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• W2803310880 abstract "•Development of direct cell-free assays to monitor enhancer-promoter looping•The SRC-3 coactivator governs ERα target gene enhancer-promoter looping•E2-dependent chromatin interactions are dynamic and involve gene body contacts•Dynamic enhancer-promoter interaction is essential for transcriptional activation Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain “poised” for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription. Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain “poised” for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription. RNA polymerase II (RNA Pol II)-transcribed genes are activated by regulatory DNA sequence elements known as enhancers that act in cis over long distances (Banerji et al., 1981Banerji J. Rusconi S. Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences.Cell. 1981; 27: 299-308Abstract Full Text PDF PubMed Scopus (920) Google Scholar, Heuchel et al., 1989Heuchel R. Matthias P. Schaffner W. Two closely spaced promoters are equally activated by a remote enhancer: evidence against a scanning model for enhancer action.Nucleic Acids Res. 1989; 17: 8931-8947Crossref PubMed Scopus (17) Google Scholar). Enhancers are evolutionarily conserved in sequence and function (Visel et al., 2009Visel A. Rubin E.M. Pennacchio L.A. Genomic views of distant-acting enhancers.Nature. 2009; 461: 199-205Crossref PubMed Scopus (441) Google Scholar); contain dense clusters of transcription factor (TF) binding sites (Spitz and Furlong, 2012Spitz F. Furlong E.E. Transcription factors: from enhancer binding to developmental control.Nat. Rev. Genet. 2012; 13: 613-626Crossref PubMed Scopus (1177) Google Scholar) and are heavily occupied by TFs, coactivators, cohesin, the mediator complex, RNA Pol II, and chromatin regulatory enzymes (Liu et al., 2014Liu Z. Merkurjev D. Yang F. Li W. Oh S. Friedman M.J. Song X. Zhang F. Ma Q. Ohgi K.A. et al.Enhancer activation requires trans-recruitment of a mega transcription factor complex.Cell. 2014; 159: 358-373Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Malik and Roeder, 2016Malik S. Roeder R.G. Mediator: a drawbridge across the enhancer-promoter divide.Mol. Cell. 2016; 64: 433-434Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, Yan et al., 2013Yan J. Enge M. Whitington T. Dave K. Liu J. Sur I. Schmierer B. Jolma A. Kivioja T. Taipale M. Taipale J. Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites.Cell. 2013; 154: 801-813Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar); and exhibit specific chromatin features (Rada-Iglesias et al., 2011Rada-Iglesias A. Bajpai R. Swigut T. Brugmann S.A. Flynn R.A. Wysocka J. A unique chromatin signature uncovers early developmental enhancers in humans.Nature. 2011; 470: 279-283Crossref PubMed Scopus (1532) Google Scholar). When bound by TFs and brought into proximity of their cognate promoters, the enhancers stimulate transcription of their target genes (Blackwood and Kadonaga, 1998Blackwood E.M. Kadonaga J.T. Going the distance: a current view of enhancer action.Science. 1998; 281: 60-63Crossref PubMed Scopus (613) Google Scholar, Marsman and Horsfield, 2012Marsman J. Horsfield J.A. Long distance relationships: enhancer-promoter communication and dynamic gene transcription.Biochim. Biophys. Acta. 2012; 1819: 1217-1227Crossref PubMed Scopus (70) Google Scholar, Ptashne, 1986Ptashne M. Gene regulation by proteins acting nearby and at a distance.Nature. 1986; 322: 697-701Crossref PubMed Scopus (526) Google Scholar) and undergo transcription to produce enhancer RNAs (eRNAs) (Li et al., 2016Li W. Notani D. Rosenfeld M.G. Enhancers as non-coding RNA transcription units: recent insights and future perspectives.Nat. Rev. Genet. 2016; 17: 207-223Crossref PubMed Scopus (452) Google Scholar). Enhancer-promoter pairs in contact over long distances have been identified using the chromosome conformation capture (3C) technique and its derivatives (Denker and de Laat, 2016Denker A. de Laat W. The second decade of 3C technologies: detailed insights into nuclear organization.Genes Dev. 2016; 30: 1357-1382Crossref PubMed Scopus (224) Google Scholar, Ong and Corces, 2011Ong C.T. Corces V.G. Enhancer function: new insights into the regulation of tissue-specific gene expression.Nat. Rev. Genet. 2011; 12: 283-293Crossref PubMed Scopus (607) Google Scholar, Spurrell et al., 2016Spurrell C.H. Dickel D.E. Visel A. The ties that bind: mapping the dynamic enhancer-promoter interactome.Cell. 2016; 167: 1163-1166Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Such studies have revealed several important features of enhancer function. (1) Pervasive enhancer-promoter contacts (EPCs), resulting from looping between distant chromatin segments, exist throughout the genome (Jin et al., 2013Jin F. Li Y. Dixon J.R. Selvaraj S. Ye Z. Lee A.Y. Yen C.A. Schmitt A.D. Espinoza C.A. Ren B. A high-resolution map of the three-dimensional chromatin interactome in human cells.Nature. 2013; 503: 290-294Crossref PubMed Scopus (810) Google Scholar, Zhang et al., 2013Zhang Y. Wong C.H. Birnbaum R.Y. Li G. Favaro R. Ngan C.Y. Lim J. Tai E. Poh H.M. Wong E. et al.Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations.Nature. 2013; 504: 306-310Crossref PubMed Scopus (316) Google Scholar). (2) Pre-formed EPCs exist at transcriptionally inert loci in the absence of any transcriptional stimulus (Andrey et al., 2013Andrey G. Montavon T. Mascrez B. Gonzalez F. Noordermeer D. Leleu M. Trono D. Spitz F. Duboule D. A switch between topological domains underlies HoxD genes collinearity in mouse limbs.Science. 2013; 340: 1234167Crossref PubMed Scopus (302) Google Scholar, Ghavi-Helm et al., 2014Ghavi-Helm Y. Klein F.A. Pakozdi T. Ciglar L. Noordermeer D. Huber W. Furlong E.E. Enhancer loops appear stable during development and are associated with paused polymerase.Nature. 2014; 512: 96-100Crossref PubMed Scopus (337) Google Scholar, Jin et al., 2013Jin F. Li Y. Dixon J.R. Selvaraj S. Ye Z. Lee A.Y. Yen C.A. Schmitt A.D. Espinoza C.A. Ren B. A high-resolution map of the three-dimensional chromatin interactome in human cells.Nature. 2013; 503: 290-294Crossref PubMed Scopus (810) Google Scholar, Phanstiel et al., 2017Phanstiel D.H. Van Bortle K. Spacek D. Hess G.T. Shamim M.S. Machol I. Love M.I. Aiden E.L. Bassik M.C. Snyder M.P. Static and dynamic DNA loops form AP-1-bound activation hubs during macrophage development.Mol. Cell. 2017; 67: 1037-1048.e6Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and are thought to keep the gene loci “poised” for transcription. (3) EPCs can form de novo upon transcriptional stimulation (Fullwood et al., 2009Fullwood M.J. Liu M.H. Pan Y.F. Liu J. Xu H. Mohamed Y.B. Orlov Y.L. Velkov S. Ho A. Mei P.H. et al.An oestrogen-receptor-alpha-bound human chromatin interactome.Nature. 2009; 462: 58-64Crossref PubMed Scopus (1229) Google Scholar, Hah et al., 2013Hah N. Murakami S. Nagari A. Danko C.G. Kraus W.L. Enhancer transcripts mark active estrogen receptor binding sites.Genome Res. 2013; 23: 1210-1223Crossref PubMed Scopus (333) Google Scholar, Li et al., 2013Li W. Notani D. Ma Q. Tanasa B. Nunez E. Chen A.Y. Merkurjev D. Zhang J. Ohgi K. Song X. et al.Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.Nature. 2013; 498: 516-520Crossref PubMed Scopus (694) Google Scholar) or upon the availability of the key TFs (Vakoc et al., 2005Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Both pre-formed and de novo EPCs participate in transcriptional regulation (Phanstiel et al., 2017Phanstiel D.H. Van Bortle K. Spacek D. Hess G.T. Shamim M.S. Machol I. Love M.I. Aiden E.L. Bassik M.C. Snyder M.P. Static and dynamic DNA loops form AP-1-bound activation hubs during macrophage development.Mol. Cell. 2017; 67: 1037-1048.e6Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). (4) EPC is required for efficient transcription from a participating promoter (Deng et al., 2012Deng W. Lee J. Wang H. Miller J. Reik A. Gregory P.D. Dean A. Blobel G.A. Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor.Cell. 2012; 149: 1233-1244Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). (5) However, maintenance of EPC is not dependent on active transcription (Palstra et al., 2008Palstra R.J. Simonis M. Klous P. Brasset E. Eijkelkamp B. de Laat W. Maintenance of long-range DNA interactions after inhibition of ongoing RNA polymerase II transcription.PLoS ONE. 2008; 3: e1661Crossref PubMed Scopus (108) Google Scholar). (6) Several classes of coregulators contribute to EPC establishment, such as tissue-specific TFs (Vakoc et al., 2005Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, Yun et al., 2014Yun W.J. Kim Y.W. Kang Y. Lee J. Dean A. Kim A. The hematopoietic regulator TAL1 is required for chromatin looping between the β-globin LCR and human γ-globin genes to activate transcription.Nucleic Acids Res. 2014; 42: 4283-4293Crossref PubMed Scopus (39) Google Scholar), the cohesin complex (Hadjur et al., 2009Hadjur S. Williams L.M. Ryan N.K. Cobb B.S. Sexton T. Fraser P. Fisher A.G. Merkenschlager M. Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus.Nature. 2009; 460: 410-413Crossref PubMed Scopus (408) Google Scholar, Kagey et al., 2010Kagey M.H. Newman J.J. Bilodeau S. Zhan Y. Orlando D.A. van Berkum N.L. Ebmeier C.C. Goossens J. Rahl P.B. Levine S.S. et al.Mediator and cohesin connect gene expression and chromatin architecture.Nature. 2010; 467: 430-435Crossref PubMed Scopus (1385) Google Scholar, Schmidt et al., 2010Schmidt D. Schwalie P.C. Ross-Innes C.S. Hurtado A. Brown G.D. Carroll J.S. Flicek P. Odom D.T. A CTCF-independent role for cohesin in tissue-specific transcription.Genome Res. 2010; 20: 578-588Crossref PubMed Scopus (291) Google Scholar), the mediator complex (Kagey et al., 2010Kagey M.H. Newman J.J. Bilodeau S. Zhan Y. Orlando D.A. van Berkum N.L. Ebmeier C.C. Goossens J. Rahl P.B. Levine S.S. et al.Mediator and cohesin connect gene expression and chromatin architecture.Nature. 2010; 467: 430-435Crossref PubMed Scopus (1385) Google Scholar, Malik and Roeder, 2016Malik S. Roeder R.G. Mediator: a drawbridge across the enhancer-promoter divide.Mol. Cell. 2016; 64: 433-434Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), specialized “bridging” factors (Chen et al., 2012Chen Y. Bates D.L. Dey R. Chen P.H. Machado A.C. Laird-Offringa I.A. Rohs R. Chen L. DNA binding by GATA transcription factor suggests mechanisms of DNA looping and long-range gene regulation.Cell Rep. 2012; 2: 1197-1206Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Krivega et al., 2014Krivega I. Dale R.K. Dean A. Role of LDB1 in the transition from chromatin looping to transcription activation.Genes Dev. 2014; 28: 1278-1290Crossref PubMed Scopus (83) Google Scholar, Ren et al., 2011Ren X. Siegel R. Kim U. Roeder R.G. Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication.Mol. Cell. 2011; 42: 342-355Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and chromatin remodelers like SWI/SNF and NuRD (Euskirchen et al., 2011Euskirchen G.M. Auerbach R.K. Davidov E. Gianoulis T.A. Zhong G. Rozowsky J. Bhardwaj N. Gerstein M.B. Snyder M. Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches.PLoS Genet. 2011; 7: e1002008Crossref PubMed Scopus (166) Google Scholar, Krivega et al., 2014Krivega I. Dale R.K. Dean A. Role of LDB1 in the transition from chromatin looping to transcription activation.Genes Dev. 2014; 28: 1278-1290Crossref PubMed Scopus (83) Google Scholar). (7) EPC also has been implicated in transcriptional pause release of genes regulated by a subset of JMJD6- and BRD4-bound enhancers (Liu et al., 2013Liu W. Ma Q. Wong K. Li W. Ohgi K. Zhang J. Aggarwal A. Rosenfeld M.G. Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release.Cell. 2013; 155: 1581-1595Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). (8) Additionally, an enhancer-silencer contact can prevent EPC formation, leading to gene repression (Jiang and Peterlin, 2008Jiang H. Peterlin B.M. Differential chromatin looping regulates CD4 expression in immature thymocytes.Mol. Cell. Biol. 2008; 28: 907-912Crossref PubMed Scopus (39) Google Scholar). Although these studies have provided important information on enhancers and their interactions with cognate promoters, our full mechanistic understanding of enhancer function remains incomplete. Addressing the specific mechanistic and functional implications of EPC in living cells has been challenging due to the complexity and dynamic nature of the cellular environment. Therefore, we developed new and highly controllable cell-free assays for EPC that are capable of interrogating transcriptional and proteomic dynamics in vitro. Here, we show that the classical Dignam HeLa cell nuclear extract (NE; Dignam et al., 1983Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9154) Google Scholar) promotes EPC in vitro, which is further enhanced when transcription ensues at both enhancer and promoter. We identified the steroid receptor coactivator-3 (SRC-3, NCOA3) as a critical and novel determinant of looping in both our cell-free systems and intact MCF-7 cells that enables dynamic chromatin interactions at the human GREB1 gene. In estrogen (E2)-depleted MCF-7 cells, we find that the enhancer holds the promoter in close proximity via direct contacts with SRC-3 binding sites located downstream of the GREB1 transcription start site (TSS). Upon E2 treatment, this connection is reorganized rapidly, leading to a temporal sequence of enhancer-promoter-intragenic looping contacts. Additionally, these gene body SRC-3 binding sites were found to be necessary for efficient transcription at both enhancer (eRNA) and promoter (mRNA) in vitro. We also present evidence that both formation and severance of chromatin interaction contacts are crucial for full transcriptional activity. We demonstrate that our looping assay is versatile, which can successfully recapitulate serum-inducible EPC and transcription activation in vitro. To investigate EPC at a mechanistic level, we developed several cell-free methodologies. We chose the human GREB1 locus as a looping model because the GREB1 gene undergoes E2-inducible EPC in MCF-7 cells that correlates with its strong activation (Fullwood et al., 2009Fullwood M.J. Liu M.H. Pan Y.F. Liu J. Xu H. Mohamed Y.B. Orlov Y.L. Velkov S. Ho A. Mei P.H. et al.An oestrogen-receptor-alpha-bound human chromatin interactome.Nature. 2009; 462: 58-64Crossref PubMed Scopus (1229) Google Scholar, Hah et al., 2013Hah N. Murakami S. Nagari A. Danko C.G. Kraus W.L. Enhancer transcripts mark active estrogen receptor binding sites.Genome Res. 2013; 23: 1210-1223Crossref PubMed Scopus (333) Google Scholar, Li et al., 2013Li W. Notani D. Ma Q. Tanasa B. Nunez E. Chen A.Y. Merkurjev D. Zhang J. Ohgi K. Song X. et al.Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.Nature. 2013; 498: 516-520Crossref PubMed Scopus (694) Google Scholar). The GREB1 enhancer was identified 41 kb upstream of the major TSS (GREB1c isoform) based on the magnitude of E2-induced ERα occupancy, ERα-anchored interaction with the promoter, enrichment of H3K4me1 and H3K27ac, and the magnitude of E2-induced eRNA synthesis (Fullwood et al., 2009Fullwood M.J. Liu M.H. Pan Y.F. Liu J. Xu H. Mohamed Y.B. Orlov Y.L. Velkov S. Ho A. Mei P.H. et al.An oestrogen-receptor-alpha-bound human chromatin interactome.Nature. 2009; 462: 58-64Crossref PubMed Scopus (1229) Google Scholar, Hah et al., 2013Hah N. Murakami S. Nagari A. Danko C.G. Kraus W.L. Enhancer transcripts mark active estrogen receptor binding sites.Genome Res. 2013; 23: 1210-1223Crossref PubMed Scopus (333) Google Scholar, Li et al., 2013Li W. Notani D. Ma Q. Tanasa B. Nunez E. Chen A.Y. Merkurjev D. Zhang J. Ohgi K. Song X. et al.Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.Nature. 2013; 498: 516-520Crossref PubMed Scopus (694) Google Scholar). Interspersed between the enhancer and the GREB1c promoter are three estrogen response elements (EREs) (Sun et al., 2007Sun J. Nawaz Z. Slingerland J.M. Long-range activation of GREB1 by estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer cells.Mol. Endocrinol. 2007; 21: 2651-2662Crossref PubMed Scopus (64) Google Scholar) that do not share the above characteristics of the enhancer. An additional ERα-bound region, ERE1up (Figure S1A), also exhibits ERα-mediated contact with the enhancer (Fullwood et al., 2009Fullwood M.J. Liu M.H. Pan Y.F. Liu J. Xu H. Mohamed Y.B. Orlov Y.L. Velkov S. Ho A. Mei P.H. et al.An oestrogen-receptor-alpha-bound human chromatin interactome.Nature. 2009; 462: 58-64Crossref PubMed Scopus (1229) Google Scholar). We designed a compact DNA construct that contained all of these elements and the GREB1c promoter while removing selected intervening DNA. We PCR-amplified these elements (Figure 1A; Figure S1A) and verified by DNA pull-down assays (Foulds et al., 2013Foulds C.E. Feng Q. Ding C. Bailey S. Hunsaker T.L. Malovannaya A. Hamilton R.A. Gates L.A. Zhang Z. Li C. et al.Proteomic analysis of coregulators bound to ERα on DNA and nucleosomes reveals coregulator dynamics.Mol. Cell. 2013; 51: 185-199Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) that ERα binds each of these fragments and recruits coregulators, including SRC-3, RAD21, MED12, and CDK8, from HeLa S3 NE (HeLa NE) (Figure S1B). These six regulatory fragments, named F1–F6, were then assembled into an 8 kb composite fragment named CompF (Figure 1A). We reasoned that if EPC occurred on the CompF, F6 would still be retained with F1 even after it is cleaved from the rest of the template with a restriction enzyme (Figure 1B). To test this possibility, we immobilized 5′ biotinylated CompF (5′Biot.CompF) on M280 streptavidin-coated Dynabeads and incubated it with HeLa NE as a source of potential looping factors along with recombinant ERα protein and ATP. HeLa NE was chosen because it lacks endogenous ERα (Figure S1B; Foulds et al., 2013Foulds C.E. Feng Q. Ding C. Bailey S. Hunsaker T.L. Malovannaya A. Hamilton R.A. Gates L.A. Zhang Z. Li C. et al.Proteomic analysis of coregulators bound to ERα on DNA and nucleosomes reveals coregulator dynamics.Mol. Cell. 2013; 51: 185-199Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), allowing us to control the availability of the key regulator of GREB1 expression. The bound complexes were digested with SacII to cleave off F6 (Figure 1B; Figure S1C). The beads were washed again, and both F6 and F1 were quantified in the remaining bound fraction by quantitative PCR (qPCR). Parallel reactions were carried out with the doubly biotinylated CompF (DBiot.CompF, biotinylated at both 5′ and 3′ ends). By comparing the amount of F6 retention on both 5′Biot.CompF and DBiot.CompF, we were able to calculate the fraction of F6 retained, which we call the “looping index.” We observed HeLa NE-dependent retention of F6 with F1 that further increased in the presence of ERα (Figure 1C). We carried out a similar “looping assay” with MCF-7 NE and E2, which revealed that ligand-activated ERα is required for maximal loop formation (Figure S1D). EPC can locally be viewed as an interaction in trans (Figure S1E). Thus, conceptually, a biotinylated enhancer fragment should be able to capture an unbiotinylated promoter fragment and vice versa (Figure S1F). We tested this possibility in our trans-interaction assay performed under identical conditions as described above. Biotinylated F1 (Biot.F1) was first immobilized on M280 beads and then incubated with unbiotinylated F6, HeLa NE, ATP, and ERα. After washes, we quantified F6 enrichment in the bound fractions relative to Biot.F1 using qPCR, which gave a measure of EPC or “trans-interaction index” (Figure 1D). Consistent with the looping assay above, we observed strong F1-F6 interaction in the presence of HeLa NE, which is significantly enhanced by ERα. Importantly, F2 and F3 did not interact with Biot.F1, whereas F4 and F5 displayed a very modest interaction with Biot.F1 (Figure S1G), indicating F1-F6 interaction specificity. The trans-interaction assay with MCF-7 NE revealed E2-enhanced F1-F6 interaction (Figure S1H). Chromatinized F1 and F6 fragments interacted with comparable efficiency as seen with “naked” DNAs (Figures S1I and S1J). Next, to make our assay more physiologically relevant, we investigated whether EPC would occur on a bacterial artificial chromosome (BAC) clone of the GREB1 locus that contains “all intervening sequences.” Conceptually, EPC on the BAC clone should be detectable using an approach based on the conventional 3C assay (Figure S1K). We developed an in vitro 3C (IV3C) assay using the BAC clone CTD-3138J7 as a template while keeping the reaction conditions identical to the looping assays described above. The reactions were crosslinked with formaldehyde, purified over a spin column, digested with PstI, diluted 20-fold, ligated and purified and the ligants (ligated fragments) were quantified by qPCR (Figure S1L details the relative locations of PstI sites and the 3C primers). The resulting values reflected the magnitude of inter-fragment crosslinking, called “Looping IndexIV3C.” To examine EPC in the context of transcription, we interrogated the enhancer’s contact with both upstream (“preTSS,” a PstI site 1.6 kb upstream of the TSS) and downstream (“postTSS,” a PstI site 1 kb downstream of TSS) regions of the promoter (Figure S1L). Both of these contacts increase in the presence of ERα, while no contact of the enhancer with a control sequence near F2 was detected (Figure 1E). We were unable to conduct IV3C on a chromatin template as we could not reconstitute the BAC into chromatin. We detected similar E2-induced enhancement of Enh-preTSS and Enh-postTSS contacts when we conducted the IV3C with MCF-7 NE (Figure S2A). We also conducted IV3C on a BAC clone (RP11-1025G18) harboring the NRIP1 gene, which revealed both Enh-preTSS and Enh-postTSS contacts in the presence of HeLa NE and ERα (Figure S2B), similar to GREB1. The high EPC detected in our assays in the absence of ERα could potentially reflect non-specific contacts. To test this, we conducted looping assay and trans-interaction assay with YY1-immunodepleted HeLa NE (YY1 likely mediates pre-formed EPCs; Weintraub et al., 2017Weintraub A.S. Li C.H. Zamudio A.V. Sigova A.A. Hannett N.M. Day D.S. Abraham B.J. Cohen M.A. Nabet B. Buckley D.L. et al.YY1 is a structural regulator of enhancer-promoter loops.Cell. 2017; 171: 1573-1588.e28Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Interestingly, YY1 depletion significantly lowered background EPC (NE only), whereas ERα-mediated EPC was unaffected (Figure S2C), suggesting that a significant proportion of the background EPC in vitro is YY1 regulated and, therefore, physiologically relevant. To test the versatility of our assay system, we prepared NE from HeLa cells serum starved for 48 hr and after 2 hr of serum stimulation. We used BAC clone CTD-2655F5, harboring the classical serum-inducible gene FOS to interrogate serum-inducible EPC in vitro. As the IV3C data in Figure 1F reveal, the two flanking enhancers Enh1 and Enh2 made contacts with the promoter and with themselves in the presence of the NE prepared from serum-starved cells (Starved). Interestingly, we observed 2- to 3-fold higher Looping IndexIV3C for these contacts in the presence of NE prepared from serum-stimulated cells (+FBS; Figure 1F). The enhancers and the promoter did not contact a control region (Ctr) in this assay. Importantly, these same contacts are observed in 3C assays in HeLa cells similarly serum starved and FBS stimulated, validating the above data (Figure S2D). Taken together, we developed novel cell-free methodologies that demonstrate that HeLa cell NEs made using a standard protocol (Dignam et al., 1983Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9154) Google Scholar) can facilitate EPC and that these contacts can be enhanced by signal-dependent TFs (e.g., ERα and serum-responsive TFs). Since EPC has been linked with transcription activation, we expected stronger EPC in vitro under conditions that support transcription. To test this, we first examined whether our CompF template could support enhancer- and activator-dependent stimulation of promoter-driven transcription. We conducted in vitro transcription (IVT) on CompF as well as CompFΔF1, which lacked the enhancer. The IVT reaction conditions were essentially the same as the other looping assays except that the reactions were shifted to 30°C after addition of 0.5 mM NTPs to allow transcription. Figure 2A demonstrates ERα-dependent activation of GREB1 promoter-driven transcription, mRNA, in vitro (and also eRNA; see Figure S2E). This activation is enhancer dependent, as ERα failed to activate mRNA synthesis on CompFΔF1. These results reveal that the reaction conditions required for EPC also are optimal for transcription activation in vitro. Importantly, the CompF exhibited a strong looping index under transcription-permissive conditions (+NTPs, 30°C), while the CompFΔF1 also failed to exhibit F6 retention in the looping assay (Figure 2B). This result demonstrates that the GREB1 promoter (F6) loops to the enhancer (F1), but not to intervening regions F2–F5. Next, we examined various cell-free requirements for EPC vis-à-vis transcription activation. We set up the looping assay with CompF and processed the reactions for both looping readout and quantitation of transcription (Figures 2C and 2D, respectively). Again, EPC is dependent on the presence of NE and is enhanced by the co-addition of ERα and ATP. However, when NTPs were added followed by incubation at 30°C (“transcription condition”), we detected maximal EPC (Figure 2C). Interestingly, we detected maximal production of both eRNA and mRNA under this condition (Figure 2D). Similar patterns of EPC enhancement by ERα under transcription conditions were observed in trans-interaction and IV3C assays (Figures 2E and 2F, respectively). We detected maximal eRNA and mRNA production in vitro at GREB1, NRIP1, and FOS loci with their respective BAC clones under conditions that generate optimal EPC (Figures S2F, S2G, and S2H, respectively). Interestingly, the enhancer’s contact with the postTSS region of the promoter weakens under these conditions (Figure 2F), possibly suggesting that efficient formation of the pre-initiation complex (PIC) and subsequent re-initiation cycles of transcription might require stronger contact between the enhancer and the preTSS promoter, while the postTSS region disengages from the enhancer, possibly to allow" @default.
• W2803310880 created "2018-06-01" @default.
• W2803310880 creator A5000250184 @default.
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• W2803310880 date "2018-05-01" @default.
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• W2803310880 title "SRC-3 Coactivator Governs Dynamic Estrogen-Induced Chromatin Looping Interactions during Transcription" @default.
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• W2803310880 doi "https://doi.org/10.1016/j.molcel.2018.04.014" @default.
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