多能干细胞：The SMAD2-3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency.pdf

The SMAD2/3 interactome reveals that TGF controls m6A mRNA methylation in pluripotencyAlessandro Bertero#1,Stephanie Brown#1,Pedro Madrigal1,2,Anna Osnato1,Daniel Ortmann1,Loukia Yiangou1,Juned Kadiwala1,Nina C.Hubner3,Igor Ruiz de los Mozos4,Christoph Sadee4,An-Sofie Lenaerts1,Shota Nakanoh1,Rodrigo Grandy1,Edward Farnell5,Jernej Ule4,Hendrik G.Stunnenberg3,Sasha Mendjan1,and Ludovic Vallier1,2,*1Wellcome Trust-MRC Cambridge Stem Cell Institute Anne McLaren Laboratory and Department of Surgery,University of Cambridge,UK 2Wellcome Trust Sanger Institute,Hinxton UK 3Department of Molecular Biology,Radboud University Nijmegen,The Netherlands 4Francis Crick Institute and Department of Molecular Neuroscience,University College London,UK 5Department of Pathology,University of Cambridge,UK#These authors contributed equally to this work.AbstractThe TGF pathway plays an essential role in embryonic development,organ homeostasis,tissue repair,and disease1,2.This diversity of tasks is achieved through the intracellular effector SMAD2/3,whose canonical function is to control activity of target genes by interacting with transcriptional regulators3.Nevertheless,a complete description of the factors interacting with SMAD2/3 in any given cell type is still lacking.Here we address this limitation by describing the interactome of SMAD2/3 in human pluripotent stem cells(hPSCs).This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription.In particular,we identify a functional interaction with the METTL3-METTL14-WTAP complex,which deposits N6-methyladenosine(m6A)4.We uncover that SMAD2/3 promotes binding of the m6A methyltransferase complex onto a subset of transcripts involved in early cell fate decisions.This mechanism destabilizes specific SMAD2/3 transcriptional targets,including the pluripotency Users may view,print,copy,and download text and data-mine the content in such documents,for the purposes of academic research,subject always to the full Conditions of use:http:/ and requests for materials should be addressed to Ludovic Vallier(lv225cam.ac.uk).Current address:Department of Pathology,University of Washington,Seattle,WA,USA.Current address:Institute of Molecular Biotechnology,Vienna,Austria.Author contributionsA.B.conceived the study,performed or contributed to most of the experiments,analysed data,and wrote the manuscript with input from the other authors.S.B.contributed to study conception,performed co-IP,NeMeRIP,and RNA-IP experiments,and analysed data.P.M.,I.R.d.l.M,and C.S.analysed NeMeRIP-seq.A.O.performed PLA and co-immunoprecipitations,and analyzed RNA-seq.D.O.,L.Y.,and J.K.assisted hPSC gene editing and differentiation;N.C.H.performed quantitative proteomics and data analysis.A.L.,S.N.,and R.G.assisted hPSC culture.E.F.optimized NeMeRIP-seq sequencing libraries.J.U.contributed to study conception and supervision.H.G.S.supervised quantitative proteomics.S.M.contributed to study conception and supervision,and assisted SMAD2/3 co-IP.L.V.conceived,supervised,and supported the study,wrote and provided final approval of the manuscript.Author informationReprints and permissions information is available at competing financial interests are declared.Europe PMC Funders GroupAuthor ManuscriptNature.Author manuscript;available in PMC 2018 August 28.Published in final edited form as:Nature.2018 March 08;555(7695):256259.doi:10.1038/nature25784.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsfactor NANOG,thereby poising them for rapid downregulation upon differentiation to enable timely exit from pluripotency.Collectively,these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulations of the epitranscriptome.These novel aspects of TGF signalling could have far-reaching implications in many other cell types and in diseases such as cancer5.Activin and Nodal,two members of the TGF superfamily,play essential roles in cell fate decision in hPSCs68.Activin/Nodal signalling is necessary to maintain pluripotency,and its inhibition drives differentiation toward the neuroectoderm lineage6,9,10.Activin/Nodal also cooperates with BMP and WNT to drive mesendoderm specification1114.Thus,we used hPSC differentiation into definitive endoderm as a model system to interrogate the SMAD2/3 interactome during a dynamic cellular process.For that we developed an optimized SMAD2/3 co-immunoprecipitation(co-IP)protocol compatible with mass-spectrometry analyses(Extended Data Fig.1a-b and Supplementary Discussion).This method allowed a comprehensive and unbiased examination of the proteins interacting with SMAD2/3 for the first time in any given cell type.By examining human embryonic stem cells(hESCs)and hESCs induced to differentiate towards endoderm(Fig.1a),we identified 89 SMAD2/3 partners(Fig.1b,Extended Data Fig.1c-d,and Supplementary Table 1).Of these,only 11 factors were not shared between hESCs and endoderm differentiating cells(Extended Data Fig.1e),suggesting that the SMAD2/3 interactome is largely conserved across these two lineages(Supplementary Discussion).Importantly,this list included known SMAD2/3 transcriptional and epigenetic cofactors(including FOXH1,SMAD4,SNON,SKI,EP300,SETDB1,and CREBBP3),which validated our method.Furthermore,we performed functional experiments on FOXH1,EP300,CREBBP,and SETDB1,which uncovered the essential function of these SMAD2/3 transcriptional and epigenetic cofactors in hPSC fate decisions(Extended Data Fig.2 and 3,and Supplementary Discussion).Interestingly,our proteomic experiments also revealed that SMAD2/3 interacts with complexes involved in functions that have never been associated with TGF signalling(Fig.1b and Extended Data Fig.1f),such as ERCC1-XPF(DNA repair)and DAPK3-PAWR(apoptosis).Most notably,we identified several factors involved in mRNA processing,modification,and degradation(Fig.1b),such as the METTL3-METTL14-WTAP complex(deposition of N6-methyladenosine,or m6A),the PABP-dependent poly(A)nuclease complex hPAN(mRNA decay),the cleavage factor complex CFIm(pre-mRNA 3 end processing),and the NONO-SFPQ-PSPC1 factors(RNA splicing and nuclear retention of defective RNAs).Overall,these results suggest that SMAD2/3 could be involved in a large number of biological processes in hPSCs,which include not only transcriptional and epigenetic regulations,but also novel“non-canonical”molecular functions.To further explore this hypothesis,we investigated the interplays between Activin/Nodal and m6A deposition.m6A is the most common RNA modification,regulating multiple aspects of mRNA biology including decay and translation4,1519.However,whether this is a dynamic event that can be modulated by extracellular cues remains to be established.Furthermore,while m6A is known to regulate hematopoietic stem cells20,21 and the transition between the nave and primed pluripotency states22,23,its function in hPSCs and Bertero et al.Page 2Nature.Author manuscript;available in PMC 2018 August 28.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsduring germ layer specification is unclear.We first validated the interaction of SMAD2/3 with METTL3-METTL14-WTAP using co-IP followed by Western Blot in both hESCs and human induced pluripotent stem cells(hiPSCs;Fig.2a and Extended Data Fig.4a-b).Interestingly,inhibition of SMAD2/3 phosphorylation blocked this interaction(Fig.2b and Extended Data Fig.4c).Proximity ligation assays(PLA)also demonstrated that the interaction occurs at the nuclear level(Fig.2c-d).These observations suggest that SMAD2/3 and the m6A methyltransferase complex interact in an Activin/Nodal signalling-dependent fashion.To investigate the functional relevance of this interaction,we assessed the transcriptome-wide effects of Activin/Nodal inhibition on the deposition of m6A by performing nuclear-enriched m6A methylated RNA immunoprecipitation followed by deep sequencing(NeMeRIP-seq;Extended Data Fig.5a-d,and Supplementary Discussion).In agreement with previous reports17,19,24,deposition of m6A onto exons was enriched around stop codons and transcription start sites,and occurred at a motif corresponding to the m6A consensus sequence(Extended Data Fig.5e-g).Assessment of differential m6A deposition revealed that Activin/Nodal inhibition predominantly resulted in reduced m6A levels in selected transcripts(Supplementary Table 2;average absolute log2 fold-change of 0.56 and 0.35 for m6A decrease and increase,respectively).Decrease in m6A deposition was predominantly observed on peaks located near to stop codons(Extended Data Fig.5h),a location which has been reported to decrease the stability of mRNAs16,24,25.Interestingly,transcripts showing reduced m6A levels after Activin/Nodal inhibition largely and significantly overlapped with genes bound by SMAD2/3(Extended Data Fig.5i),including well-known transcriptional targets such as NANOG,NODAL,LEFTY1,and SMAD7(Fig.2e and Extended Data Fig.5j).Accordingly,Activin/Nodal-sensitive m6A deposition was largely associated with transcripts rapidly decreasing during the exit from pluripotency triggered by Activin/Nodal inhibition(Extended Data Fig.6a).Transcripts behaving in this fashion were enriched in pluripotency regulators and in factors involved in the Activin/Nodal signalling pathway(Supplementary Table 3).On the other hand,the expression of a large number of developmental regulators associated to Activin/Nodal-sensitive m6A deposition remained unchanged following Activin/Nodal inhibition(Extended Data Fig.6a-c and Supplementary Table 3).Considered together,these findings establish that Activin/Nodal signalling can regulate m6A deposition on a number of specific transcripts.We then examined the underlying molecular mechanisms.RNA immunoprecipitation experiments on nuclear RNAs showed that inhibition of Activin/Nodal signalling impaired binding of WTAP to multiple m6A-marked transcripts including NANOG and LEFTY1(Fig.2f and Extended Data Fig.4d-e),while SMAD2/3 itself interacted with such transcripts in the presence of Activin/Nodal signalling(Fig.2g and Extended Data Fig.4e).Thus,SMAD2/3 appears to promote the recruitment of the m6A methyltransferase complex onto nuclear RNAs.Interestingly,recent reports have established that m6A deposition occurs co-transcriptionally and involves nascent pre-RNAs16,26,20.Considering the broad overlap between SMAD2/3 transcriptional targets and transcripts showing Activin/Nodal-sensitive m6A deposition(Extended Data Fig 5i),we therefore hypothesized that SMAD2/3 could facilitate co-transcriptional recruitment of the m6A methyltransferase complex onto nascent transcripts.Supporting this notion,inhibition of Activin/Nodal signalling mainly resulted in Bertero et al.Page 3Nature.Author manuscript;available in PMC 2018 August 28.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsdownregulation of m6A not only on exons,but also onto pre-mRNA-specific features such as introns and exon-intron junctions(Extended Data Fig.6d-i and Supplementary Table 2).Moreover,we observed a correlation in Activin/Nodal sensitivity for m6A peaks within the same transcript(Extended Data Fig.6j),suggesting that SMAD2/3 regulates m6A deposition at the level of a genomic locus rather than on a specific mRNA peak.Nevertheless,a stable and direct binding of the m6A methyltransferase complex to the DNA could not be detected(Extended Data Fig.4f).Thus,co-transcriptional recruitment might rely on indirect and dynamic interactions with the chromatin.Considering all these results,we propose a model in which Activin/Nodal signalling promotes co-transcriptional m6A deposition by facilitating the recruitment of the m6A methyltransferase complex onto nascent mRNAs(Fig.2h).To understand the functional relevance of these regulations in the context of hPSC cell fate decisions,we performed inducible knockdown experiments for the various subunits of the m6A methyltransferase complex27(Extended Data Fig.7a-b).As expected,decrease in WTAP,METTL14,or METTL3 expression reduced the deposition of m6A(Extended Data Fig 7c-d).Interestingly,prolonged knockdown did not affect pluripotency(Extended Data Fig.7e-f).However,expression of m6A methyltransferase complex subunits was necessary for neuroectoderm differentiation induced by the inhibition of Activin/Nodal signalling,while it was dispensable for Activin-driven endoderm specification(Fig.3a and Extended Data Fig.8a-c).Activin/Nodal is known to block neuroectoderm induction by promoting NANOG expression28,while NANOG is required for the early stages of endoderm specification13.Therefore,we monitored the levels of this factor during neuroectoderm differentiation.We observed that both transcript and protein were upregulated following impairment of m6A methyltransferase activity(Fig.3b and Extended Data Fig.9a-b),while mRNA stability was increased(Extended Data Fig.9c).These results show that m6A deposition decreases the stability of the NANOG mRNA to facilitate its downregulation upon loss of Activin/Nodal signalling,thus facilitating exit from pluripotency and neuroectoderm specification(Extended Data Fig.9d).Additional transcriptomic analyses showed that WTAP knockdown resulted in a global upregulation of genes transcriptionally activated by SMAD2/3 in hESCs,while it impaired the upregulation of genes induced by Activin/Nodal inhibition during neuroectoderm differentiation(Fig.3b,Extended Data Fig.10a-e,Supplementary Table 4,and Supplementary Discussion).Importantly,the decrease in WTAP expression also led to the upregulation of mRNAs marked by m6A(Extended Data Fig.10f),confirming that WTAP-dependent m6A deposition destabilises mRNAs16,24,25.Moreover,transcripts rapidly downregulated after Activin/Nodal inhibition were enriched in m6A-marked mRNAs(Extended Data Fig.10f).Finally,simultaneous knockdown of METTL3,METTL14,and WTAP in hESCs resulted in an even stronger dysregulation of Activin/Nodal target transcripts(Fig.3c-d and Extended Data Fig.8d)and defective neuroectoderm differentiation(Fig.3d and Extended Data Fig.8e-f).Taken together,these results indicate that the interaction of SMAD2/3 with METTL3-METTL14-WTAP can promote m6a deposition on a subset of transcripts,including a number of pluripotency regulators that are also transcriptionally activated by Activin/Nodal signalling.The resulting negative feedback destabilizes these mRNAs and causes their rapid degradation following Bertero et al.Page 4Nature.Author manuscript;available in PMC 2018 August 28.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsinhibition of Activin/Nodal signalling.This mechanism allows timely exit from pluripotency and induction of neuroectoderm differentiation(Extended Data Fig.9d).To conclude,this first analysis of the SMAD2/3 interactome reveals novel interplays between TGF signalling and a diversity of cellular processes.Our results su