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ALKBH5-mediated m6A mRNA methylation governs human embryonic stem cell cardiac commitment

2021; Cell Press; Volume: 26; Linguagem: Inglês

10.1016/j.omtn.2021.05.019

ISSN

2162-2531

Autores

Zhenbo Han, Zihang Xu, Ying Yu, Yang Cao, Zhengyi Bao, Xinlu Gao, Danyu Ye, Gege Yan, Rui Gong, Juan Xu, Lai Zhang, Wenya Ma, Xiuxiu Wang, Fan Yang, Lei Hong, Ye Tian, Shijun Hu, Djibril Bamba, Ying Li, Desheng Li, Changzhu Li, Ning Wang, Ying Zhang, Zhenwei Pan, Baofeng Yang, Benzhi Cai,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

N6-methyladenosine (m6A), as the most abundant modification of mammalian messenger RNAs, is essential for tissue development and pathogenesis. However, the biological significance of m6A methylation in cardiac differentiation and development remains largely unknown. Here, we identify that the downregulation of m6A demethylase ALKBH5 is responsible for the increase of m6A methylation and cardiomyocyte fate determination of human embryonic stem cells (hESCs) from mesoderm cells (MESs). In contrast, ALKBH5 overexpression remarkably blocks cardiomyocyte differentiation of hESCs. Mechanistically, KDM5B and RBBP5, the components of H3K4 modifying enzyme complexes, are identified as downstream targets for ALKBH5 in cardiac-committed hESCs. Loss of function of ALKBH5 alters the expression of KDM5B and RBBP5 through impairing stability of their mRNAs, which in turn promotes the transcription of GATA4 by enhancing histone H3 Lys4 trimethylation (H3K4me3) at the promoter region of GATA4. Taken together, we reveal a previously unidentified role of m6A demethylase ALKBH5 in determining cardiac lineage commitment of hESCs. N6-methyladenosine (m6A), as the most abundant modification of mammalian messenger RNAs, is essential for tissue development and pathogenesis. However, the biological significance of m6A methylation in cardiac differentiation and development remains largely unknown. Here, we identify that the downregulation of m6A demethylase ALKBH5 is responsible for the increase of m6A methylation and cardiomyocyte fate determination of human embryonic stem cells (hESCs) from mesoderm cells (MESs). In contrast, ALKBH5 overexpression remarkably blocks cardiomyocyte differentiation of hESCs. Mechanistically, KDM5B and RBBP5, the components of H3K4 modifying enzyme complexes, are identified as downstream targets for ALKBH5 in cardiac-committed hESCs. Loss of function of ALKBH5 alters the expression of KDM5B and RBBP5 through impairing stability of their mRNAs, which in turn promotes the transcription of GATA4 by enhancing histone H3 Lys4 trimethylation (H3K4me3) at the promoter region of GATA4. Taken together, we reveal a previously unidentified role of m6A demethylase ALKBH5 in determining cardiac lineage commitment of hESCs. Chemical modifications have a well-defined role in regulating the activity of mammalian messenger RNAs (mRNAs). Among over 100 types of chemical modifications,1Cantara W.A. Crain P.F. Rozenski J. McCloskey J.A. Harris K.A. Zhang X. Vendeix F.A. Fabris D. Agris P.F. The RNA Modification Database, RNAMDB: 2011 update.Nucleic Acids Res. 2011; 39: D195-D201Crossref PubMed Scopus (572) Google Scholar N6-methyladenosine (m6A) represents the most abundant form.2Sibbritt T. Patel H.R. Preiss T. Mapping and significance of the mRNA methylome.Wiley Interdiscip. Rev. 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Watabe T. et al.Cell-sheet therapy with omentopexy promotes arteriogenesis and improves coronary circulation physiology in failing heart.Mol. Ther. 2015; 23: 374-386Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) has an unmet need for cell replacement therapy.22Zhu K. Wu Q. Ni C. Zhang P. Zhong Z. Wu Y. Wang Y. Xu Y. Kong M. Cheng H. et al.Lack of Remuscularization Following Transplantation of Human Embryonic Stem Cell-Derived Cardiovascular Progenitor Cells in Infarcted Nonhuman Primates.Circ. Res. 2018; 122: 958-969Crossref PubMed Scopus (68) Google Scholar,23Yoshida Y. Yamanaka S. Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications.Circ. Res. 2017; 120: 1958-1968Crossref PubMed Scopus (130) Google Scholar Transplantation of hPSC-derived cardiomyocytes has been shown to improve cardiac function of infarcted non-human primate heart.24Shiba Y. Gomibuchi T. Seto T. Wada Y. Ichimura H. Tanaka Y. Ogasawara T. Okada K. Shiba N. Sakamoto K. et al.Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.Nature. 2016; 538: 388-391Crossref PubMed Scopus (383) Google Scholar In addition, hPSC-derived cardiomyocytes have been extensively used in drug cardiotoxicity screening, disease model establishment, and exploration of human heart development.25Lam C.K. Wu J.C. Disease modelling and drug discovery for hypertrophic cardiomyopathy using pluripotent stem cells: how far have we come?.Eur. Heart J. 2018; 39: 3893-3895Crossref PubMed Scopus (10) Google Scholar,26Bao Z. Han Z. Zhang B. Yu Y. Xu Z. Ma W. Ding F. Zhang L. Yu M. Liu S. et al.Arsenic trioxide blocked proliferation and cardiomyocyte differentiation of human induced pluripotent stem cells: Implication in cardiac developmental toxicity.Toxicol. Lett. 2019; 309: 51-58Crossref PubMed Scopus (12) Google Scholar However, the molecular mechanism underlying cardiac differentiation remains largely unknown. Cardiogenesis of hPSCs is controlled by temporal expression of transcription factors critical for mesoderm cells (MESs) and cardiac lineage specification.27Burridge P.W. Matsa E. Shukla P. Lin Z.C. Churko J.M. Ebert A.D. Lan F. Diecke S. Huber B. Mordwinkin N.M. et al.Chemically defined generation of human cardiomyocytes.Nat. Methods. 2014; 11: 855-860Crossref PubMed Google Scholar Recent studies have suggested that histone epigenetic modifications play a crucial role in cardiomyocyte differentiation.28Ohtani K. Zhao C. Dobreva G. Manavski Y. Kluge B. Braun T. Rieger M.A. Zeiher A.M. Dimmeler S. Jmjd3 controls mesodermal and cardiovascular differentiation of embryonic stem cells.Circ. Res. 2013; 113: 856-862Crossref PubMed Scopus (61) Google Scholar, 29Wamstad J.A. Alexander J.M. Truty R.M. Shrikumar A. Li F. Eilertson K.E. Ding H. Wylie J.N. Pico A.R. Capra J.A. et al.Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.Cell. 2012; 151: 206-220Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 30Lee J. Shao N.Y. Paik D.T. Wu H. Guo H. Termglinchan V. Churko J.M. Kim Y. Kitani T. Zhao M.T. et al.SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation.Cell Stem Cell. 2018; 22: 428-444.e5Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Nevertheless, whether RNA m6A epigenetic modification regulates cardiomyocyte differentiation is currently unknown. In this study, we first explored the role of m6A modification in cardiac differentiation and found that m6A methylation was significantly upregulated upon the differentiation of MESs into CMs, which is accompanied by the decrease of m6A demethylase ALKBH5. Moreover, overexpression of ALKBH5 dramatically inhibited the differentiation of MESs into CMs, indicating that m6A methylation caused by the downregulation of ALKBH5 plays a critical role in cardiac differentiation. Mechanistically, m6A methylation targeted mRNAs of lysine demethylase 5B (KDM5B) and retinoblastoma-binding protein 5 (RBBP5), two known H3K4me3-modifying enzymes, and regulated their activity, which in turn promoted the expression of GATA4 by enhancing histone H3 Lys4 trimethylation (H3K4me3) at its promoter region, thereby driving the cardiac differentiation of MESs. Together, our study defined the critical role of ALKBH5-mediated m6A RNA modification in cardiogenesis and revealed the molecular mechanism underlying cardiac lineage commitment of human ESCs (hESCs). To define the role of m6A methylation in cardiomyocyte commitment of hESCs, we induced the differentiation of hESCs into CMs in vitro according to a previous report27Burridge P.W. Matsa E. Shukla P. Lin Z.C. Churko J.M. Ebert A.D. Lan F. Diecke S. Huber B. Mordwinkin N.M. et al.Chemically defined generation of human cardiomyocytes.Nat. Methods. 2014; 11: 855-860Crossref PubMed Google Scholar (Figure S1A). Beating CMs appeared from day 7 after induction, which are highly expressed with cardiac-specific markers such as cardiac troponin T (cTnT) and α-actinin (Figures S1C and S1D). According to gene-expression patterns, the differentiation of hESCs into CMs was divided into four stages: hESCs (day 0), MESs (day 2), CPCs (cardiac progenitor cells, day 5), and CMs (day 8; Figure S1B). We performed m6A dot blot analysis of mRNAs from all stages of differentiation and found that m6A methylation was not affected from hESC stage to MES stage, whereas it significantly increased during the differentiation of MESs into CMs (Figure 1A). This finding was also supported by m6A-ELISA assay (Figure 1B). Since the cellular level of m6A methylation is determined by the activities of m6A methyltransferases and demethylases, we next investigated the expression of these enzymes. We found that the expression of m6A demethylase ALKBH5 was significantly decreased during the differentiation of MESs into CMs, while m6A methyltransferase METTL3, METTL14, WTAP, and demethylase FTO did not alter in this stage (Figures 1C and 1D). Together, these data raise the possibility that ALKBH5-mediated m6A modification may play a regulatory role in the differentiation of MESs into CMs. We next examined the effect of ALKBH5-mediated m6A demethylation on the lineage commitment of CMs by overexpressing this enzyme in MESs. As shown in Figures 2A–2D, transfection of ALKBH5-expressing plasmid at day 2 of differentiation dramatically elevated the RNA and protein levels of ALKBH5, which significantly decreased the global m6A levels in differentiating hESCs. Moreover, the exogenous expression of ALKBH5 significantly reduced the percentage of cTnT+ and α-actinin+ cells and spontaneously contracting colonies derived from hESCs (Figures 2E and 2F). Consistent with this finding, cells expressing exogenous ALKBH5 produced much fewer cardiac genes and ion channel genes on indicated days of differentiation (Figures 2G and 2H). Together, these data suggest that ALKBH5-mediated m6A demethylation inhibits cardiac differentiation of MESs. To further verify the role of ALKBH5 in cardiac differentiation, we inhibited its expression in MESs by using small interfering RNA (siRNA)-mediated knockdown. As shown in Figures 3A and 3B , transfections of two siRNAs targeting different regions of ALKBH5 significantly reduced both mRNA and protein levels of ALKBH5 in cells. ALKBH5 knockdown strongly elevated the level of global m6A methylation in MESs (Figures 3C and 3D) and upregulated the expression of cardiac genes and ion channel genes in differentiated cells (Figures 3F and 3H). Moreover, MESs with less ALKBH5 had a higher propensity to generate cTnT+ and α-actinin+ cells and spontaneously contracting CMs (Figures 3E and 3G). Together, these data indicate that the blockage of ALKBH5-mediated m6A demethylation promotes the differentiation of MESs into CMs. To understand how ALKBH5-mediated m6A demethylation regulates the cardiac commitment of MESs, we next performed m6A transcriptomic microarray analysis to detect m6A methylation level on which genes have changed in ALKBH5-OE differentiating hESCs compared to control. The gene ontology (GO) analysis revealed that targets of m6A methylation were enriched in calcium signaling pathway and transcription corepression pathway (Figure 4A). Given the critical role of transcription factors such as NKX2-5, MESP1, and GATA4 in cardiac lineage specification,30Lee J. Shao N.Y. Paik D.T. Wu H. Guo H. Termglinchan V. Churko J.M. Kim Y. Kitani T. Zhao M.T. et al.SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation.Cell Stem Cell. 2018; 22: 428-444.e5Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar hereby we focused on those targets that regulate transcription corepressor activity. Among them, we confirmed that the mRNA level of histone demethylase KDM5B was significantly elevated upon overexpression of ALKBH5 (Figure 4B). We next determined whether other components of histone methylation complexes were also altered by ALKBH5-mediated m6A demethylation and found that the mRNA level of RBBP5 but not MLL1, ASH2L, and WDR5 was dramatically decreased upon ALKBH5 overexpression (Figure 4C). RBBP5 and KDM5B are two key components modulating H3K4 methylation and demethylation, respectively.31Nayak A. Viale-Bouroncle S. Morsczeck C. Muller S. The SUMO-specific isopeptidase SENP3 regulates MLL1/MLL2 methyltransferase complexes and controls osteogenic differentiation.Mol. Cell. 2014; 55: 47-58Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,32Roesch A. Fukunaga-Kalabis M. Schmidt E.C. Zabierowski S.E. Brafford P.A. Vultur A. Basu D. Gimotty P. Vogt T. Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.Cell. 2010; 141: 583-594Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar Western-blot analysis also showed that the protein level of KDM5B was significantly elevated while RBBP5 was reduced upon overexpression of ALKBH5 (Figure 4D). To the contrary, the protein expression of KDM5B was downregulated while RBBP5 was upregulated upon ALKBH5 knockdown (Figure 4E). These data imply that m6A methylation may regulate cardiogenesis through KDM5B- and RBBP5-mediated histone modifications. To explore how ALKBH5-mediated m6A demethylation regulates histone methylation, we carried out methylated RNA immunoprecipitation (MeRIP)-quantitative polymerase chain reaction (qPCR) assays and found that the alterations of m6A methylation were highly enriched on mRNAs of KDM5B and RBBP5 at day 3 of differentiation (Figure 5A). Given that m6A modification regulates gene expression mainly through altering mRNA stability, we then evaluated potential changes in RNA stability of KDM5B and RBBP5. We compared the half-lives of mRNAs of target genes with or without ALKBH5 overexpression. We found that overexpression of ALKBH5 noticeably extended the half-life of KDM5B mRNAs albeit shortened the half-life of RBBP5 mRNAs (Figure 5B). We next examined various H3 lysine methyl modifications upon ALKBH5 overexpression or knockdown. We found that the global methylation level of H3K4me3 was dramatically downregulated upon ALKBH5 overexpression and upregulated upon ALKBH5 knockdown in contrast to that of H3K9me3, H3K27me3, or H3K36me3 (Figures 5C and 5D). Immunostaining analysis also confirmed that H3K4me3 level was decreased by overexpression of ALKBH5 (Figure S2A). Together, these data suggest that ALKBH5-mediated m6A demethylation reduced H3K4me3 methylation through altering expression of KDM5B and RBBP5. To confirm that RBBP5 and KDM5B are two key downstream effectors of m6A in cardiac differentiation, we first knocked down RBBP5 by using siRNAs at day 2 of differentiation and found that it significantly decreased the methylation level of H3K4me3 (Figures 6A and 6B ) and reduced the percentage of beating colonies and cTnT+ and α-actinin+ cells at day 10 of cardiac differentiation (Figures 6C and 6D). On the other hand, overexpression of RBBP5 was able to promote the generation of cTnT+, α-actinin+, and spontaneously beating CMs, even with ALKBH5 overexpression (Figures 6E–6H). We then used the specific inhibitor of KDM5B, AS8351, to verify its role in m6A methylation-driven cardiac differentiation and found that AS8351 treatment significantly increased global H3K4me3 methylation in differentiating hESCs (Figure 6I) and recovered the cardiac differentiation propensity of hESCs with low m6A methylation, as well as the expression of cardiac genes in differentiated cells (Figure 6J; Figure S3). These results suggest that ALKBH5-mediated m6A demethylation inhibits cardiac differentiation through blocking RBBP5- and KDM5B-mediated methylation of H3K4me3. We next addressed how m6A methylation drove H3K4me3 methylation in cardiac differentiation. Cardiomyocyte lineage commitment is mainly modulated by temporal expression of transcription factors.27Burridge P.W. Matsa E. Shukla P. Lin Z.C. Churko J.M. Ebert A.D. Lan F. Diecke S. Huber B. Mordwinkin N.M. et al.Chemically defined generation of human cardiomyocytes.Nat. Methods. 2014; 11: 855-860Crossref PubMed Google Scholar We thus checked whether KDM5B and RBBP5 were recruited to the promoter regions of these key factors and thereby regulated their expression by adding or erasing active H3K4me3 marks. We performed chromatin immunoprecipitation (ChIP)-qPCR with specific primers targeting promoter regions of key transcription factors EOMES (Eomesodermin), MESP1 (mesoderm posterior BHLH transcription factor 1), GATA4, NKX2-5 (NK2 homeobox 5), and ISL-1 (islet-1), and found that overexpression of ALKBH5 caused a noticeable downregulation of H3K4me3 methylation at the promoter region of GATA4 but not that of other factors. (Figure 7A). The co-immunoprecipitation (coIP) assays also revealed that both KDM5B and RBBP5 were associated with the GATA4 in differentiating hESCs (Figures S4A and S4B). As expected, the expression of GATA4 was dramatically decreased in cells overexpressing ALKBH5 (Figure 7B; Figure S4C). In addition, the introduction of GATA4 recovered their propensity of cardiac differentiation (Figures 7C and 7D). Altogether, these results indicate that ALKBH5-mediated m6A demethylation impairs the H3K4me3 methylation at GATA4 promoter regions and thereby inhibits its expression. Recent studies have revealed that m6A methylation is linked to pluripotency regulation of ESCs/induced PSCs (iPSCs). Batista et al.18Batista P.J. Molinie B. Wang J. Qu K. Zhang J. Li L. Bouley D.M. Lujan E. Haddad B. Daneshvar K. et al.m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.Cell Stem Cell. 2014; 15: 707-719Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar showed that METTL3 impaired ESC exit from self-renewal toward differentiation through prolonged Nanog expression. Geula and colleagues revealed that METTL3 knockout preimplantation epiblasts and naive ESCs fail to terminate their naive pluripotent state.33Geula S. Moshitch-Moshkovitz S. Dominissini D. Mansour A.A. Kol N. Salmon-Divon M. Hershkovitz V. Peer E. Mor N. Manor Y.S. et al.Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.Science. 2015; 347: 1002-1006Crossref PubMed Scopus (822) Google Scholar In addition, dysregulation of m6A modification could impair hematopoietic stem cell differentiation34Weng H. Huang H. Wu H. Qin X. Zhao B.S. Dong L. Shi H. Skibbe J. Shen C. Hu C. et al.METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m6A Modification.Cell Stem Cell. 2018; 22: 191-205.e9Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar and affect embryonic neural stem cell self-renewal.35Wang Y. Li Y. Yue M. Wang J. Kumar S. Wechsler-Reya R.J. Zhang Z. Ogawa Y. Kellis M. Duester G. Zhao J.C. N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications.Nat. Neurosci. 2018; 21: 195-206Crossref PubMed Scopus (157) Google Scholar Moreover, m6A modification was also involved in spermatogonial differentiation and meiosis initiation.36Xu K. Yang Y. Feng G.H. Sun B.F. Chen J.Q. Li Y.F. Chen Y.S. Zhang X.X. Wang C.X. Jiang L.Y. et al.Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation.Cell Res. 2017; 27: 1100-1114Crossref PubMed Scopus (158) Google Scholar However, the role of m6A RNA modification in cardiac differentiation of stem cells remains unknown. Our study indicated that ALKBH5-mediated m6A modification was significantly upregulated during the differentiation of MESs into CMs, and forced expression of ALKBH5 results in markedly inhibition of cardiac differentiation from MESs. Furthermore, ALKBH5 loss-of-function enhances the capacity of differentiation. Thus, our data demonstrate for the first time, to the best of our knowledge, that m6A RNA modification plays a key role in human stem cell cardiogenesis. Accumulating evidence has shown that epigenetic modifications play a critical role in cardiogenesis.28Ohtani K. Zhao C. Dobreva G. Manavski Y. Kluge B. Braun T. Rieger M.A. Zeiher A.M. Dimmeler S. Jmjd3 controls mesodermal and cardiovascular differentiation of embryonic stem cells.Circ. Res. 2013; 113: 856-862Crossref PubMed Scopus (61) Google Scholar, 29Wamstad J.A. Alexander J.M. Truty R.M. Shrikumar A. Li F. Eilertson K.E. Ding H. Wylie J.N. Pico A.R. Capra J.A. et al.Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.Cell. 2012; 151: 206-220Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 30Lee J. Shao N.Y. Paik D.T. Wu H. Guo H. Termglinchan V. Churko J.M. Kim Y. Kitani T. Zhao M.T. et al.SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation.Cell Stem Cell. 2018; 22: 428-444.e5Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,37Willems E. Mercola M. Jumonji and cardiac fate.Circ. Res. 2013; 113: 837-839Crossref PubMed Scopus (4) Google Scholar,38Guo X. Xu Y. Wang Z. Wu Y. Chen J. Wang G. Lu C. Jia W. Xi J. Zhu S. et al.A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis.Cell Stem Cell. 2018; 22: 893-908.e6Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar However, little is known regarding the contribution of histone-modifying enzymes in cardiac differentiation. Previous studies have shown that KDM5B and RBBP5 were widely associated with stem cell stemness maintenance and differentiation.31Nayak A. Viale-Bouroncle S. Morsczeck C. Muller S. The SUMO-specific isopeptidase SENP3 regulates MLL1/MLL2 methyltransferase complexes and controls osteogenic differentiation.Mol. Cell. 2014; 55: 47-58Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar,39Kidder B.L. Hu G. Zhao K. KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation.Genome Biol. 2014; 15: R32Crossref PubMed Scopus (82) Google Scholar,40He R. Kidder B.L. H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells.Nucleic Acids Res. 2017; 45: 6427-6441Crossref PubMed Scopus (28) Google Scholar However, the function of KDM5B or RBBP5 in cardiac lineage commitment was unknown. Our study revealed the integral role of KDM5B and RBBP5 in cardiogenesis. coIP combined with ChIP analysis indicated that during cardiac differentiation, KDM5B and RBBP5 inhibit the transcription of GATA4 by removing active H3K4me3 marks at its promoter regions. And RBBP5 overexpression and KDM5B inhibitor AS8351 treatment ameliorated the decreased property of cardiac differentiation arising from m6A reduction. Transcription factor GATA4 is a known master regulator of cardiogenesis.41Ang Y.S. Rivas R.N. Ribeiro A.J.S. Srivas R. Rivera J. Stone N.R. Pratt K. Mohamed T.M.A. Fu J.D. Spencer C.I. et al.Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.Cell. 2016; 167 (e1722): 1734-1749Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Our study identified ALKBH5-mediated m6A modification as a regulator of GATA4 and thus uncovered the key crosstalk between m6A and GATA4. This outside-in regulatory axis of ALKBH5-m6A methylation-H3K4me3-GATA4 provides a novel insight into an epigenetic mechanism that controls the fate of hESC cardiac differentiation. A recent study reported that m6A methyltransferase METTL14 modulates embryonic neural stem cell self-renewal through histone H3K27 modifications.35Wang Y. Li Y. Yue M. Wang

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