YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

Abstract

The Yes-associated protein (YAP), one of the major effectors of the Hippo pathway together with its related protein WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ), mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) regulate a large number of target genes, acting as coactivators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis, we identified two molecules that could have a role in the altered genome-wide methylation profile: the long noncoding RNA ephemeron, whose rapid upregulation is crucial for the transition of ESCs into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency. The Yes-associated protein (YAP), one of the major effectors of the Hippo pathway together with its related protein WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ), mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) regulate a large number of target genes, acting as coactivators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis, we identified two molecules that could have a role in the altered genome-wide methylation profile: the long noncoding RNA ephemeron, whose rapid upregulation is crucial for the transition of ESCs into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency. One of the molecular machineries that play crucial roles during embryo development is that involving the Yes-associated protein (YAP) and the related protein WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ) (1Pocaterra A. Romani P. Dupont S. YAP/TAZ functions and their regulation at a glance.J. Cell. Sci. 2020; 133: jcs230425Crossref PubMed Scopus (49) Google Scholar, 2Moya I.M. Halder G. Hippo-YAP/TAZ signalling in organ regeneration and regenerative medicine.Nat. Rev. Mol. Cell. Biol. 2019; 20: 211-226Crossref PubMed Scopus (163) Google Scholar). These two proteins play a fundamental role in the so-called Hippo pathway, as they, through a cytosol–nucleus shuttling regulated by nucleus-excluding phosphorylation, govern the transcription of various genes involved in sensing mechanical stress (3Dupont S. Morsut L. Aragona M. Enzo E. Giulitti S. Cordenonsi M. Zanconato F. Le Digabel J. Forcato M. Bicciato S. Elvassore N. Piccolo S. Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (2573) Google Scholar), cell proliferation and apoptosis (4Ma S. Meng Z. Chen R. Guan K.L. The Hippo pathway: biology and pathophysiology.Annu. Rev. Biochem. 2019; 88: 577-604Crossref PubMed Scopus (148) Google Scholar, 5Hashimoto M. Sasaki H. Epiblast formation by TEAD-YAP-dependent expression of pluripotency factors and competitive elimination of unspecified cells.Dev. Cell. 2019; 50: 139-154.e5Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and organ size (6Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease.Development. 2014; 141: 1614-1626Crossref PubMed Scopus (343) Google Scholar). YAP and TAZ function as coactivators of the Transcriptional Enhanced Associate Domains (TEADs) (1Pocaterra A. Romani P. Dupont S. YAP/TAZ functions and their regulation at a glance.J. Cell. Sci. 2020; 133: jcs230425Crossref PubMed Scopus (49) Google Scholar), but the multitasking ability of YAP/TAZ is demonstrated by many results indicating TEAD-independent functions even outside the nucleus (7Azzolin L. Panciera T. Soligo S. Enzo E. Bicciato S. Dupont S. Bresolin S. Frasson C. Basso G. Guzzardo V. Fassina A. Cordenonsi M. Piccolo S. YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response.Cell. 2014; 158: 157-170Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). The critical role of these two proteins during the very early steps of development is recapitulated by the phenotype of YAP/TAZ double knockout (KO): these embryos are arrested in the premorula stage (8Sasaki H. Roles and regulations of Hippo signaling during preimplantation mouse development.Dev. Growth Differ. 2017; 59: 12-20Crossref PubMed Scopus (52) Google Scholar), likely because of the induced repression of Sox2 preventing the appearance of the inner cell mass phenotype. At the morula stage, YAP is responsible for the activation of trophoblast master genes, like Cdx2, in the cells of the external layer, thus governing the acquisition of the trophoblast cell identity (9Nishioka N. Inoue K. Adachi K. Kiyonari H. Ota M. Ralston A. Yabuta N. Hirahara S. Stephenson R.O. Ogonuki N. Makita R. Kurihara H. Morin-Kensicki E.M. Nojima H. Rossant J. et al.The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.Dev. Cell. 2009; 16: 398-410Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Besides, YAP is expressed in the in in embryonic stem cells at the function of YAP/TAZ in results that the of YAP or in the of of and the downregulation of and with expression of like and J. H. J. J. A. R. S. Guan K.L. The role of YAP transcription in stem cell and PubMed Scopus Google Scholar). of a protein to the of ESC the C. N. C. of mouse embryonic stem cell by a signaling pathway of Cell. Sci. 2011; PubMed Scopus Google Scholar). that activity pluripotency in the inner cell cells with are M. Sasaki H. Epiblast formation by TEAD-YAP-dependent expression of pluripotency factors and competitive elimination of unspecified cells.Dev. Cell. 2019; 50: 139-154.e5Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). results that YAP/TAZ the stemness of ESCs in and in these YAP/TAZ downregulation that the β-catenin pathway (7Azzolin L. Panciera T. Soligo S. Enzo E. Bicciato S. Dupont S. Bresolin S. Frasson C. Basso G. Guzzardo V. Fassina A. Cordenonsi M. Piccolo S. YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response.Cell. 2014; 158: 157-170Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). with these the or the of YAP the of the H. N. J. J. is for but for of mouse embryonic stem PubMed Scopus Google Scholar). in YAP knockdown results in of like and although and are H. N. J. J. is for but for of mouse embryonic stem PubMed Scopus Google Scholar). These results could at in the of YAP/TAZ in the various steps of ESC differentiation, whose in could to this we the of a very transient downregulation of YAP the of mouse although the YAP are during the YAP cells show a methylation from that of the control at the expression of YAP we downregulation of and the long noncoding These that YAP expression in ESCs is to the responsible for the remodeling of the methylation place at the exit of ESCs from the to the of a transient YAP in this control of the of the gene S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar), with a of a of YAP expression YAP cells induced to differentiate the S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar), and expression at various during the in the number of cells in YAP the very of with to control with negative control at the in YAP with a number of cells The expression of genes also that the transient of YAP The expression of YAP steps the of YAP as its and protein at at of and the of YAP the of the suggesting that YAP could play a role in ESCs or in the early steps of their and that this activity is for the At this we the ability of YAP cells to differentiate the to whether the transient downregulation of YAP expression in ESCs with the pathway of a of the of this we with YAP the in the expression of is control of the of gene G. C. S. G. F. P. F. F. L. formation development from mouse embryonic stem 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). YAP cells induced to differentiate into by formation in A. A. A. G. A. T. S. is for exit of embryonic stem cells from the Biol. PubMed Scopus Google Scholar). the of differentiation, the number of cells in YAP cells although from cells the in YAP cells These results suggest that the transient downregulation of YAP expression the of ESCs to properly. The inability of ESCs to could be the one to of pluripotency the to the of stemness and the of differentiation. whether YAP plays some role in the of the of we YAP expression in cells by or cells in the of for a and RNA to the expression of stemness The results in that the expression of the stemness by the transient of These cells also at and for in the of and to the formation of in and YAP cells to with a a in the number of from YAP that of cell a of cell death YAP but to the that the observed number of and cells because of cell YAP cell during the early steps of differentiation, YAP cells induced to differentiate by the formation of A. A. A. G. A. T. S. is for exit of embryonic stem cells from the Biol. PubMed Scopus Google Scholar). number of cells in YAP with to responsible for the in the size of as by the of with YAP these results suggest that transient YAP in ESCs a of cellular homeostasis leading to a of The in the in the number of cells are to or differentiation. cells to be to differentiate suggesting that the transient downregulation of YAP the of of The induced by the transient of YAP are likely because of that to their also of YAP expression are One is that transient YAP could be responsible for in genome-wide de novo is known to place the of ESC L. M. P. R. H. epigenetic de novo DNA methylation at a of pluripotency gene during embryonic stem cell PubMed Scopus Google Scholar, G. S. A. M. of methylation and of during embryonic development in the Biol. 2014; PubMed Scopus Google Scholar). this we by sequencing the methylation pattern of and YAP cell in ESCs cells as of we the in genomic DNA methylation in cells of differentiation, with cells at with a we the in YAP cell at with to the number of in the two the transient of YAP a in of the observed in the of cells to a of with of methylation in the observed in YAP the of the observed in YAP cells of methylation in with some as in the of and the and YAP cells and in the with of methylation in YAP cells and in cells and of methylation in cells and in YAP cells with a of at are the of methylation in YAP cells and in cells and the is a of methylation in cells and in YAP the of genes to the and by the protein through for gene H. A. X. C. X. for and gene function with the 2019; PubMed Scopus Google Scholar). we observed very of the Wnt signaling pathway, and the related pathway, for genes the of methylation in YAP cells These data demonstrated that the transient downregulation of YAP the of de novo methylation in the very early steps of the of might to the of governance of the epigenetic dynamics at the exit from pluripotency. that the phenotype we observed is caused by a transient downregulation of we to at the expression of ESCs the with YAP YAP the RNA from for YAP and and by in the expression of genes in YAP cells with of in downregulation we also the gene expression of ESCs with a or with the this or as a of YAP expression and The the and data that genes to with in YAP cells and in YAP and the through of the genes in YAP cells revealed a in related to development and the genes YAP and in YAP we two genes that could have a role in the altered genome-wide methylation we observed in YAP The one is that the dynamics of exit from pluripotency P. 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T. et the dynamics of a cell transition involving and de novo DNA 2017; PubMed Scopus Google Scholar). expression of YAP cells a but of in in ESCs also observed although induced in cells this in YAP cells the expression of and altered in YAP with downregulation of at and at we observed also for Dnmt3a and Dnmt3b in YAP cells cells data and show in the expression of Dnmt3a or we that their Dnmt3l, YAP and in YAP a protein although cooperates during the embryo development with Dnmt3a and to the de novo DNA methylation that governs the early steps of ESC J. S. H. Sasaki H. T. M. and dynamics of DNA methylation during early mouse PubMed Scopus Google Scholar). is also expressed in ESCs N. S. S. K. J. H. H. Chen T. DNA methylation by in mouse embryonic stem 2019; PubMed Scopus Google Scholar), it the methylation of gene by interacting with the complex F. A. M. C. F. F. S. 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Transcriptional of and mechanical by and 2017; PubMed Scopus Google thus we to whether transcription factors could with YAP in the to the gene by of either or in we the of YAP to the in the of the the of DNA in with to revealed that this The YAP and also for a of from to the of and to the phenotype induced by YAP cells with a of Dnmt3l, or expression for target gene cells induced to differentiate the in and transient to a phenotype to that induced by YAP number of cells at stage of differentiation. the expression of genes also that the downregulation of or expression the that the expression of a YAP target could be to the YAP we the of mouse gene into the S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google in to the in YAP cells cells in to the of in or YAP By the of cells at stage of differentiation, we that of functions by YAP could be by the expression of observed in the expression of genes in YAP with or Here we show results indicating that YAP is in ESCs to to the epigenetic that are for the exit from the we observed that transient downregulation of YAP the in the DNA methylation pattern and mouse phenotype is by a in the of methylation is and a in the of de novo methylation we observed that are many the exit from pluripotency DNA methylation in YAP and the are a of methylation observed in These the that transient of YAP in ESCs to a dysregulation of DNA methylation in of a in de novo methylation and of These to be because of the very of or of de novo methylation at is by the of genes involved in the Wnt signaling pathway with the is that we observed that of genes whose expression is as a of transient YAP are also of is of the of embryonic stem PubMed Scopus Google Scholar). 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P. in and of de novo methylation in the mouse PubMed Scopus Google Scholar). results that downregulation induced a phenotype to that observed in YAP its in YAP cells to the thus indicating that are genes, whose expression is by YAP transient downregulation that contribute to the of the observed phenotype. gene whose altered in YAP cells is that for a observed that is the exit of ESCs from pluripotency and its by either gene or by a downregulation of genes P. M. T. M. S. F. M. F. Chen C. J. T. et the dynamics of a cell transition involving and de novo DNA 2017; PubMed Scopus Google Scholar). One of the in cells is methylation at the gene the methylation observed in gene cells P. M. T. M. S. F. M. F. Chen C. J. T. et the dynamics of a cell transition involving and de novo DNA 2017; PubMed Scopus Google Scholar). the of it is that in YAP the of and the of any ESC have a negative the de novo of but their functions are in still have roles in ESC M. J. M. G. G. R. L. X. A. A. et in the pluripotency and 2011; PubMed Scopus Google Scholar), and many of have a role in the regulation of the Hippo pathway C. K. L. J. L. Z. The and the Hippo signalling pathway in 2020; PubMed Scopus Google Scholar). is known the regulation of gene expression by the Hippo we demonstrated that the transcription of gene is the control of to a is the of the gene and that this is the mechanisms through YAP downregulation a of could be The of YAP in ESCs a number of likely because of the of ESCs YAP is expressed in the we genes of YAP as a show any of of YAP in the of it is that its downregulation in YAP acting either at at protein V. target in Scopus Google any in the of Dnmt3l, but of this the that a mouse ESCs and in the ESC with 1 from and and of the and cell have S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar, G. C. S. G. F. P. F. F. L. formation development from mouse embryonic stem 2011; Full Text Full Text PDF PubMed Scopus Google Scholar). The by the of mouse YAP into the S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar). The by the of mouse into the S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar). of and the mouse as for YAP for as and for and for and for as as and for for as and and for negative ESCs at in and for and in for The by for at with the differentiation, ESCs induced to differentiate either in S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar), by ESCs in cell cell or by formation of A. A. A. G. A. T. S. is for exit of embryonic stem cells from the Biol. PubMed Scopus Google Scholar), by 1 ESCs in in the with 1 and at of ESC ESC into induced by formation of and A. A. A. G. A. T. S. is for exit of embryonic stem cells from the Biol. PubMed Scopus Google Scholar). M. M. M. E. L. R. F. F. of gene expression data of 2014; PubMed Scopus Google Scholar). RNA by and DNA to the with the The as for for are in data are as to the expression a and ESCs in and and by The mouse and mouse with the from of DNA by incorporation to cell death in cells by ESCs and in and in a and at for in the cell with a from the by the the DNA of the a and the of the in the analysis, ESCs and with and S. F. L. R. L. T. is involved in of mouse embryonic stem Cell. Sci. PubMed Scopus Google Scholar). with and as A. A. A. G. A. T. S. is for exit of embryonic stem cells from the Biol. PubMed Scopus Google Scholar). The and or and the with a with the the and to in from for YAP YAP and by and to sequencing with have A. F. J. C. S. P. M. PubMed Scopus Google the The from the of M. C. M. H. 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