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• W2110492802 abstract "•The splicing factor SFRS2 supports human PSC self-renewal and is a target of OCT4•SFRS2 mediates splicing of MBD2 isoforms that play opposing roles in pluripotency•MBD2a, but not MBD2c, can interact with the NuRD chromatin remodeling complex•OCT4, SFRS2, and MBD2 comprise a positive feedback loop in human PSCs Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate that the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG binding protein MBD2, whose isoforms play opposing roles in maintenance of and reprogramming to pluripotency. Although both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity in support of hPSC self-renewal and reprogramming. Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate that the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG binding protein MBD2, whose isoforms play opposing roles in maintenance of and reprogramming to pluripotency. Although both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity in support of hPSC self-renewal and reprogramming. The transcription factors OCT4, NANOG, and SOX2 are master regulators of pluripotency in embryonic stem cells (ESCs) (De Los Angeles et al., 2012De Los Angeles A. Loh Y.-H. Tesar P.J. Daley G.Q. Accessing naïve human pluripotency.Curr. Opin. Genet. Dev. 2012; 22: 272-282Crossref PubMed Scopus (78) Google Scholar) and, along with Klf4 and c-Myc, facilitate reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) (Park et al., 2008Park I.-H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Reprogramming of human somatic cells to pluripotency with defined factors.Nature. 2008; 451: 141-146Crossref PubMed Scopus (2374) Google Scholar, 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). ESCs are indispensable models of early development, whereas iPSCs hold great promise as cell-based therapeutics that circumvent the immunologic and ethical hurdles of embryo-derived cells. As a result, significant effort has been invested in elucidating the mechanisms that underlie stem cell function, with a particular emphasis on these core pluripotent genes. Despite the requirement of OCT4, SOX2, and NANOG in stem cell function (De Los Angeles et al., 2012De Los Angeles A. Loh Y.-H. Tesar P.J. Daley G.Q. Accessing naïve human pluripotency.Curr. Opin. Genet. Dev. 2012; 22: 272-282Crossref PubMed Scopus (78) Google Scholar), discrepancies between ostensibly identical pluripotent cell lines (Gore et al., 2011Gore A. Li Z. Fung H.-L. Young J.E. Agarwal S. Antosiewicz-Bourget J. Canto I. Giorgetti A. Israel M.A. Kiskinis E. et al.Somatic coding mutations in human induced pluripotent stem cells.Nature. 2011; 471: 63-67Crossref PubMed Scopus (998) Google Scholar), in addition to the divergent lineage commitment properties of iPSCs derived from different adult tissues (Kim et al., 2010Kim K. Doi A. Wen B. Ng K. Zhao R. Cahan P. Kim J. Aryee M.J. Ji H. Ehrlich L.I.R. et al.Epigenetic memory in induced pluripotent stem cells.Nature. 2010; 467: 285-290Crossref PubMed Scopus (1714) Google Scholar), illustrate that the molecular network balancing self-renewal, pluripotency, and lineage commitment is not yet resolved. Recently, functional genomics and molecular profiling approaches have been used to explore the broader role of the core pluripotent factors in stem cell biology. These studies expanded the set of genes that support pluripotency (Chia et al., 2010Chia N.-Y. Chan Y.-S. Feng B. Lu X. Orlov Y.L. Moreau D. Kumar P. Yang L. Jiang J. Lau M.-S. et al.A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.Nature. 2010; 468: 316-320Crossref PubMed Scopus (364) Google Scholar) and defined a biochemical network centered around OCT4, NANOG, and SOX2 that is highly enriched for genes essential for development and stem cell function (Kim et al., 2008Kim J. Chu J. Shen X. Wang J. Orkin S.H. An extended transcriptional network for pluripotency of embryonic stem cells.Cell. 2008; 132: 1049-1061Abstract Full Text Full Text PDF PubMed Scopus (1074) Google Scholar). Furthermore, use of chromatin immunoprecipitation (ChIP)-chip (Boyer et al., 2005Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. et al.Core transcriptional regulatory circuitry in human embryonic stem cells.Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3503) Google Scholar) and ChIP sequencing (Guenther et al., 2010Guenther M.G. Frampton G.M. Soldner F. Hockemeyer D. Mitalipova M. Jaenisch R. Young R.A. Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells.Cell Stem Cell. 2010; 7: 249-257Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar) has established the landscape of genetic targets for several key pluripotent factors and defined correlations between promoter co-occupancy and transcriptional activation. In parallel, genome-scale molecular measurement technologies have been used to quantify differences in epigenetic modifications (Gifford et al., 2013Gifford C.A. Ziller M.J. Gu H. Trapnell C. Donaghey J. Tsankov A. Shalek A.K. Kelley D.R. Shishkin A.A. Issner R. et al.Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.Cell. 2013; 153: 1149-1163Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, Xie et al., 2013Xie W. Schultz M.D. Lister R. Hou Z. Rajagopal N. Ray P. Whitaker J.W. Tian S. Hawkins R.D. Leung D. et al.Epigenomic analysis of multilineage differentiation of human embryonic stem cells.Cell. 2013; 153: 1134-1148Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar), gene expression (Tang et al., 2010Tang F. Barbacioru C. Bao S. Lee C. Nordman E. Wang X. Lao K. Surani M.A. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis.Cell Stem Cell. 2010; 6: 468-478Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), and protein translation (Ingolia et al., 2011Ingolia N.T. Lareau L.F. Weissman J.S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.Cell. 2011; 147: 789-802Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar) in addition to protein expression and phosphorylation (Brill et al., 2009Brill L.M. Xiong W. Lee K.-B. Ficarro S.B. Crain A. Xu Y. Terskikh A. Snyder E.Y. Ding S. Phosphoproteomic analysis of human embryonic stem cells.Cell Stem Cell. 2009; 5: 204-213Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Phanstiel et al., 2011Phanstiel D.H. Brumbaugh J. Wenger C.D. Tian S. Probasco M.D. Bailey D.J. Swaney D.L. Tervo M.A. Bolin J.M. Ruotti V. et al.Proteomic and phosphoproteomic comparison of human ES and iPS cells.Nat. Methods. 2011; 8: 821-827Crossref PubMed Scopus (217) Google Scholar) between pluripotent stem cells and other cell types. These data provide a rich resource of molecular information, although it remains challenging to generate specific hypotheses from these disparate data types or establish mechanistic links between these molecular profiles and the core pluripotent factors. Recently, alternative splicing (AS) has garnered attention as a possible means by which stem cells regulate the expression of gene and protein isoforms in order to support pluripotency and self-renewal. Indeed, functional roles for alternatively spliced gene products of NANOG, FOXP1, and Tcf7l1 have been demonstrated (Das et al., 2011Das S. Jena S. Levasseur D.N. Alternative splicing produces Nanog protein variants with different capacities for self-renewal and pluripotency in embryonic stem cells.J. Biol. Chem. 2011; 286: 42690-42703Crossref PubMed Scopus (55) Google Scholar, Gabut et al., 2011Gabut M. Samavarchi-Tehrani P. Wang X. Slobodeniuc V. O’Hanlon D. Sung H.-K. Alvarez M. Talukder S. Pan Q. Mazzoni E.O. et al.An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.Cell. 2011; 147: 132-146Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, Salomonis et al., 2010Salomonis N. Schlieve C.R. Pereira L. Wahlquist C. Colas A. Zambon A.C. Vranizan K. Spindler M.J. Pico A.R. Cline M.S. et al.Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.Proc. Natl. Acad. Sci. USA. 2010; 107: 10514-10519Crossref PubMed Scopus (169) Google Scholar). In addition, the muscleblind-like family of RNA binding proteins was found to repress pluripotency by mediating expression of several somatic cell-specific protein isoforms, including FOXP1 (Han et al., 2013Han H. Irimia M. Ross P.J. Sung H.-K. Alipanahi B. David L. Golipour A. Gabut M. Michael I.P. Nachman E.N. et al.MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.Nature. 2013; 498: 241-245Crossref PubMed Scopus (219) Google Scholar). These data illustrate a general role for AS in pluripotent cells; however, the specific splicing factors and mechanistic links to the core pluripotent genes, which work in concert to reinforce a ground state of self-renewal, remain unresolved. The splicing factor SFRS2 (also known as SC35) is essential for embryonic development (Xiao et al., 2007Xiao R. Sun Y. Ding J.-H. Lin S. Rose D.W. Rosenfeld M.G. Fu X.-D. Li X. Splicing regulator SC35 is essential for genomic stability and cell proliferation during mammalian organogenesis.Mol. Cell. Biol. 2007; 27: 5393-5402Crossref PubMed Scopus (130) Google Scholar) and regulates transcription (Lin et al., 2008Lin S. Coutinho-Mansfield G. Wang D. Pandit S. Fu X.-D. The splicing factor SC35 has an active role in transcriptional elongation.Nat. Struct. Mol. Biol. 2008; 15: 819-826Crossref PubMed Scopus (269) Google Scholar). Although several splicing substrates have been identified (Lin et al., 2008Lin S. Coutinho-Mansfield G. Wang D. Pandit S. Fu X.-D. The splicing factor SC35 has an active role in transcriptional elongation.Nat. Struct. Mol. Biol. 2008; 15: 819-826Crossref PubMed Scopus (269) Google Scholar), no pluripotency-specific role has been established for SFRS2. The methyl-DNA binding protein methyl-CpG binding domain protein 2 (MBD2) comprises two predominant isoforms, MBD2a and MBD2c (Hendrich and Bird, 1998Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1068) Google Scholar), that share the same MBD domain but differ in the C-terminal region as a result of AS. MBD2 silences gene expression by binding to methylated DNA and recruiting the nucleosome remodeling and deacetylation (NuRD) complex (Zhang et al., 1999Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (931) Google Scholar). Although NuRD has well-established roles in development (Reynolds et al., 2012Reynolds N. Latos P. Hynes-Allen A. Loos R. Leaford D. O’Shaughnessy A. Mosaku O. Signolet J. Brennecke P. Kalkan T. et al.NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment.Cell Stem Cell. 2012; 10: 583-594Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), the function of MBD2 in stem cells is not well understood. In fact, data from two recent studies are inconsistent with respect to the impact of MBD2 in somatic cell reprogramming (Lee et al., 2013Lee M.R. Prasain N. Chae H.-D. Kim Y.-J. Mantel C. Yoder M.C. Broxmeyer H.E. Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming.Stem Cells. 2013; 31: 666-681Crossref PubMed Scopus (75) Google Scholar, Onder et al., 2012Onder T.T. Kara N. Cherry A. Sinha A.U. Zhu N. Bernt K.M. Cahan P. Marcarci B.O. Unternaehrer J. Gupta P.B. et al.Chromatin-modifying enzymes as modulators of reprogramming.Nature. 2012; 483: 598-602Crossref PubMed Scopus (499) Google Scholar), although the possibility of isoform-specific function was not considered. In this study, we establish mechanistic links between OCT4 and SFRS2 and demonstrate that these factors work in concert to regulate AS of MBD2. Expression of specific MBD2 isoforms is further regulated by the microRNA (miRNA) machinery, and we find that the resulting gene products play opposing functional roles with respect to self-renewal of human PSCs (hPSCs) and reprogramming of fibroblasts. Consistent with these observations, MBD2 isoforms target the promoters of OCT4 and NANOG in human ESCs (hESCs) but differ dramatically in their ability to biochemically interact with chromatin remodeling proteins. Collectively, our results suggest a positive feedback loop comprised of OCT4, SFRS2, and splice products of MBD2 that regulates proteome diversity in order to support a self-renewing ground state. First, we sought to identify a molecular signature for pluripotency that integrated gene and protein expression, in addition to protein phosphorylation in cells representing a broad range of genetic backgrounds and cell fates (Figures S1A and S2 and Table S1 available online). Independent hierarchical clustering of each data type revealed that hPSCs from different tissue types exhibit protein phosphorylation, gene transcription, and protein expression profiles that are clearly distinct from differentiated fibroblasts (DFs; Figure 1A), and each molecular class contributes a subset of unique genes to the signature (Figure S1B). Notably, the molecular divergence observed between pluripotent cells and DFs was considerably higher than it was in hPSCs (Figure S1C); in addition we confirmed that the phosphorylation signature was strongly linked to cell type rather than specific culture conditions (Figure S1D). As is typical of high-throughput measurements (Brill et al., 2009Brill L.M. Xiong W. Lee K.-B. Ficarro S.B. Crain A. Xu Y. Terskikh A. Snyder E.Y. Ding S. Phosphoproteomic analysis of human embryonic stem cells.Cell Stem Cell. 2009; 5: 204-213Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Phanstiel et al., 2011Phanstiel D.H. Brumbaugh J. Wenger C.D. Tian S. Probasco M.D. Bailey D.J. Swaney D.L. Tervo M.A. Bolin J.M. Ruotti V. et al.Proteomic and phosphoproteomic comparison of human ES and iPS cells.Nat. Methods. 2011; 8: 821-827Crossref PubMed Scopus (217) Google Scholar, Tang et al., 2010Tang F. Barbacioru C. Bao S. Lee C. Nordman E. Wang X. Lao K. Surani M.A. Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis.Cell Stem Cell. 2010; 6: 468-478Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), classification of gene function within the pluripotency signature based on gene ontology (GO) biological process revealed enrichment of several disparate pathways (Figure 1B, left). There is growing appreciation that the principles of network theory are applicable to human physiology whereby extended physical, genetic, or metabolic relationships between biomolecules may have predictive power with respect to biological outcomes (Balázsi et al., 2011Balázsi G. van Oudenaarden A. Collins J.J. Cellular decision making and biological noise: from microbes to mammals.Cell. 2011; 144: 910-925Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, Vidal et al., 2011Vidal M. Cusick M.E. Barabási A.-L. Interactome networks and human disease.Cell. 2011; 144: 986-998Abstract Full Text Full Text PDF PubMed Scopus (1183) Google Scholar). Consistent with this notion, we next asked whether interpretation of our molecular signature data within the context of physical interaction networks would highlight specific cellular functions that support self-renewal. Accordingly, we assessed the number of physical interactions between constituent genes of the pluripotency signature and three positive reference sets (PRSs) of pluripotent factors derived from (1) literature survey, (2) a recent functional genomics study, and (3) proteins defined as biochemical interactors of Oct4 or Nanog (Figure S1E, Supplemental Experimental Procedures, and Table S2). This analysis revealed that only members of the RNA splicing pathway are consistently enriched across each measurement class (Figure 1B, right, and Table S3). Additional analysis (Supplemental Experimental Procedures) of the splicing factors in our pluripotent molecular signature suggested that the splicing factor SFRS2 might be an important mediator of pluripotency (Figure 1C and Table S3). Given the role of SFRS2 in AS, we next compared the levels of spliced isoforms for 16,084 genes in hESCs and DFs and found that the spliced products from 2,974 genes differed between these cell types (Figure S3A and Table S4). Strikingly, we observed that 1,424 of these were not otherwise represented in the set of pluripotency signature genes (Figure S3A). As with other molecular classes of the pluripotent signature (Figure 1B), gene products subject to AS in hESCs are enriched for physical interactions with the PRSs (Figure 1D and Table S3). Extension of this analysis to GO annotation revealed a consistent enrichment of factors related to transcription regulation and chromatin modification (Figure S3B and Table S3); in total, we observed 236 alternatively spliced genes that spanned these pathways. Within the exon junction microarray data (Table S4), MBD2 had the highest prediction score for AS between hESCs and DFs (Figure 1E and Table S4). Next, we sought to establish specific links between SFRS2, MBD2, and the machinery supporting pluripotency. Depletion of endogenous SFRS2 disrupted self-renewal in hESCs as gauged by cell morphology (Figure 2A), expression of OCT4 and NANOG (Figure 2B), alkaline phosphatase staining (Figure S4A), and cell colony integrity (Figure S4B). We observed a coordinate decrease in expression level of SFRS2 upon OCT4 depletion in hESCs (Figure 2C); importantly, this effect was specific to SFRS2 and not observed for other splicing factors (Figure S4C). Furthermore, we found that OCT4 bound directly to the promoter of SFRS2 in hESCs (Figure 2D) and drove expression of luciferase downstream of the native SFRS2 promoter in vitro (Figure 2E). The specificity of this interaction was confirmed by mutation or deletion within the predicted OCT4 binding site of the SFRS2 promoter (Figure 2F). These data provide evidence for functional and genetic links between OCT4 and SFRS2 in hPSCs. MBD2 comprises multiple isoforms (Hendrich and Bird, 1998Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1068) Google Scholar) (Figure 3A). We detected preferential gene- and protein-level expression of the MBD2c and MBD2a isoforms in H1 ESCs and BJ DFs, respectively (Figures 3B and 3C). Interestingly, depletion of endogenous SFRS2 or OCT4 in hESCs led to a dramatic increase in expression of MBD2a and a reduction in MBD2c (Figures 3D and S4D). Next, we probed for a direct biochemical interaction between SFRS2 and MBD2 pre-mRNA by assaying RNA that coprecipitated with exogenously expressed SFRS2-FLAG-HA. We observed that SFRS2 bound to MBD2 pre-mRNA specifically at intron 2, preceding exon 3, which is unique to the ESC-predominant MBD2c isoform (Figure 3E), suggesting that SFRS2 may mediate alternative splicing of this methyl-DNA binding protein in hPSCs. In addition, close inspection of the 3′ untranslated region (3′ UTR) of SFRS2 and MBD2a (but not MBD2c) revealed potential binding motifs for miR-301 and miR-302, miRNA families that are functionally associated with lineage commitment and self-renewal (Figure S4E) (Bar et al., 2008Bar M. Wyman S.K. Fritz B.R. Qi J. Garg K.S. Parkin R.K. Kroh E.M. Bendoraite A. Mitchell P.S. Nelson A.M. et al.MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries.Stem Cells. 2008; 26: 2496-2505Crossref PubMed Scopus (261) Google Scholar). We confirmed that overexpression of miR-301b and miR-130b reduced luciferase driven by the wild-type SFRS2 3′ UTR, whereas mutation of the miR-301 motif restored luciferase expression (Figure 3F). Similarly, miR-302 specifically targeted the 3′ UTR of MBD2a (Figure 3G) but not that of MBD2c (Figure 3H). Indeed, we confirmed that exogenous expression of miR-302 reduced levels of MBD2a in vivo (Figure 3I). These data suggest that the miR-301 and miR-302 families may independently regulate SFRS2 and MBD2 in order to fine-tune the expression of MBD2 isoforms. Next, we investigated the functional roles of MBD2 isoforms in hPSCs. Overexpression of MBD2a (Figures 4A and 4B ) disrupted pluripotency, as evidenced by cell morphology (Figure 4C) in addition to reduced expression of OCT4, NANOG, and SOX2 (Figure 4D). In contrast, increased MBD2c levels had no effect in hESC on the basis of these measures. However, the addition of the ESC-specific MBD2c isoform (Figure 4E) to a cocktail of reprogramming factors enhanced reprogramming efficiency in BJ DFs, whereas exogenous expression of MBD2a had no effect (Figures 4F and 4G). These data suggested that MBD2a and MBD2c play opposing roles in pluripotency. ChIP indicated that MBD2a and MBD2c were enriched at OCT4 and NANOG promoter regions in 293T cells as well as H1 ESCs (Figure 4H). Interestingly, co- and reverse immunoprecipitation followed by western blotting (Figures 4I and 4J) revealed that the somatic cell-specific MBD2a isoform exhibits much higher affinity for interaction with members of the transcriptionally repressive NuRD complex, including HDAC1, HDAC2, RbAp46, MTA2, and Mi-2 (Zhang et al., 1999Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (931) Google Scholar). The specificity of the MBD2a-NURD interaction was further confirmed by probing for SIN3A, a corepressor (Zhang et al., 2005Zhang Y. Fatima N. Dufau M.L. Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription.Mol. Cell. Biol. 2005; 25: 7929-7939Crossref PubMed Scopus (80) Google Scholar) independent of NuRD that did not biochemically interact with either MBD2 isoform (Figure 4I). PSCs are phenotypically well defined but exhibit significant molecular heterogeneity (Cahan and Daley, 2013Cahan P. Daley G.Q. Origins and implications of pluripotent stem cell variability and heterogeneity.Nat. Rev. Mol. Cell Biol. 2013; 14: 357-368Crossref PubMed Scopus (231) Google Scholar). These observations suggest that the core pluripotent factors OCT4, SOX2, and NANOG must balance a stochastic transcriptional ground state but yet respond rapidly to exogenous cues in order to properly orchestrate the cell lineages required for life, all from a relatively modest number of protein-coding genes (Wu et al., 2010Wu J.Q. Habegger L. Noisa P. Szekely A. Qiu C. Hutchison S. Raha D. Egholm M. Lin H. Weissman S. et al.Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing.Proc. Natl. Acad. Sci. USA. 2010; 107: 5254-5259Crossref PubMed Scopus (140) Google Scholar). Alternative splicing represents a likely pathway whereby the core pluripotency factors can dynamically regulate proteome diversity to support high-fidelity lineage commitment (Wang et al., 2008Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Crossref PubMed Scopus (3567) Google Scholar). Although several examples of alternatively spliced gene products have been functionally validated in pluripotent cells (Das et al., 2011Das S. Jena S. Levasseur D.N. Alternative splicing produces Nanog protein variants with different capacities for self-renewal and pluripotency in embryonic stem cells.J. Biol. Chem. 2011; 286: 42690-42703Crossref PubMed Scopus (55) Google Scholar, Gabut et al., 2011Gabut M. Samavarchi-Tehrani P. Wang X. Slobodeniuc V. O’Hanlon D. Sung H.-K. Alvarez M. Talukder S. Pan Q. Mazzoni E.O. et al.An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.Cell. 2011; 147: 132-146Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, Han et al., 2013Han H. Irimia M. Ross P.J. Sung H.-K. Alipanahi B. David L. Golipour A. Gabut M. Michael I.P. Nachman E.N. et al.MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.Nature. 2013; 498: 241-245Crossref PubMed Scopus (219) Google Scholar, Salomonis et al., 2010Salomonis N. Schlieve C.R. Pereira L. Wahlquist C. Colas A. Zambon A.C. Vranizan K. Spindler M.J. Pico A.R. Cline M.S. et al.Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.Proc. Natl. Acad. Sci. USA. 2010; 107: 10514-10519Crossref PubMed Scopus (169) Google Scholar), a general framework that mechanistically links OCT4, NANOG, or SOX2 with specific splicing factors, pre-mRNA substrates, and canonical regulators of gene transcription has yet to be described. We found that the splicing factor SFRS2 was strongly represented within the pluripotent molecular signature and, moreover, that OCT4 bound to SFRS2 promoters in vivo and drove luciferase expression in vitro. These data establish interdependent genetic and functional links between OCT4 and SFRS2 in hPSCs. We confirmed a cell-type-specific expression pattern for MBD2 isoforms, and found that SFRS2 biochemically targets the pre-mRNA of this methyl-DNA binding protein. We also observed a reciprocal link between OCT4 and MBD2a, manifested at the level of gene expression and pluripotent phenotype. Interestingly, hESCs displayed distinct morphologies in response to depletion of SRFR2 or overexpression of MBD2a, suggesting that the splicing factor most likely targets additional gene products; indeed, it is intriguing to speculate that the pool of pluripotent-specific, alternatively spliced transcripts in our exon-junction microarray data may be rich in previously unrecognized gene isoforms that support self-renewal. Similarly, use of next-generation DNA sequencing technologies may provide an exhaustive set of pluripotent-specific gene isoforms and splicing factor gene targets. Notwithstanding a comprehensive analysis of SFRS2 gene targets, our current results provide compelling mechanistic evidence that the functional role of OCT4 in pluripotent cells extends to the pathways that regulate gene splicing. Although the editing of pre-mRNA transcripts can be reconstituted in vitro, it has become clear that gene splicing in vivo is intimately linked to transcription, chromatin structure, and histone modifications (Braunschweig et al., 2013Braunschweig U. Gueroussov S. Plocik A.M. Graveley B.R. Blencowe B.J. Dynamic integration of splicing within gene regulatory pathways.Cell. 2013; 152: 1252-1269Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, Lin et al., 2008Lin S. Coutinho-Mansfield G. Wang D. Pandit S. Fu X.-D. The splicing factor SC35 has an active role in transcriptional elongation.Nat. Struct. Mol. Biol. 2008; 15: 819-826Crossref PubMed Scopus (269) Google Scholar). NuRD is a chromatin remodeling complex that is thought to promote lineage commitment of ESCs via silencing of pluripotency genes (Reynolds et al., 2012Reynolds N. Latos P. Hynes-Allen A. Loos R. Leaford D. O’Shaughnessy A. Mosaku O. Signolet J. Brennecke P. Kalkan T. et al.NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment.Cell Stem Cell. 2012; 10: 583-594Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Although previous work suggested that NuRD was recruited to methylated DNA by MBD2 (Zhang et al., 1999Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bir" @default.
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• W2110492802 date "2014-07-01" @default.
• W2110492802 modified "2023-10-17" @default.
• W2110492802 title "Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells" @default.
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