Report YTHDF2/3 Are Required for Somatic Reprogramming through Different RNA Deadenylation Pathways Graphical Abstract Authors Jiadong Liu, Mingwei Gao, Shuyang Xu, ..., Weiwei Liu, Xichen Bao, Jiekai Chen Correspondence [email protected] In Brief Liu et al. show that the YTHDF2-CCR4- NOT complex promotes the mRNA clearance of somatic genes, especially Tead2, paralleling the activity of the YTHDF3-PAN2-PAN3 deadenylase complex during reprogramming. Furthermore, Ythdf2/3 deficiency suppresses the mesenchymal-to- epithelial transition (MET) process and MEF-related chromatin loci silencing in the early stage of reprogramming and then decreases reprogramming efficiency. Highlights d Knockdown of Ythdf2/3, but not Ythdf1, inhibits somatic cell reprogramming d YTHDF2/3 recruits different deadenylase complexes to regulate mRNA clearance d YTHDF2/3 regulates the MET process through Hippo signaling pathway effector Tead2 d Ythdf2/3 deficiency represses silencing of MEF-related chromatin loci Liu et al., 2020, Cell Reports 32, 108120 September 8, 2020 ª 2020 The Author(s). https://doi.org/10.1016/j.celrep.2020.108120 ll ll OPEN ACCESS Report YTHDF2/3 Are Required for Somatic Reprogramming through Different RNA Deadenylation Pathways Jiadong Liu,1,2,3,7 Mingwei Gao,1,2,3,7 Shuyang Xu,1,2 Yaping Chen,1,2,3 Kaixin Wu,1,2,5 He Liu,1,2,4,5 Jie Wang,1,2 Xuejie Yang,4 Junwei Wang,1,2 Weiwei Liu,6 Xichen Bao,1,2,3,5 and Jiekai Chen1,2,3,4,5,8,* 1CAS Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China 2Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou 510530, China 3University of Chinese Academy of Sciences, Beijing 100049, China 4Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou 511436, China 5Guangzhou Regenerative Medicine and Health GuangDong Laboratory (GRMH-GDL), Guangzhou 510005, China 6CAS Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 7These authors contributed equally 8Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2020.108120 SUMMARY N6-methyladenosine (m6A), the most abundant reversible modification on eukaryote messenger RNA, is recognized by a series of readers, including the YT521-B homology domain family (YTHDF) proteins, which are coupled to perform physiological functions. Here, we report that YTHDF2 and YTHDF3, but not YTHDF1, are required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Mechanistically, we found that YTHDF3 recruits the PAN2-PAN3 deadenylase complex and conduces to reprogramming by promoting mRNA clearance of somatic genes, including Tead2 and Tgfb1, which parallels the activity of the YTHDF2-CCR4-NOT deadenylase complex. Ythdf2/3 deficiency represses mesenchymal-to-epithelial transition (MET) and chromatin silencing at loci containing the TEAD motif, contributing to decreased reprog- ramming efficiency. Moreover, RNA interference of Tgfb1 or the Hippo signaling effectors Yap1, Taz, and Tead2 rescues Ythdf2/3-defective reprogramming. Overall, YTHDF2/3 couples RNA deadenylation and regu- lation with the clearance of somatic genes and provides insights into iPSC reprogramming at the posttran- scriptional level. INTRODUCTION 2, have been identified as directly recognizing m6A modification within RNAs (Li et al., 2014; Xu et al., 2014; Zhu et al., 2014). In N6-methyladenosine (m6A), the most abundant modification of the nucleus, YTHDC1 regulates target mRNA splicing, nuclear messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) export, and X chromosome inactivation (Patil et al., 2016; in eukaryotes (Adams and Cory, 1975; Desrosiers et al., 1974), is Roundtree et al., 2017; Xiao et al., 2016). In the cytosol, YTHDF1 responsible for posttranscriptional regulation. m6A modifications interacts with initiation factors to facilitate the translational effi- are often located within the conserved sequence RR(m6A)CH (in ciency of target mRNAs (Wang et al., 2015), whereas YTHDF2 which R = G/A and H = A/C/U) in RNAs (Dominissini et al., 2012; accelerates target mRNA decay by recruiting the CCR4-NOT Meyer et al., 2012), are especially enriched in long exons and 30 deadenylase complex (Du et al., 2016; Wang et al., 2014a). In untranslated regions (30 UTRs), and are near the stop codons of addition, both YTHDF3 and YTHDC2 play a role in promoting mRNAs (Fu et al., 2014; Meyer and Jaffrey, 2014; Zhao et al., target mRNA translation and degradation (Hsu et al., 2017; Li 2017a). In mammals, m6A is catalyzed by a large methyltransfer- et al., 2017a; Shi et al., 2017). In recent years, accumulating ev- ase complex containing methyltransferase-like (METTL) 3, idence has demonstrated that the m6A modification is crucial in METTL14, and Wilms’ tumor 1-associating protein (WTAP) in regulating stem cell self-renewal, development, and somatic cell the nucleus (Bokar et al., 1997; Liu et al., 2014; Ping et al., reprogramming, playing a crucial role in cell fate transition 2014) and removed by the demethylase fat mass and obesity- (Aguilo et al., 2015; Batista et al., 2014; Chen et al., 2015; Geula associated protein (FTO) or the alkylated DNA repair protein et al., 2015; Wang et al., 2014b). alkB homolog 5 (ALKBH5) (Jia et al., 2011; Zheng et al., 2013). Somatic cell reprogramming by Yamanaka factors to induced In addition, the proteins containing an YTH domain, including pluripotent stem cells (iPSCs) is a system for studying the molec- YT521-B homology domain family (YTHDF) 1-3 and YTHDC1- ular mechanism of cell fate transition (Takahashi and Yamanaka, Cell Reports 32, 108120, September 8, 2020 ª 2020 The Author(s). 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS Report 2006). However, the effects of m6A modification in the genera- butions of YTHDF2 and YTHDF3 to reprogramming (Figures 1E tion of iPSCs are inconsistent, which might result from compli- and 1H). Then, we constructed YTHDF2 W432A and YTHDF3 cated biological functions of m6A(Aguilo et al., 2015; Chen W438A mutants in which a single amino acid residue in the et al., 2015). mRNA decay mediated by the m6A reader YTHDF2 YTH domain had been mutated (Figure 1F), which significantly was shown to be a critical regulator of hematopoietic stem and reduced the m6A RNA binding affinity (Li et al., 2014; Zhu progenitor cell specification (Zhang et al., 2017). In addition, in- et al., 2014). The decreased reprogramming efficiency could hibition of YTHDF2 promoted hematopoietic stem cell regenera- not be rescued by these point mutants, indicating that m6A tion and expansion (Li et al., 2018; Wang et al., 2018). The studies modification is important in this process (Figures 1G and 1H). imply that the m6A reader proteins YTHDFs may play a specific Ectopic protein expression was validated by western blotting role in cell fate transition. (Figures S1H and S1I). In conclusion, defects in Ythdf2/3 impair Here, we reported that knockdown of YTHDF2 or YTHDF3, but reprogramming by upregulating developmental genes and not YTHDF1, in mouse embryonic fibroblasts (MEFs) led to a sig- reduced cell proliferation, and these functions depend on m6A nificant m6A-dependent decrease of reprogramming efficiency. modification. We showed that YTHDF3 recruits the PAN2-PAN3 deadenylase complex to regulate target mRNA deadenylation, paralleling the Ythdf2/3 Deficiency Impairs Reprogramming through activity of the YTHDF2-CCR4-NOT deadenylase complex. Different mRNA Deadenylation Pathways Mechanistically, we found that Ythdf2/3 deficiency suppressed In mammals, three major deadenylase complexes have been the mRNA clearance of MEF-related genes, especially Tead2, characterized as decay enzymes involved in mRNA degradation: an effector of the Hippo signaling pathway that facilitates the the nine-subunit CCR4-NOT complex, the PAN2-PAN3 hetero- epithelial-to-mesenchymal transition (EMT), which is known to multimeric complex, and the PARN homodimer (Lau et al., repress reprogramming (Diepenbruck et al., 2014; Li et al., 2010). 2009; Mangus et al., 2004; Wu et al., 2005). YTHDF2 physically interacts with CNOT1 to recruit the CCR4-NOT complex and in- RESULTS duces mRNA deadenylation (Du et al., 2016). A recent study showed that YTHDF3 binds YTHDF2 to promote mRNA decay Ythdf2/3 Deficiency Impairs Somatic Cell (Shi et al., 2017). However, there is no evidence to illustrate Reprogramming in an m6A-Dependent Manner which pathway is responsible for YTHDF3-mediated deadenyla- To explore the possible features of YTHDFs during reprogram- tion (Figure 2A). To address this, we knocked down the mRNA ming, we used our previously described reprogramming system, levels of CCR4-NOT complex subunits CNOT1 or CCR4A to which includes ectopically expressed Oct4, Klf4, and Sox2 study their effects on reprogramming (Figures S2A and S2B). It (OKS) transcription factors; the optimized chemically defined is showed that knockdown of Cnot1 or Ccr4a reduced reprog- medium iCD1; and MEFs harboring the Oct4-GFP reporter ramming efficiency, which could not be rescued by overexpres- (Chen et al., 2011)(Figure 1A). We first knocked down the sion of YTHDF2 (Figures 2B, 2E, S2C, and S2D). In contrast, the mRNA of Ythdf1, Ythdf2,orYthdf3 throughout the reprogram- decreased reprogramming efficiency could be recovered by ming process using short