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• W1981375302 abstract "•Pol II is paused at pluripotency genes during reprogramming•P-TEFb induces transcriptional elongation at pluripotency genes in reprogramming•BRD4 and HEXIM1 have opposite roles in reprogramming•KLF4 helps recruit P-TEFb to pluripotency genes in reprogramming and ESCs Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release. Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release. The enforced expression of defined transcription factors can change cell fate (Sindhu et al., 2012Sindhu C. Samavarchi-Tehrani P. Meissner A. Transcription factor-mediated epigenetic reprogramming.J. Biol. Chem. 2012; 287: 30922-30931Crossref PubMed Scopus (23) Google Scholar). A striking example of this phenomenon is the reprogramming of induced pluripotent stem cells (iPSCs) from somatic cells by the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC; OSKM) (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar). Because of their ability to differentiate into diverse cell lineages, iPSCs provide a potential supply of cells for regenerative medicine and also an excellent platform for in vitro disease modeling and toxicology screening (Saha and Jaenisch, 2009Saha K. Jaenisch R. Technical challenges in using human induced pluripotent stem cells to model disease.Cell Stem Cell. 2009; 5: 584-595Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Reprogramming is characterized by the existence of roadblocks that tend to derail the process (Apostolou and Hochedlinger, 2013Apostolou E. Hochedlinger K. Chromatin dynamics during cellular reprogramming.Nature. 2013; 502: 462-471Crossref PubMed Scopus (284) Google Scholar). Gene expression analyses of bulk populations have helped clarify these roadblocks and contributed to dividing reprogramming into distinct phases, e.g., initiation, maturation, and stabilization (Samavarchi-Tehrani et al., 2010Samavarchi-Tehrani P. Golipour A. David L. Sung H.K. Beyer T.A. Datti A. Woltjen K. Nagy A. Wrana J.L. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.Cell Stem Cell. 2010; 7: 64-77Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). The notion that gene transcription in reprogramming is phased has also been validated using single-cell profiling at different time points (Buganim et al., 2012Buganim Y. Faddah D.A. Cheng A.W. Itskovich E. Markoulaki S. Ganz K. Klemm S.L. van Oudenaarden A. Jaenisch R. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase.Cell. 2012; 150: 1209-1222Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) and with expression arrays of defined intermediate cell populations (Polo et al., 2012Polo J.M. Anderssen E. Walsh R.M. Schwarz B.A. Nefzger C.M. Lim S.M. Borkent M. Apostolou E. Alaei S. Cloutier J. et al.A molecular roadmap of reprogramming somatic cells into iPS cells.Cell. 2012; 151: 1617-1632Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). Elucidating the rate-limiting steps regulating the different phases of gene transcription in reprogramming is important because this may help improve the methodology. Gene transcription in metazoans is highly regulated and comprises multiple steps (Min et al., 2011Min I.M. Waterfall J.J. Core L.J. Munroe R.J. Schimenti J. Lis J.T. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.Genes Dev. 2011; 25: 742-754Crossref PubMed Scopus (244) Google Scholar). Formation of the preinitiation complex involves the recruitment of several general transcription factors in addition to Pol II and has traditionally been considered the major rate-limiting step of gene transcription. Consistent with this idea, overexpressing specific subunits of the general transcription factor TFIID potentiates reprogramming by facilitating pluripotency gene transcription (Pijnappel et al., 2013Pijnappel W.W. Esch D. Baltissen M.P. Wu G. Mischerikow N. Bergsma A.J. van der Wal E. Han D.W. Bruch Hv. Moritz S. et al.A central role for TFIID in the pluripotent transcription circuitry.Nature. 2013; 495: 516-519Crossref PubMed Scopus (60) Google Scholar). Pausing of activated Pol II ∼30–50 nucleotides downstream of +1 and its release by the P-TEFb complex constitutes another rate-limiting step of gene transcription that has received growing attention in recent years (Levine, 2011Levine M. Paused RNA polymerase II as a developmental checkpoint.Cell. 2011; 145: 502-511Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Although Pol II pause release was originally thought to represent a rather specialized mechanism of transcriptional regulation, analysis of nascent RNAs using global run-on sequencing (GRO-sequencing) (Min et al., 2011Min I.M. Waterfall J.J. Core L.J. Munroe R.J. Schimenti J. Lis J.T. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.Genes Dev. 2011; 25: 742-754Crossref PubMed Scopus (244) Google Scholar) has shown that around one-third of genes in the metazoan genome experience this mode of regulation at some point during the organism’s lifetime (Levine, 2011Levine M. Paused RNA polymerase II as a developmental checkpoint.Cell. 2011; 145: 502-511Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In this regard, genes regulated through pause release are particularly prevalent in stress responses and during embryonic development (Smith and Shilatifard, 2013Smith E. Shilatifard A. Transcriptional elongation checkpoint control in development and disease.Genes Dev. 2013; 27: 1079-1088Crossref PubMed Scopus (53) Google Scholar). Potentially, this is because Pol II pausing and its subsequent release enables a method of rapid and synchronous activation of gene expression programs. Pol II pausing might also be a mechanism to hold the activation of specialized gene programs during cell fate conversions if not stimulated properly through pause release. Here, we demonstrate that the transition of Pol II from a paused to a productive elongation stage is a rate-limiting step for inducing pluripotency genes in the late phase of reprogramming. This transition is promoted by the recruitment of active P-TEFb to pluripotency gene promoters, which is simultaneously coordinated by BRD4 and KLF4. Our study thus proposes a revised model for the reactivation of the pluripotency network in reprogramming. It has been reported that reprogramming comprises two major waves of gene transcription (Polo et al., 2012Polo J.M. Anderssen E. Walsh R.M. Schwarz B.A. Nefzger C.M. Lim S.M. Borkent M. Apostolou E. Alaei S. Cloutier J. et al.A molecular roadmap of reprogramming somatic cells into iPS cells.Cell. 2012; 151: 1617-1632Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). The first wave involves the upregulation of genes related to proliferation, metabolism, and cytoskeletal organization, while the second wave is mostly genes related to the pluripotency network. Because mouse embryonic fibroblasts (MEFs) are very different from pluripotent cells, we envisaged that variations in the mode of transcriptional regulation occur during reprogramming. To test this, we initially contrasted the genes upregulated in the two waves of reprogramming with a previous GRO-sequencing data set of MEFs and embryonic stem cells (ESCs) (Min et al., 2011Min I.M. Waterfall J.J. Core L.J. Munroe R.J. Schimenti J. Lis J.T. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.Genes Dev. 2011; 25: 742-754Crossref PubMed Scopus (244) Google Scholar). This study divided genes into four classes: (1) not paused and transcribed, which show concentration of GRO-sequencing reads along the gene body (class I, Figure 1A); (2) paused and transcribed, which show concentration of GRO-sequencing reads in the proximal promoter and less pronouncedly along the gene body (class II, Figure 1A); (3) paused and not transcribed; and (4) not paused and not transcribed. Roughly ∼35%–40% of all RefSeq genes were shown to belong to class II in both MEFs and ESCs, but the kind of genes varied in each cell type. Our comparative analysis showed that genes upregulated in the first wave of reprogramming mostly belong to class II in both ESCs and MEFs (606 genes out of 816) (Figure 1B and Figure S1A available online). Conversely, the second wave is substantially depleted of class II genes in both MEFs and ESCs and is enriched in class II genes in ESCs only (Figure 1B and Figure S1A). Average density of nascent RNA along the promoter and gene body regions of all class II genes induced in the second wave of reprogramming confirmed higher levels of Pol II pausing and pause release in ESCs than in MEFs (Figure S1B). We conclude that many genes activated in the second wave of reprogramming must experience a change in their mode of transcriptional regulation compared to MEFs. This change consists in the pausing of Pol II at their proximal promoters and the subsequent pause release to enter productive elongation. We then hypothesized that productive transcriptional elongation of pluripotency genes could represent a rate-limiting step of reprogramming. To explore this, we performed ChIP-sequencing for Pol II in MEFs reprogrammed with OSKM at days 5 and 8; ESCs and untransduced MEFs were used as controls. The Pol II traveling ratio (TR), which compares the average density of Pol II in the proximal promoter and gene body (Figure 1C), was employed to calculate the degree of pausing among different genes. Consistent with previous reports, we used a TR > 4 as indicative of Pol II pausing (Zeitlinger et al., 2007Zeitlinger J. Stark A. Kellis M. Hong J.-W. Nechaev S. Adelman K. Levine M. Young R.A. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.Nat. Genet. 2007; 39: 1512-1516Crossref PubMed Scopus (575) Google Scholar). Notably, approximately half of the genes upregulated in the second wave of reprogramming displayed a TR > 4 in OSKM reprogramming at day 8 (Figure 1D), but the same genes showed a significantly lower value at day 5 and in untransduced MEFs (Figures 1E and 1F). There was good correlation between second wave genes paused at day 8 of reprogramming and those classified as class II in ESCs in the GRO-sequencing data analysis (Figure S1C). In addition, comparison with our Pol II ChIP-sequencing data in ESCs showed that a large fraction (55%) of second wave genes has lower TR in ESCs than at day 8 of reprogramming, indicating that they experience productive elongation in ESCs (Figures 1G–1I). Among those genes there are key pluripotency regulators including Oct4 and Nanog (Figure 1G). Interestingly, Oct4 showed substantial pausing at day 5 as well, indicating that for some pluripotency regulators Pol II is recruited early to their promoters but remains paused until the late phase (Figure 1J). Notably, the noncoding pluripotency regulator Mir290 also exhibited Pol II pausing at day 8 of reprogramming (Figure S1D). The Pol II binding pattern of a class II gene in MEFs, Thy1, which is downregulated early in reprogramming, is shown as a control (Figure 1K). Therefore, Pol II pausing at pluripotency promoters characterizes reprogramming, strongly suggesting that productive transcriptional elongation is limiting for this process. To demonstrate that transcriptional pause release of pluripotency genes is limiting for reprogramming, we focused on CDK9, the catalytic subunit of the P-TEFb complex, because it stimulates elongation by phosphorylating Pol II and the negative elongation factors DSIF and NELF (Zhou and Yik, 2006Zhou Q. Yik J.H. The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar). We observed increased CDK9 expression (assessed by western blotting and quantitative PCR [qPCR]) in ESCs compared to that in MEFs, and we also observed such during the reprogramming of MEFs with OSKM factors delivered as retroviruses (Figure 2A and Figures S2A and S2B). To reduce CDK9 activity, we prepared a retrovirus producing a dominant-negative form (a kinase inactive mutant) of CDK9 (CDK9-DN) and two independent retroviral shRNA vectors (Figure S2C), which were transduced along with OSKM into MEFs bearing an Oct4-GFP transgenic reporter. Reducing CDK9 activity (tested using CDK9-DN) did not alter exogenous OSKM expression level and only impaired cell proliferation moderately (Figures S2D and S2E). Notably, OSKM reprogramming with CDK9-DN or the two shRNAs allowed the formation of AP+ colonies (an early marker of reprogramming) (Buganim et al., 2013Buganim Y. Faddah D.A. Jaenisch R. Mechanisms and models of somatic cell reprogramming.Nat. Rev. Genet. 2013; 14: 427-439Crossref PubMed Scopus (326) Google Scholar), but these were mostly GFP− compared to the controls (Figures 2B–2E), indicating incomplete reprogramming. We achieved the same result using a different reprogramming system, Oct4-GFP transgenic MEFs with an integrated OSKM cassette in the Col1a1 locus, hereafter termed secondary MEFs (Figures S2F and S2G). We also overexpressed CDK9 during OSKM reprogramming but observed no change in the number of GFP+ colonies (Figure S2H). To confirm the selective role of CDK9 in the late phase of reprogramming, we employed a doxycycline-inducible lentivirus producing CDK9-DN (Figure S2I) and a known CDK9 inhibitor (flavopiridol) (Rahl et al., 2010Rahl P.B. Lin C.Y. Seila A.C. Flynn R.A. McCuine S. Burge C.B. Sharp P.A. Young R.A. c-Myc regulates transcriptional pause release.Cell. 2010; 141: 432-445Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar). Both approaches impaired the appearance of GFP+ colonies if induced/administered in the late phase (day 7 to day 11) of reprogramming, but not in the early phase (day 0 to day 5) (Figures 2F and 2G and Figures S2J and S2K). We also performed RNA-sequencing of MEFs reprogrammed with OSKM in the presence or absence of CDK9-DN. Interestingly, CDK9-DN induced rather modest changes in gene transcription in the early phase of reprogramming compared to the control (Figures 2H and 2I). Of note, the well-known CDK9 target Hexim1 was among the downregulated genes (He et al., 2006He N. Pezda A.C. Zhou Q. Modulation of a P-TEFb functional equilibrium for the global control of cell growth and differentiation.Mol. Cell. Biol. 2006; 26: 7068-7076Crossref PubMed Scopus (88) Google Scholar) (Table S1). Conversely, CDK9-DN led to the downregulation of a large number of genes at day 10, many of which belong to the second transcriptional wave of reprogramming (Figures 2H–2J and Table S1). The selective effect of ablating CDK9 function on the late phase of reprogramming was validated by qPCR of mesenchymal and epithelial genes in MEFs reprogrammed with OSKM and CDK9-DN at day 5 (Figure S2L) and by qPCR of pluripotency genes at day 9 (Figure 2K). Overall our data support the idea that high levels of P-TEFb activity are dispensable in the early phase of reprogramming but instrumental for reactivating the pluripotency network in the late phase. The experiments above showing that CDK9 overexpression does not enhance the formation of GFP+ colonies led us to speculate that CDK9 enzymatic activity, but not its overall expression level, is a limiting factor for reprogramming. Supporting this possibility, a big proportion of CDK9 is held inactive in a complex containing HEXIM1 in many cell types (Zhou and Yik, 2006Zhou Q. Yik J.H. The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar). It is also well known that the bromodomain and extraterminal (BET) family member BRD4 activates CDK9 by displacing HEXIM1. BRD4 thus appeared as a candidate for further exploration. There are two different splice isoforms of BRD4 (short and long). They share the amino terminal domain (two tandem bromodomains [BDs]), responsible for interacting with acetylated histones, and the extraterminal (ET) domain (Belkina and Denis, 2012Belkina A.C. Denis G.V. BET domain co-regulators in obesity, inflammation and cancer.Nat. Rev. Cancer. 2012; 12: 465-477Crossref PubMed Scopus (512) Google Scholar). However, only the carboxy terminal domain (CTD) of the long isoform allows interaction with CDK9 and its activation. The short isoform of BRD4 displayed low expression level by qPCR (based on the qPCR cycle threshold [Ct] value) in MEFs and ESCs (Figure S3A), so we did not study it further. Notably, qPCR and western blot of the long isoform of BRD4 (hereafter named BRD4) showed higher expression in ESCs compared to MEFs and an increase of such during OSKM reprogramming (Figure 3A and Figures S3A and S3B). We then reduced BRD4 levels in OSKM reprogramming with two independent retroviral shRNA vectors (Figure S3C). The two shRNAs enabled the formation of AP+ colonies but greatly diminished the appearance of GFP+ colonies (Figures 3B and 3C), matching the pattern previously observed for CDK9 inhibition. Moreover, we could not detect any obvious change in cell proliferation (Figure S3D). The negative effect on reprogramming efficiency of Brd4 knockdown was confirmed using secondary MEFs (Figure S3E). In addition, we observed that the BRD4 inhibitor JQ1 (Belkina and Denis, 2012Belkina A.C. Denis G.V. BET domain co-regulators in obesity, inflammation and cancer.Nat. Rev. Cancer. 2012; 12: 465-477Crossref PubMed Scopus (512) Google Scholar) did not affect reprogramming efficiency when administered in the early phase, but significantly reduced reprogramming efficiency in the late phase (Figures 3D and 3E). Next, we reprogrammed MEFs with OSKM and a retroviral vector producing BRD4. This increased the number of GFP+ colonies (∼3-fold) without noticeably changing the appearance of AP+ colonies (Figures 3F and 3G). Exogenous BRD4 also accelerated the formation of GFP+ colonies compared to the control (Figure 3H) but did not affect cell proliferation (Figure S3F). The enhancing effect on reprogramming of BRD4 overexpression was confirmed using secondary MEFs (Figure S3G). Moreover, iPSC colonies generated with BRD4 and OSKM contained the Brd4 retrovirus in their genome and were fully pluripotent (Figure 3I and Figures S3H–S3K). These data indicate that BRD4 is a positive regulator of the late phase of reprogramming. Because BRD4 has CDK9-independent functions (Belkina and Denis, 2012Belkina A.C. Denis G.V. BET domain co-regulators in obesity, inflammation and cancer.Nat. Rev. Cancer. 2012; 12: 465-477Crossref PubMed Scopus (512) Google Scholar), we tested whether it regulates reprogramming specifically through the activation of CDK9. We prepared retroviral vectors producing a series of truncations that correspond to the major functional domains of BRD4 (Figure 4A). Only those BRD4 constructs retaining the CTD, including BRD4-CTD, were able to pull down endogenous CDK9 when overexpressed in HEK293T cells (Figure 4B). We overexpressed these different truncations together with OSKM in MEFs. Interestingly, BRD4-BD acted as a dominant-negative for endogenous BRD4, reducing GFP+ colonies (Figure 4C) but without obviously changing the number of AP+ colonies (Figure S4A). This effect is likely related to the ability of this construct to diminish the interaction of endogenous BRD4 with chromatin. As predicted, BRD4-Δ-CTD also failed to enhance, and even reduced, the number of GFP+ colonies compared to the control (Figure 4C). In addition, we observed that BRD4-CTD reproducibly enhanced the formation of GFP+ colonies ∼3-fold over intact BRD4 (Figure 4C). This suggested that the ET domain of BRD4 might contain a module that impairs reprogramming, perhaps through interaction with negative regulators. Supporting this idea, a truncated form of BRD4 (BRD4-Δ-ET) that only lacks the ET domain (Figure 4A) increased the number of GFP+ colonies to a level closer to that of BRD4-CTD (Figure 4C). One possible candidate mediating this negative effect is CHD4, which belongs to the MBD3/NuRD complex with repressive function in reprogramming (Rais et al., 2013Rais Y. Zviran A. Geula S. Gafni O. Chomsky E. Viukov S. Mansour A.A. Caspi I. Krupalnik V. Zerbib M. et al.Deterministic direct reprogramming of somatic cells to pluripotency.Nature. 2013; 502: 65-70Crossref PubMed Scopus (411) Google Scholar), because it is known to interact with BRD4 through the ET domain (Rahman et al., 2011Rahman S. Sowa M.E. Ottinger M. Smith J.A. Shi Y. Harper J.W. Howley P.M. The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3.Mol. Cell. Biol. 2011; 31: 2641-2652Crossref PubMed Scopus (364) Google Scholar). We also detected that overexpressing BRD4-ET inhibited rather than enhanced the number of GFP+ colonies (Figure 4C). This implies that overexpressed BRD4-ET might interact as well with positive regulators of reprogramming, thus titrating down their interaction with endogenous BRD4. To further show that the effect of BRD4-CTD is mediated through CDK9, we prepared two mutant forms (CTD-FEE/AAA and CTD-R/P) that cannot interact with CDK9 (Figure 4D). Consistent with previous studies (Bisgrove et al., 2007Bisgrove D.A. Mahmoudi T. Henklein P. Verdin E. Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription.Proc. Natl. Acad. Sci. USA. 2007; 104: 13690-13695Crossref PubMed Scopus (273) Google Scholar), these mutants could not pull down endogenous CDK9 in immunoprecipitation assays (Figure 4E) and failed to increase the formation of GFP+ colonies over the baseline in MEFs reprogrammed with OSKM (Figure 4F). Introducing the same mutations into full-length BRD4 (BRD4-FEE/AAA and BRD4-R/P) had the same outcome (Figure 4G). We also validated the effect of all BRD4 constructs in secondary MEFs (Figures S4B and S4C). Likewise, we employed a polycistronic OSKM lentiviral vector and BRD4-CTD to reprogram a noninvasive reprogrammable human cell source, urinary cells (Zhou et al., 2012Zhou T. Benda C. Dunzinger S. Huang Y. Ho J.C. Yang J. Wang Y. Zhang Y. Zhuang Q. Li Y. et al.Generation of human induced pluripotent stem cells from urine samples.Nat. Protoc. 2012; 7: 2080-2089Crossref PubMed Scopus (398) Google Scholar). BRD4-CTD significantly increased the number of human ESC-like colonies using cells from three independent donors (Figure 4H). We picked eight independent colonies from one donor; they could be expanded retaining ESC-like morphology (Figure 4I), had integrated the BRD4-CTD transgene, and were fully pluripotent (Figure 4I and Figures S4D–S4F). Collectively, these experiments demonstrate that BRD4 enhances reprogramming (mouse and human) through CDK9. To demonstrate that BRD4 facilitates the activation of the pluripotency network in the late phase of reprogramming, we performed qPCR of MEFs reprogrammed with OSKM and BRD4-CTD at day 9. As expected, BRD4-CTD induced a substantial increase in the expression of pluripotency genes belonging to the second transcriptional wave of reprogramming (Figure 5A), but there was no significant change in epithelial or mesenchymal genes at day 5 (Figure 5B). Next, we studied whether the effect of BRD4 on pluripotency genes is due to stimulation of transcriptional elongation. For this purpose, we selected eight candidates induced in the second wave of reprogramming: Nanog, Oct4, Sall4, Sox2, Dppa2, Rex1, Esrrb, and Utf1. ChIP-qPCR along the promoter and coding region of these genes showed that BRD4-CTD increases Pol II binding to the gene body compared to the control, indicating more productive elongation (Figure 5C and Figure S5A). Similar results were observed performing ChIP for Pol II phosphorylated on serine 2 (Ser2P), a posttranslational modification triggered by CDK9 that specifically identifies elongating Pol II (Zhou and Yik, 2006Zhou Q. Yik J.H. The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar) (Figure 5C and Figure S5A). If our hypothesis that BRD4 is a major regulator of pluripotency genes during reprogramming were true, one might also expect that reducing BRD4 expression should blunt pluripotency genes in ESCs. However, a previous genome-wide siRNA screening in ESCs showed cell death but not differentiation upon Brd4 knockdown (Fazzio et al., 2008Fazzio T.G. Huff J.T. Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.Cell. 2008; 134: 162-174Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). To clarify this issue, we reduced Brd4 expression in ESCs with two independent lentiviral shRNA vectors (Figure S5B). This caused quick (within 4 days) acquisition of a differentiated morphology and concomitant loss of AP staining and OCT4-GFP signal, which was associated with reduced expression of pluripotency genes and enhanced expression of lineage-specific genes (Figures 5D and 5E and Figure S5C). In addition, we overexpressed BRD4-CTD in ESCs before letting them differentiate by withdrawing LIF. BRD4-CTD had a moderate effect in both sustaining pluripotency genes and preventing the increase of lineage-specific genes, but it could not prevent the quick (within 4 days) loss of ESC morphology and AP staining (Figures S5D and S5E). Finally, to further demonstrate that BRD4 regulates pluripotency genes, we analyzed a BRD4 ChIP-sequencing data set from ESCs. This showed strong binding of BRD4 at the promoter regions of multiple pluripotency genes including Nanog and Oct4 (Figure 5F). Hence, BRD4 regulates pluripotency gene expression in reprogramming and in ESCs. Differences between our work and the study by Fazzio et al., 2008Fazzio T.G. Huff J.T. Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.Cell. 2008; 134: 162-174Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar may rely on the extent of Brd4 knockdown, which is likely stronger with the siRNA, and may point to an additional function of BRD4 in regulating the ESC cell cycle. Because the reprogramming booster BRD4 enhances P-TEFb activity by displacing HEXIM1 bound to CDK9 (Zhou and Yik, 2006Zhou Q. Yik J.H. The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar), we postulated that HEXIM1 is a roadblock to reprogramming. To evaluate this, we reprogrammed MEFs with OSKM and a retroviral vector producing HEXIM1. As expected, this reduced the interaction between endogenous BRD4 and CDK9, as assessed by immunoprecipitation of CDK9 at day 9 of reprogramming (Figure 6A). HEXIM1 overexpression also reduced cell proliferation (Figure S6A), in agreement with previous work in other contexts (Hong et al., 2012Hong P. Chen K. Huang B. Liu M. Cui M. Rozenberg I. Chaqour B. Pan X. Barton E.R. Jiang X.C. Siddiqui M.A. HEXIM1 controls satellite cell expansion after injury to regulate skeletal muscle regeneration.J. Clin. Invest. 2012; 122: 3873-3887Crossref PubMed Scopus (9) Google Scholar). Nevertheless, a significant number of AP+ colonies still formed, though these were mostly GFP− (Figures 6B and 6C). A similar effect was observed with HEXIM1 overexpression in secondary MEFs (F" @default.
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• W1981375302 title "Transcriptional Pause Release Is a Rate-Limiting Step for Somatic Cell Reprogramming" @default.
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• W1981375302 doi "https://doi.org/10.1016/j.stem.2014.09.018" @default.
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