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

The Yes-associated protein (YAP), one of the major effectors Hippo pathway together with its related WW-domain-containing transcription regulator 1 (WWTR1; also known as TAZ), mediates a range cellular processes from proliferation and death to morphogenesis. YAP TAZ) regulate large number target genes, acting coactivators DNA-binding factors or negative regulators by interacting nucleosome remodeling histone deacetylase complexes. is expressed in self-renewing embryonic stem cells (ESCs), although it still debated whether plays any crucial roles control either stemness differentiation. Here we show that transient downregulation mouse ESCs perturbs homeostasis, leading inability differentiate properly. Bisulfite genomic sequencing revealed this knockdown caused genome-wide alteration DNA methylation takes place during early steps differentiation, suggesting phenotype observed might be due dysregulation some mechanisms involved regulation ESC exit pluripotency. By gene expression analysis, identified two molecules could have role altered profile: long noncoding RNA ephemeron, whose rapid upregulation for transition into epiblast, methyltransferase-like Dnmt3l, which, embryo development, cooperates Dnmt3a Dnmt3b contribute de novo governs These data suggest new governance epigenetic dynamics One molecular machineries play development involving (YAP) (1Pocaterra A. Romani P. Dupont S. YAP/TAZ functions their at glance.J. Cell. Sci. 2020; 133: jcs230425Crossref PubMed Scopus (49) Google Scholar, 2Moya I.M. Halder G. Hippo-YAP/TAZ signalling organ regeneration regenerative medicine.Nat. Rev. Mol. Biol. 2019; 20: 211-226Crossref (163) Scholar). proteins fundamental so-called pathway, they, through cytosol–nucleus shuttling regulated nucleus-excluding phosphorylation, govern various genes sensing mechanical stress (3Dupont Morsut L. Aragona M. Enzo E. Giulitti Cordenonsi Zanconato F. Le Digabel J. Forcato Bicciato Elvassore N. Piccolo Role mechanotransduction.Nature. 2011; 474: 179-183Crossref (2573) Scholar), cell apoptosis (4Ma Meng Z. Chen R. Guan K.L. pathway: biology pathophysiology.Annu. Biochem. 88: 577-604Crossref (148) 5Hashimoto Sasaki H. Epiblast formation TEAD-YAP-dependent pluripotency competitive elimination unspecified cells.Dev. 50: 139-154.e5Abstract Full Text PDF (39) size (6Varelas X. TAZ homeostasis disease.Development. 2014; 141: 1614-1626Crossref (343) function Transcriptional Enhanced Associate Domains (TEADs) but multitasking ability demonstrated many results indicating TEAD-independent even outside nucleus (7Azzolin Panciera T. Soligo Bresolin Frasson C. Basso Guzzardo V. Fassina incorporation ?-catenin destruction complex orchestrates Wnt response.Cell. 158: 157-170Abstract (576) critical these very recapitulated double knockout (KO): embryos are arrested premorula stage (8Sasaki Roles regulations signaling preimplantation development.Dev. Growth Differ. 2017; 59: 12-20Crossref (52) likely because induced repression Sox2 preventing appearance inner mass phenotype. At morula stage, responsible activation trophoblast master like Cdx2, external layer, thus governing acquisition identity (9Nishioka Inoue K. Adachi Kiyonari Ota Ralston Yabuta Hirahara Stephenson R.O. Ogonuki Makita Kurihara Morin-Kensicki E.M. Nojima Rossant et al.The components Lats Yap pattern Tead4 activity distinguish trophectoderm mass.Dev. 2009; 16: 398-410Abstract (630) Besides, blastocyst and, vitro, (ESCs). Several works aimed characterizing ESCs. Some indicated suppression TEAD resulted decreased intensity alkaline phosphatase (AP) staining colonies Oct4 Sox2, concomitant differentiation markers, T, alpha-fetoprotein, Gata4 (10Lian I. Kim Okazawa Zhao B. Yu Chinnaiyan Israel M.A. Goldstein L.S. Abujarour Ding coactivator regulating self-renewal differentiation.Genes Dev. 2010; 24: 1106-1118Crossref (482) Accordingly, overexpression (OE) dominant-negative led induction toward endodermal lineage (11Tamm Böwer Annerén Regulation Yes-YAP-TEAD2 downstream LIF.J. 124: 1136-1144Crossref (146) In vivo analysis showed high sustains mass, whereas low levels eliminated (5Hashimoto However, conflicting depletion had no effects on grown 2i medium that, conditions, mimics GSK3 inhibitor blocks Consistent results, silencing KO has maintenance undifferentiated state (12Chung Lee B.K. Uprety Shen W. Yap1 dispensable required proper (ES) cells.EMBO Rep. 2016; 17: 519-529Crossref (35) Furthermore, differentiation-inducing (KD) an insufficient accumulation Gata6, Gata3, Nanog normally suppressed apparently least part depend multifaceted activities different experimental conditions lead diverse consequences. To address point, explored We found normal completely rescued process, KD genome profile significantly cells. Looking cells, appreciated significant Dnmt3l ephemeron (lncRNA). findings indicate necessary sustain appropriate machinery patterns taking naive state. decided explore possible long-lasting aim, E14Tg2a clones, stably expressing GFP under neural-specific promoter ?1-tubulin (?1T–GFP) (13Parisi Passaro Aloia Manabe Nagai Pastore Russo Klf5 cells.J. 2008; 121: 2629-2634Crossref (105) were transiently transfected mixture siRNAs eliciting robust (Fig. S1). About 48 h after transfection, neuroectodermal fate was monitored time points process. As shown Fig. 1A, GFP-positive since first step (day 4), respect (CTR KD) siRNA duplex. later stages 14), process incomplete, reduced ?3-tubulin 1B). late marker confirmed severely affects 1C). over transiency silencing, mRNA returned steady day 4 1, D E). Nevertheless, consequences lasted until end protocol, strongly determinant following events. mesendodermal interferes specifically or, instead, induces general perturbation potential siRNAs, clone MLC2v–GFP, which cardiomyocyte-specific MLC2v (14Testa Tarantino Parisi Galizia Della-Morte D. Abete Rengo Salvatore Serum withdrawal embryoid body does not impair cardiomyocyte cells.Cytotherapy. 13: 350-356Abstract (2) mesodermal derivatives bodies culture (15Navarra Musto Gargiulo Petrosino Pierantoni G.M. Fusco Hmga2 Otx2-dependent pluripotent ground state.BMC 14: 24Crossref (13) Again, 8 days 1F), interestingly, CTR expected beating phenotype, almost areas 1G). compromises undertake partial due, hand, premature loss contrary, persistence despite ESCs, silenced shRNAs S2A). After h, plated absence leukemia inhibitory factor (LIF) further samples analyzed measure genes. 2A demonstrate main markers affected YAP. density cultured 7 more presence LIF serum examine AP+ colonies. 2B, both able form AP staining. difference emerged total derived lower than 2B). Fluorescence-activated sorting (FACS) certain degree 2C) so support possibility differentiated exclusively death. assess viability serum-free (SFEBs) Trypan blue exclusion assay increased nonvital among those SFEBs 2D), probably differences assessed measuring diameter 2E), appearing smaller SFEBs. Altogether, causes survival latter can explain decrease when they replated surviving seem unable properly, affect signature selected subpopulations phenotypes events continue exert restored. deregulation methylation, take upon (16Petell C.J. Alabdi He San Miguel Rose Gowher An switch regulates subset enhancers differentiation.Nucleic Acids Res. 44: 7605-7617Crossref (36) 17Auclair Guibert Bender Weber Ontogeny CpG island specificity DNMT3 methyltransferases mouse.Genome 15: 545Crossref (84) possibility, bisulfite (BS-Seq) (T0) versus (T4). First all, changes occurring compared T0, finding 6661 differentially methylated regions (DMRs) q value ?0.05 (Table Then, performed same T4 identifying 6898 DMRs Although similar absolute terms between dramatic effect methylation. 3A, about 30% gain only 13% whereas, other most (86.9% 69.8% cells), chromosomes particularly affected, cases 2, 3, 4, 13, 14 3B). then examined loci showing particular differentiation-dependent Considering 0.05, there 2325 where evident 1218 where, retrieved sets closest them using evolutionary relationships classification system platform ontology (GO) (18Mi Muruganujan Huang Ebert Mills Guo Thomas P.D. Protocol Update large-scale PANTHER (v.14.0).Nat. Protoc. 703-721Crossref (300) Interestingly, overrepresentation cadherin 3C). repatterning This ultimately lack YAP, look profiles transfection reaches minimum levels. Total isolated three independent each RNA-Seq. 4A, 1196 deregulated (p < 0.005; false discovery rate <0.05), 54% S2). parallel, YAP-encoding vector (OE YAP) empty CTR) 4B), again biological samples. case, 488 transcripts overexpressed (73%) underexpressed (27%), consequence ectopic S2 S3). comparison OE 215 common sets, 134 coherently downregulated upregulated 46 opposite behavior 4C). Protein (0.6 fold change [FC] > 1.7) statistically enrichment (false 0.05) GO morphogenesis 4D). Among focused our attention (Eprn—D630045M09Rik), encodes lncRNA modulates (19Li Amaral P.P. Cheung Bergmann J.H. Kinoshita Kalkan Ralser Robson von Meyenn Paramor Yang Nichols Spector D.L. Kouzarides al.A fine tunes Lin28, let-7 methylation.eLife. 6e23468Crossref (16) Indeed, recently described Eprn Upon removal LIF, deletion delays extinction reducing Lin28a expression, consequent microRNAs. methyltransferase Dnmt3a/b delayed, retards slight = 0.0042) 5A). 12 removal, abolished entirely 5B). addition, Lin28b, withdrawal, T12 Lin28b T48. significant, RNA-Seq quantitative PCR (qPCR) assays did b, activating cofactor, lacking enzyme activity, Dnmt3b, contributing (20Borgel Li Y. Chiba Schübeler Forné Targets development.Nat. Genet. 42: 1093-1100Crossref (427) Of interest, (21Veland Lu Hardikar Gaddis Zeng Liu Estecio M.R. Takata Lin Tomida M.W. Saha DNMT3L facilitates partly maintaining DNMT3A stability cells.Nucleic 47: 152-167Crossref (37) hampers bivalent promoters polycomb repressive 2 (22Neri Krepelova Incarnato Maldotti Parlato Galvagni Matarese Stunnenberg H.G. Oliviero Dnmt3L antagonizes favors ESCs.Cell. 2013; 155: 121-134Abstract (114) qPCR 5C). case Eprn, withdrawal. remained all 5D). addressed question and/or direct targets explored, fraction clearly present relatively (about 20%), nuclear undetectable most, if S4, A B). Thus, should chromatin immunoprecipitation (ChIP-Seq) experiments run duplicate 428 bona fide peaks S3) start site located within 500 kbps S5A Table putative clustered based associated well assembly disassembly S5B). crossing ChIP-Seq data, 43 6A), proximity (500 kbps) ChIP peaks. Neither nor site. relaxing stringency threshold, peak second intron S6A). 154 correspond published collections (23Croci O. De Fazio Biagioni Donato Caganova Curti Doni Sberna Aldeghi Biancotto Verrecchia Olivero Amati Campaner integration mitogenic signals Myc YAP.Genes 31: 2017-2022Crossref Scholar) S4), interact binding Epnr assay, TEAD1 TEAD2 S5C). 6B, gene. Moreover, amount immunoprecipitated TEADs dependent. interaction S6B). order define contribution downregulation, ?1T–GFP producing S7A). Two used. fate. 7A, indeed expre