A., Ryan, C.A., Murray, D.C., Rickels, R.A., Yilmaz, B.D., et al. (2019).
• CATACOMB An endogenous inducible gene that antagonizes H3K27 methylation activity of Polycomb repressive complex 2 via an H3K27M-like mechanism. Sci. Adv.5, eaax2887. Ptashne, M., and Gann, A. (1997). Transcriptional activation by recruitment.
• RelB a new Rel family transcription activator that can interact with p50-NF-kappa B. Mol. Cell. Biol. 12, 674–684. Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988).
• GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564.
• Novel SH3 protein encoded by the AF3p21 gene is fused to the mixed lineage leukemia protein in a therapy-related leukemia with t(3;11) (p21;q23).
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• IXL a new subunit of the mammalian Mediator complex, functions as a transcriptional suppressor. Biochem. Biophys. Res. Commun.325, 1330–1338. Wang, Y., Chen, J., Hu, J.-L., Wei, X.-X., Qin, D., Gao, J., Zhang, L., Jiang, J., Li, J.-S., Liu, J., et al. (2011). Reprogramming of mouse and human somatic cells by high-performance engineered factors. EMBO Rep.12, 373–378.

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