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• W2983176933 endingPage "1985.e6" @default.
• W2983176933 startingPage "1974" @default.
• W2983176933 abstract "•The altered 3D genome of 2i ESCs is due to polycomb re-distribution•Stopping DNA hypomethylation and genome re-organization does not affect 2i cell state•Hypomethylated mouse blastocysts have a similar 3D chromatin organization to 2i ESCs The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs. The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs. The extent to which epigenetic modifications and three-dimensional (3D) chromatin structure are linked and contribute to cell state and cell function is unresolved. Two key and inter-related epigenetic modifiers in the mammalian genome are DNA methylation and polycomb. Polycomb complexes are implicated in the maintenance of repression of key developmental genes (Blackledge et al., 2015Blackledge N.P. Rose N.R. Klose R.J. Targeting Polycomb systems to regulate gene expression: modifications to a complex story.Nat. Rev. Mol. Cell Biol. 2015; 16: 643-649Crossref PubMed Scopus (234) Google Scholar). Whereas polycomb repressive complex PRC2 deposits H3K27me3, the canonical PRC1 complex promotes compact local chromatin structures and longer-range chromatin interactions (Boettiger et al., 2016Boettiger A.N. Bintu B. Moffitt J.R. Wang S. Beliveau B.J. Fudenberg G. Imakaev M. Mirny L.A. Wu C.T. Zhuang X. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.Nature. 2016; 529: 418-422Crossref PubMed Scopus (499) Google Scholar, Eskeland et al., 2010Eskeland R. Leeb M. Grimes G.R. Kress C. Boyle S. Sproul D. Gilbert N. Fan Y. Skoultchi A.I. Wutz A. Bickmore W.A. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.Mol. Cell. 2010; 38: 452-464Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Joshi et al., 2015Joshi O. Wang S.Y. Kuznetsova T. Atlasi Y. Peng T. Fabre P.J. Habibi E. Shaik J. Saeed S. Handoko L. et al.Dynamic Reorganization of Extremely Long-Range Promoter-Promoter Interactions between Two States of Pluripotency.Cell Stem Cell. 2015; 17: 748-757Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Kundu et al., 2017Kundu S. Ji F. Sunwoo H. Jain G. Lee J.T. Sadreyev R.I. Dekker J. Kingston R.E. Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation.Mol. Cell. 2017; 65: 432-446.e5Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Schoenfelder et al., 2015Schoenfelder S. Sugar R. Dimond A. Javierre B.M. Armstrong H. Mifsud B. Dimitrova E. Matheson L. Tavares-Cadete F. Furlan-Magaril M. et al.Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.Nat. Genet. 2015; 47: 1179-1186Crossref PubMed Scopus (242) Google Scholar, Williamson et al., 2012Williamson I. Eskeland R. Lettice L.A. Hill A.E. Boyle S. Grimes G.R. Hill R.E. Bickmore W.A. Anterior-posterior differences in HoxD chromatin topology in limb development.Development. 2012; 139: 3157-3167Crossref PubMed Scopus (50) Google Scholar). Chromatin compaction and developmental gene repression are independent of the E3 ligase catalytic activity of Ring1B in canonical PRC1 (Cohen et al., 2018Cohen I. Zhao D. Bar C. Valdes V.J. Dauber-Decker K.L. Nguyen M.B. Nakayama M. Rendl M. Bickmore W.A. Koseki H. et al.PRC1 Fine-tunes Gene Repression and Activation to Safeguard Skin Development and Stem Cell Specification.Cell Stem Cell. 2018; 22: 726-739.e7Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, Eskeland et al., 2010Eskeland R. Leeb M. Grimes G.R. Kress C. Boyle S. Sproul D. Gilbert N. Fan Y. Skoultchi A.I. Wutz A. Bickmore W.A. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.Mol. Cell. 2010; 38: 452-464Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Illingworth et al., 2015Illingworth R.S. Moffat M. Mann A.R. Read D. Hunter C.J. Pradeepa M.M. Adams I.R. Bickmore W.A. The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.Genes Dev. 2015; 29: 1897-1902Crossref PubMed Scopus (111) Google Scholar, Kundu et al., 2017Kundu S. Ji F. Sunwoo H. Jain G. Lee J.T. Sadreyev R.I. Dekker J. Kingston R.E. Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation.Mol. Cell. 2017; 65: 432-446.e5Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Williamson et al., 2014Williamson I. Berlivet S. Eskeland R. Boyle S. Illingworth R.S. Paquette D. Dostie J. Bickmore W.A. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.Genes Dev. 2014; 28: 2778-2791Crossref PubMed Scopus (178) Google Scholar). In mammalian cells, the polycomb system is primarily targeted to the unmethylated CpG islands (CGIs) of non- or weakly expressed genes (Blackledge et al., 2015Blackledge N.P. Rose N.R. Klose R.J. Targeting Polycomb systems to regulate gene expression: modifications to a complex story.Nat. Rev. Mol. Cell Biol. 2015; 16: 643-649Crossref PubMed Scopus (234) Google Scholar, Li et al., 2017Li H. Liefke R. Jiang J. Kurland J.V. Tian W. Deng P. Zhang W. He Q. Patel D.J. Bulyk M.L. et al.Polycomb-like proteins link the PRC2 complex to CpG islands.Nature. 2017; 549: 287-291Crossref PubMed Scopus (169) Google Scholar, Riising et al., 2014Riising E.M. Comet I. Leblanc B. Wu X. Johansen J.V. Helin K. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide.Mol. Cell. 2014; 55: 347-360Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Consistent with this, loss of DNA methylation—by exposing new CpG sites—leads to a redistribution of H3K27me3, to satellite and dispersed repeat sequences, while titrating it away from its normal CGI targets (Brinkman et al., 2012Brinkman A.B. Gu H. Bartels S.J. Zhang Y. Matarese F. Simmer F. Marks H. Bock C. Gnirke A. Meissner A. Stunnenberg H.G. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.Genome Res. 2012; 22: 1128-1138Crossref PubMed Scopus (290) Google Scholar, Jermann et al., 2014Jermann P. Hoerner L. Burger L. Schübeler D. Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation.Proc. Natl. Acad. Sci. USA. 2014; 111: E3415-E3421Crossref PubMed Scopus (103) Google Scholar, Reddington et al., 2013Reddington J.P. Perricone S.M. Nestor C.E. Reichmann J. Youngson N.A. Suzuki M. Reinhardt D. Dunican D.S. Prendergast J.G. Mjoseng H. et al.Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes.Genome Biol. 2013; 14: R25Crossref PubMed Scopus (158) Google Scholar, Reddington et al., 2014Reddington J.P. Sproul D. Meehan R.R. DNA methylation reprogramming in cancer: does it act by re-configuring the binding landscape of Polycomb repressive complexes?.BioEssays. 2014; 36: 134-140Crossref PubMed Scopus (30) Google Scholar). This is consistent with a model in which PRC2 can associate transiently and weakly with a large fraction of the genome (Schuettengruber et al., 2017Schuettengruber B. Bourbon H.M. Di Croce L. Cavalli G. Genome Regulation by Polycomb and Trithorax: 70 Years and Counting.Cell. 2017; 171: 34-57Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). One notable instance in which this occurs is in mouse embryonic stem cells (mESCs) cultured with two small molecule inhibitors of MEK1 and glycogen synthase kinase 3 (GSK3); 2i conditions (Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). mESCs cultured conventionally in the presence of fetal calf serum and LIF (leukemia inhibitory factor) are functionally heterogeneous, with a fraction of cells resembling a state of “naive pluripotency” with unbiased developmental potential and high expression of pluripotency genes. Other cells in the culture more closely resemble a “primed” state, in which they begin expressing early lineage markers and downregulate pluripotency genes (Canham et al., 2010Canham M.A. Sharov A.A. Ko M.S. Brickman J.M. Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript.PLoS Biol. 2010; 8: e1000379Crossref PubMed Scopus (188) Google Scholar, Hackett and Surani, 2014Hackett J.A. Surani M.A. Regulatory principles of pluripotency: from the ground state up.Cell Stem Cell. 2014; 15: 416-430Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, Hayashi et al., 2008Hayashi K. de Sousa Lopes S.M.C. Tang F. Lao K. Surani M.A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states.Cell Stem Cell. 2008; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, Wongtawan et al., 2011Wongtawan T. Taylor J.E. Lawson K.A. Wilmut I. Pennings S. Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells.J. Cell Sci. 2011; 124: 1878-1890Crossref PubMed Scopus (67) Google Scholar). These two states are metastable, with cells in the population fluctuating between the two. By contrast, culturing mESCs serum free, in the presence of 2i blocks differentiation signals and promotes the pluripotency network, resulting in homogeneous expression of pluripotency factors and reduced expression of early lineage-specific genes (Morgani et al., 2013Morgani S.M. Canham M.A. Nichols J. Sharov A.A. Migueles R.P. Ko M.S. Brickman J.M. Totipotent embryonic stem cells arise in ground-state culture conditions.Cell Rep. 2013; 3: 1945-1957Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, Wray et al., 2011Wray J. Kalkan T. Gomez-Lopez S. Eckardt D. Cook A. Kemler R. Smith A. Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation.Nat. Cell Biol. 2011; 13: 838-845Crossref PubMed Scopus (388) Google Scholar, Ying and Smith, 2017Ying Q.L. Smith A. The Art of Capturing Pluripotency: Creating the Right Culture.Stem Cell Reports. 2017; 8: 1457-1464Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The epigenetic properties of 2i-cultured mESCs closely resemble those of cells in the pre-implantation inner cell mass (ICM) of the mouse embryo. This includes global DNA hypomethylation (Ficz et al., 2013Ficz G. Hore T.A. Santos F. Lee H.J. Dean W. Arand J. Krueger F. Oxley D. Paul Y.L. Walter J. et al.FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.Cell Stem Cell. 2013; 13: 351-359Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Naive pluripotency is associated with global DNA hypomethylation.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (358) Google Scholar, Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, Wray et al., 2011Wray J. Kalkan T. Gomez-Lopez S. Eckardt D. Cook A. Kemler R. Smith A. Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation.Nat. Cell Biol. 2011; 13: 838-845Crossref PubMed Scopus (388) Google Scholar). Expression levels of the de novo methyltransferases Dnmt3a, Dnmt3b, and the non-catalytic cofactor Dnmt3l are reduced under 2i conditions. Uhrf1 (a Dnmt1 co-factor) is also downregulated at the protein level (Ficz et al., 2013Ficz G. Hore T.A. Santos F. Lee H.J. Dean W. Arand J. Krueger F. Oxley D. Paul Y.L. Walter J. et al.FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.Cell Stem Cell. 2013; 13: 351-359Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, Grabole et al., 2013Grabole N. Tischler J. Hackett J.A. Kim S. Tang F. Leitch H.G. Magnúsdóttir E. Surani M.A. Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation.EMBO Rep. 2013; 14: 629-637Crossref PubMed Scopus (120) Google Scholar, Graf et al., 2017Graf U. Casanova E.A. Wyck S. Dalcher D. Gatti M. Vollenweider E. Okoniewski M.J. Weber F.A. Patel S.S. Schmid M.W. et al.Pramel7 mediates ground-state pluripotency through proteasomal-epigenetic combined pathways.Nat. Cell Biol. 2017; 19: 763-773Crossref PubMed Scopus (26) Google Scholar, Habibi et al., 2013Habibi E. Brinkman A.B. Arand J. Kroeze L.I. Kerstens H.H. Matarese F. Lepikhov K. Gut M. Brun-Heath I. Hubner N.C. et al.Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells.Cell Stem Cell. 2013; 13: 360-369Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Naive pluripotency is associated with global DNA hypomethylation.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (358) Google Scholar, von Meyenn et al., 2016von Meyenn F. Iurlaro M. Habibi E. Liu N.Q. Salehzadeh-Yazdi A. Santos F. Petrini E. Milagre I. Yu M. Xie Z. et al.Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.Mol. Cell. 2016; 62: 848-861Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Yamaji et al., 2013Yamaji M. Ueda J. Hayashi K. Ohta H. Yabuta Y. Kurimoto K. Nakato R. Yamada Y. Shirahige K. Saitou M. PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells.Cell Stem Cell. 2013; 12: 368-382Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). However, coupling these DNA methylation differences to gene expression changes using triple-knockout (TKO) cells that lack all the active Dnmts reveals that only a small (but significant) proportion of gene expression changes under 2i can be directly attributed to DNA methylation loss (Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Naive pluripotency is associated with global DNA hypomethylation.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (358) Google Scholar). Importantly, although global levels of H3K27me3 are not altered in 2i-cultured cells, there is a marked reduction (up to 75%) of H3K27me3 at polycomb targets, including at the Hox clusters (Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). This is accompanied by reduced occupancy of Suz12 and Ezh2 (PRC2) and Ring1B (PRC1) (Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, Joshi et al., 2015Joshi O. Wang S.Y. Kuznetsova T. Atlasi Y. Peng T. Fabre P.J. Habibi E. Shaik J. Saeed S. Handoko L. et al.Dynamic Reorganization of Extremely Long-Range Promoter-Promoter Interactions between Two States of Pluripotency.Cell Stem Cell. 2015; 17: 748-757Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The consequences of such a dramatically altered epigenome on 3D genome organization have not been explored. Given the epigenetic alterations that occur in 2i, and the role of polycomb in shaping the 3D genome, we sought to investigate whether 2i culturing conditions impact on 3D chromatin organization in mESCs. Using fluorescence in situ hybridization (FISH) and Hi-C, we show that both local chromatin compaction at polycomb-target Hox loci and long-range polycomb interactions are profoundly altered in 2i, and we demonstrate that this is directly attributable to the loss of DNA methylation. By restoring the epigenetic landscape (DNA methylation and H3K27me3 targeting) of cells in 2i, we show that 3D genome organization can be reset to resemble that of mESCs grown in serum. Strikingly, this has a limited impact on gene expression. mESCs cultured in a chemically defined medium in the presence of LIF and two inhibitors (2i) of the Erk and Gsk-3 signaling pathways achieve a homogeneous ground state of pluripotency, thought to closely resemble that of the ICM (Ying and Smith, 2017Ying Q.L. Smith A. The Art of Capturing Pluripotency: Creating the Right Culture.Stem Cell Reports. 2017; 8: 1457-1464Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523Crossref PubMed Scopus (2534) Google Scholar). In doing so, 2i mESCs acquire a distinct epigenomic landscape, including global DNA hypomethylation and an altered genomic distribution of H3K27me3 (Ficz et al., 2013Ficz G. Hore T.A. Santos F. Lee H.J. Dean W. Arand J. Krueger F. Oxley D. Paul Y.L. Walter J. et al.FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.Cell Stem Cell. 2013; 13: 351-359Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, Habibi et al., 2013Habibi E. Brinkman A.B. Arand J. Kroeze L.I. Kerstens H.H. Matarese F. Lepikhov K. Gut M. Brun-Heath I. Hubner N.C. et al.Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells.Cell Stem Cell. 2013; 13: 360-369Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Naive pluripotency is associated with global DNA hypomethylation.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (358) Google Scholar, Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. The transcriptional and epigenomic foundations of ground state pluripotency.Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar) This includes a loss of H3K27me3 enrichment at classic polycomb targets such as Hox loci (Figure 1A). Since polycomb is a powerful mediator of higher-order chromatin structure (Boettiger et al., 2016Boettiger A.N. Bintu B. Moffitt J.R. Wang S. Beliveau B.J. Fudenberg G. Imakaev M. Mirny L.A. Wu C.T. Zhuang X. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states.Nature. 2016; 529: 418-422Crossref PubMed Scopus (499) Google Scholar, Eskeland et al., 2010Eskeland R. Leeb M. Grimes G.R. Kress C. Boyle S. Sproul D. Gilbert N. Fan Y. Skoultchi A.I. Wutz A. Bickmore W.A. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.Mol. Cell. 2010; 38: 452-464Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Francis et al., 2004Francis N.J. Kingston R.E. Woodcock C.L. Chromatin compaction by a polycomb group protein complex.Science. 2004; 306: 1574-1577Crossref PubMed Scopus (627) Google Scholar, Joshi et al., 2015Joshi O. Wang S.Y. Kuznetsova T. Atlasi Y. Peng T. Fabre P.J. Habibi E. Shaik J. Saeed S. Handoko L. et al.Dynamic Reorganization of Extremely Long-Range Promoter-Promoter Interactions between Two States of Pluripotency.Cell Stem Cell. 2015; 17: 748-757Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Kundu et al., 2017Kundu S. Ji F. Sunwoo H. Jain G. Lee J.T. Sadreyev R.I. Dekker J. Kingston R.E. Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation.Mol. Cell. 2017; 65: 432-446.e5Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Schoenfelder et al., 2015Schoenfelder S. Sugar R. Dimond A. Javierre B.M. Armstrong H. Mifsud B. Dimitrova E. Matheson L. Tavares-Cadete F. Furlan-Magaril M. et al.Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.Nat. Genet. 2015; 47: 1179-1186Crossref PubMed Scopus (242) Google Scholar, Williamson et al., 2012Williamson I. Eskeland R. Lettice L.A. Hill A.E. Boyle S. Grimes G.R. Hill R.E. Bickmore W.A. Anterior-posterior differences in HoxD chromatin topology in limb development.Development. 2012; 139: 3157-3167Crossref PubMed Scopus (50) Google Scholar), it is possible that the redistribution of H3K27me3/polycomb across the genome results in an alteration to 3D chromatin organization in ESCs grown in 2i culture conditions, but this has not been investigated. The murine HoxD locus is a large canonical polycomb target in mESCs, demarked by a domain of H3K27me3, PRC2, and PRC1 deposition across the 100-kb cluster (Illingworth et al., 2012Illingworth R.S. Botting C.H. Grimes G.R. Bickmore W.A. Eskeland R. PRC1 and PRC2 are not required for targeting of H2A.Z to developmental genes in embryonic stem cells.PLoS ONE. 2012; 7: e34848Crossref PubMed Scopus (35) Google Scholar). Under serum/LIF culture conditions the HoxD locus is maintained in a compact chromatin conformation in mESCs, and this is dependent on the presence of PRC1 (Eskeland et al., 2010Eskeland R. Leeb M. Grimes G.R. Kress C. Boyle S. Sproul D. Gilbert N. Fan Y. Skoultchi A.I. Wutz A. Bickmore W.A. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.Mol. Cell. 2010; 38: 452-464Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Williamson et al., 2014Williamson I. Berlivet S. Eskeland R. Boyle S. Illingworth R.S. Paquette D. Dostie J. Bickmore W.A. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.Genes Dev. 2014; 28: 2778-2791Crossref PubMed Scopus (178) Google Scholar). To investigate higher-order chromatin compaction at HoxD in mESCs grown under serum and 2i conditions, we used 3D FISH to measure the separation of hybridization signals from probe pairs at opposite ends of the HoxD locus (Hoxd3 and Hoxd13) under the different conditions. We compared these measurements to those from control probes at a nearby genomic region (3′ of Lnp) that is not coated by H3K27me3 but that is highly DNA methylated in serum-grown ESCs (Figure 1A). Under 2i/LIF culture conditions, the HoxD locus significantly decompacts relative to cells cultured in serum/LIF; median inter-probe distances increase from ∼300 to ∼400 nm, p = < 0.0001 (Figures 1B, 1C, and S1A; Tables S1 and S2). This decompaction occurs to the same extent when either PRC1 (Ring1B−/−) or PRC2 (Eed−/−) is absent in mESCs grown under serum conditions (Figures 1B, 1C, and S1A). No further decompaction is observed when PRC1 or PRC2 mutant mESCs are grown under 2i conditions, showing that decompaction of a polycomb target in 2i can be primarily accounted for by the titration of H3K37me3/polycomb away from these genomic regions. We confirmed these data for two other Hox clusters: HoxB (Figures S1B, S1D, and S1F) and HoxC (Figures S1C, S1E, and S1G). As a control, we examined a locus not marked by H3K27me3, and highly DNA methylated, in serum-grown ESCs, that is adjacent to HoxD (Figure 1A). Inter-probe distances at this control locus were not significantly different between wild-type (WT) or polycomb mutant mESCs, or between mESCs grown in the different culture conditions (Figures 1C and S1A), even though this region is subject to DNA hypomethylation in 2i (Figure 1A). This suggests that the chromatin decompaction we detect in 2i conditions at polycomb target loci is not a result of a general/global alteration in the 3D chromatin organization of naive 2i/LIF cells, and that global loss of DNA methylation across genomic regions may have no direct effect on chromatin compaction, as assayed at a cytological level. Next, we investigated whether the chromatin decompaction observed in 2i-cultured mESCs is also present in the cells of the mouse blastocyst, which are hypomethylated during normal development (Messerschmidt et al., 2014Messerschmidt D.M. Knowles B.B. Solter D. DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.Genes Dev. 2014; 28: 812-828Crossref PubMed Scopus (461) Google Scholar). To compare chromatin states between in vitro mESCs and their in vivo counterparts, we measured distances between HoxD probes in embryonic day (E) 3.5 mouse blastocysts using 3D FISH (Figure 2A). These data indicate that the HoxD locus in the pre-implantation blastocyst is decompact relative to that in conventionally cultured serum/LIF mESCs, and closely resembles the compaction state of the locus under 2i/LIF conditions (Figures 2B and 2C; Tables S1 and S2). There is a large amount of variability between and within blastocysts, which is likely because these blastocysts will contain distinct cell lineages (trophectoderm, ICM, and primitive endoderm), all of which are hypomethylated (Rossant et al., 1986Rossant J. Sanford J.P. Chapman V.M. Andrews G.K. Undermethylation of structural gene sequences in extraembryonic lineages of the mouse.Dev. Biol. 1986; 117: 567-573Crossref PubMed Scopus (77) Google Scholar). In contrast, inter-probe distances at the control locus were much more similar between blastocysts and cultured cells (Figure 2B), suggesting the decompaction at HoxD in the blastocyst cannot be explained by the in vivo population having a generally more open chromatin structure. Polycomb is responsible for forming self-interacting topologically associated domains (TADs) at Hox loci as detected by chromosome conformation capture methods (Kundu et al., 2017Kundu S. Ji F. Sunwoo H. Jain G. Lee J.T. Sadreyev R.I. Dekker J. Kingston R.E. Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation.Mol. Cell. 2017; 65: 432-446.e5Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Noordermeer et al., 2011Noordermeer D. Leleu M. Splinter E. Rougemont J. De Laat W. Duboule D. The dynamic architecture of Hox gene clusters.Science. 2011; 334: 222-225Crossref PubMed Scopus (299) Google Scholar, Williamson et al., 2014Williamson I. Berlivet S. Eskeland R. Boyle S. Illingworth R.S. Paquette D. Dostie J. Bickmore W.A. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.Genes Dev. 2014; 28: 2778-2791Crossref PubMed Scopus (178) Google Scholar). To assess whether changes in 3D chromatin organization occur in 2i cells at regions other than Hox loci, we employed in situ Hi-C (Lieberman-Aiden et al., 2009Lieberman-Aiden E. van Berkum N.L. Williams L. Imakaev M. Ragoczy T. Telling A. Amit I. Lajoie B.R. Sabo P.J. Dorschner M.O. et al.Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (4868) Google Scholar, Rao et al., 2014Rao S.S. Huntley M.H. Durand N.C. Stamenova E.K. Boch" @default.
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• W2983176933 title "DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency" @default.
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