YTHDF2/3 Are Required for Somatic Reprogramming Through Different RNA Deadenylation Pathways

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

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 reprogramming 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 regulation with the clearance of somatic genes and provides insights into iPSC reprogramming at the posttranscriptional 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 hairpin RNAs (shRNAs) (Figures S1A YTHDF3 overexpression (Figures 2B and 2E). These results indi- and S1B). Interestingly, Ythdf2 or Ythdf3 deficiency, but not cate that the effect of YTHDF2 during somatic cell reprogram- Ythdf1 deficiency, significantly decreased the number of Oct4- ming relied on the CCR4-NOT complex, whereas YTHDF3 would GFP-positive iPSC colonies (Figures 1B and 1C). This finding recruit other poly(A) nuclease complexes to compensate for the was verified with the four classical Yamanaka factors OKSM defect in Cnot1 or Ccr4a. (OKS plus c-Myc)-mediated reprogramming under serum/leuke- To determine the deadenylase and deadenylases directly re- mia inhibitory factor (LIF) culture conditions (Figure S1C). Ythdf2/ cruited by YTHDF3 in mammals, we analyzed the YTHDF3-inter- 3 deficiency also reduced cell proliferation during reprogram- acting proteins from previously reported protein mass spectrom- ming, but there was no similar phenotype in MEFs, suggested etry data (Shi et al., 2017). The results revealed that CNOT1 and that it was reprogramming specifically (Figures 1D and S1D). PAN3 are protein partners of YTHDF3. To confirm this finding, we We tested the effects of YTHDF overexpression in both systems performed coimmunoprecipitation (coIP) assays to detect inter- and found that overexpression of YTHDF2 or YTHDF3 caused actions between YTHDF3 and subunits of these potential dead- only a slight boost in reprogramming (Figure S1E). The gene enylation complexes in cells at day 1 of reprogramming. A similar expression profiles at day 7 measured by RNA sequencing result was observed by affinity pull-down followed by western (RNA-seq) revealed that a portion of genes involved in develop- blotting, which showed that YTHDF2 interacts with CNOT1, ment and cell adhesion were upregulated after Ythdf2/3 knock- but not PAN3, in an RNA-independent manner (Figure 2C) (Du down (Figures S1F and S1G). et al., 2016). However, YTHDF3 was not co-purified with To determine whether the functions of YTHDF2 and YTHDF3 CNOT1, probably because of the indirect and weak interaction during reprogramming occur in an m6A-dependent manner, we between YTHDF3 and CNOT1 (Figure 2C). In addition, YTHDF3 overexpressed the human YTHDF1/2/3 proteins (homologs of coimmunoprecipitated with PAN3 (Figure 2C), demonstrating mouse YTHDF1/2/3 but one that could not be targeted by the that YTHDF3 recruits the PAN2-PAN3 complex to digest poly(A) corresponding mouse shRNAs) in Ythdf2/3-defective reprog- via an RNA decay pathway different from that used by YTHDF2. ramming to perform functional rescue experiments. The overex- We next examined the impact of YTHDF3-PAN2-PAN3 on repression of only YTHDF2 or YTHDF3 rescued the effects of programming. As expected, although knockdown of Pan2 or Ythdf2 or Ythdf3 knockdown, respectively, confirming the contri- Pan3 decreased reprogramming efficiency, there was no

2 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS

ABC

F H

Figure 1. Ythdf2/3 Deficiency Impairs Somatic Cell Reprogramming in an m6A-Dependent Manner (A) Schematic showing the time course and collection of data regarding MEF reprogramming. (B) Oct4-GFP-positive colonies of cells with Ythdf1/2/3 deficiency at day 7 of OKS-driven reprogramming. Luc, luciferase. (C) Whole-well scans from 24-well plates at day 7 of reprogramming in (B) using the GFP (Oct4-GFP) channel. Scale bar, 5 mm. (D) Growth curves of Ythdf2/3-knockdown cells or control cells during reprogramming. (E) Oct4-GFP-positive colonies of Ythdf2/3-knockdown cells with overexpressed YTHDF1/2/3 at day 7 of reprogramming. (F) Schematic of the Ythdf2 and Ythdf3 mutants. (G) Oct4-GFP-positive colonies of Ythdf2-orYthdf3-knockdown cells with overexpressed YTHDF2 W432A or YTHDF3 W438A at day 7 of reprogramming, respectively. (H) Oct4-GFP fluorescent scanning image of whole 24-well plates at day 7 of reprogramming related to (E) and (G). Scale bar, 5 mm. Data are presented as the mean ± SD of three (B, E, and G) or two (D) independent experiments. **p < 0.01, ***p < 0.001; NS, no significance; two-tailed Student’s t test. significant change in reprogramming efficiency after Parn knock- could rescue the phenotype (Figures 2D, 2E, and S2H), suggest- down (Figures S2E–S2G). Then, we overexpressed YTHDF2 or ing that the function of YTHDF3 depends on the PAN2-PAN3 YTHDF3 with Pan2 or Pan3 knockdown and assessed the complex. Moreover, simultaneous YTHDF2 and YTHDF3 knock- impact on reprogramming. Overexpression of only YTHDF2 down further damaged reprogramming to pluripotency (Figures

Cell Reports 32, 108120, September 8, 2020 3 ll OPEN ACCESS Report

E F

Figure 2. Ythdf2/3 Deﬁciency Impairs Reprogramming through Different mRNA Deadenylation Pathways (A) Schematics of the deadenylation of m6A-containing RNAs. The pathway used by YTHDF3 is unclear. (B) Oct4-GFP-positive colonies of Cnot1-orCcr4a-knockdown cells with overexpressed YTHDF1/2/3 at day 7 of reprogramming. (C) coIP assays between YTHDF2 or YTHDF3 and deadenylases. (D) Oct4-GFP-positive colonies of Pan2-orPan3-knockdown cells with overexpressed YTHDF1/2/3 at day 7 of reprogramming. (E) Whole-well ﬂuorescent scans from 24-well plates of cells related to (B) and (D). Scale bar, 5 mm. (F) Fluorescent images of the iPSCs generated in (G). Scale bar, 5 mm. (G) iPSCs generated from the knockdown of Ythdf2 or Ythdf3 alone or the simultaneous knockdown of Ythdf2 and Ythdf3. All data are presented as the mean ± SD of three independent experiments (B, D, and G). *p < 0.05, ***p < 0.001, two-tailed Student’s t test.

2F and 2G). These results veriﬁed that the deadenylation path- that m6A-marked somatic transcripts are readily removed by ways mediated by YTHDF2 or YTHDF3 have synergistic effects YTHDF2/3-mediated deadenylation during somatic cell reprog- during somatic cell reprogramming. ramming. Without YTHDF2/3, the unwanted presence of somatic transcripts will disrupt proper reprogramming progression. To Tead2 mRNA Is a Major Target of YTHDF2/3-Mediated verify this hypothesis, we ﬁrst treated the cells with shRNAs at Deadenylation in the Early Stage of Reprogramming different time points during reprogramming. Knockdown at Based on a model in which m6A marks transcripts for faster turn- earlier stages (day 0 to day 2) had a noticeable impact on reprog- over in cell fate transition (Batista et al., 2014), we hypothesized ramming, whereas treatment from day 3 to day 7 had little effect,

4 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS revealing that YTHDF2 and YTHDF3 are mainly required during revealed that TEAD2 predominantly controls genes that regu- the initial period of reprogramming (Figures 3A, S3A, and S3B). late cell migration and response to transforming growth factor We next performed RNA lifetime profiling of Ythdf2/3-knock- b (TGF-b) stimulus (Figure 4B). Based on these data, we down and control samples treated with actinomycin D at day 1 conclude that TEAD2 can promote the EMT process during of reprogramming by RNA-seq. Indeed, knockdown of Ythdf2/ reprogramming. 3 prolonged the lifetimes of MEF-related mRNAs that impede re- The mesenchymal-to-epithelial transition (MET) is an impor- programming, such as Tgfb1 (Figure 3B). Most somatic-specific tant and necessary process for the initiation of reprogramming transcripts were rapidly degraded, except Tead2 (Figures 3B (Li et al., 2010). A previous study showed that TEAD2 protein and S3C), an effector of Hippo signaling pathway that plays level upregulation led to the nuclear accumulation of YAP and important roles in stem cell self-renewal, development, growth TAZ and triggered EMT (Diepenbruck et al., 2014), which control, and tumor suppression (Halder and Johnson, 2011; impeded the MET process and repressed fibroblast reprogram- Ota and Sasaki, 2008; Pan, 2010; Ramos and Camargo, 2012). ming (Li et al., 2010). Therefore, we examined TEAD2-targeted Moreover, the protein level of TEAD2 was upregulated with EMT-related gene expression in our reprogramming system by knockdown of Ythdf2/3 (Figure 3C). Other genes involved in RNA-seq. As expected, EMT-related genes were upregulated Hippo pathway, such as Yap1 and Taz, exhibited no significant in Ythdf2/3-defective reprogramming (Figure 4C). In addition, change in lifetime (Figure 3B). In addition, the binding of YTHDF2 transcriptome analysis of MEFs, mESCs (mouse embryonic or YTHDF3 on Tead2 or Tgfb1 mRNA was affirmed by RIP (RNA stem cells), and iPSCs with Ythdf2/3 deficiency showed that immunoprecipitation) following qPCR (Figures 3D and S3D), and knockdown of Ythdf2/3 in MEFs, but not in mESCs and iPSCs, m6A-RIP assay showed that both Tead2 and Tgfb1 mRNA were could upregulate EMT-related genes, such as genes involved marked by m6A modification (Figure 3E). We examined the bind- in extracellular matrix organization and cell substrate adhesion ing strength of YTHDF2 or YTHDF3 on Tead2 mRNA with Ythdf3 (Figure S4C). Furthermore, we evaluated the EMT-related char- or Ythdf2 deficiency, respectively, but there was no significant acteristics of Ythdf2/3-defective cells in reprogramming via difference (Figure S3E). Knockdown of Cnot1 or Pan3 could pro- wound healing assay. The results showed that knockdown of long the lifetime of Tead2 mRNA, which could be recovered by Ythdf2/3 accelerated cell migration and wound healing (Figures overexpressing YTHDF3 or YTHDF2, respectively (Figure 3F). 4D and 4E). To confirm the results presented earlier, we also These results indicated that when YTHDF2 was knocked knocked down Tead2, Yap1, Taz, Tgfb1, and Hoxa9 during re- down, endogenous YTHDF3 was not enough to compensate programming in cells upon Ythdf2 or Ythdf3 knockdown. Knock- for the functions of YTHDF2 during reprogramming, but the over- down of Tead2, Yap1, Taz,orTgfb1 could rescue the decreased expressed YTHDF3 could strengthen the YTHDF3-PAN2/3 reprogramming efficiency mediated by Ythdf2 or Ythdf3 knock- pathway to compensate for the YTHDF2-CCR4/NOT pathway down (Figures 4F, 4G, S4D, and S4E). These results may be in mRNA degradation and rescue reprogramming efficiency, due to knockdown of Tead2 or Tgfb1 inhibiting the TEAD2-medi- and vice versa, indicating that YTHDF2-CCR4/NOT pathway ated EMT process and rescuing the decreased efficiency of and YTHDF3-PAN2/3 pathway regulated mRNA deadenylation Ythdf2/3-defective reprogramming. Moreover, the transcrip- in parallel but were not redundant during reprogramming. tional coactivators YAP1 and TAZ are required for transcriptional Furthermore, the overexpression of Tead2 and Tgfb1, but not activation of TEADs by physical interaction (Mahoney et al., Yap1, Taz, and Hoxa9, inhibited reprogramming (Figures 3G 2005; Vassilev et al., 2001). Therefore, even though the mRNA and S3G), and Tead2 overexpression decreased cell prolifera- lifetimes of Yap1 and Taz were not prolonged in Ythdf2/3-defec- tion during reprogramming, similar to knockdown of Ythdf2/3 tive reprogramming (Figure 3B), knockdown of these genes (Figures 1D and S3F). These results suggest that YTHDF2 and could suppress transcriptional activation of TEAD2 and rescue YTHDF3 are necessary for the degradation of MEF-associated the decreased reprogramming efficiency. Altogether, these re- mRNAs and that Tead2 mRNA is a major target of YTHDF2/3- sults demonstrate that Ythdf2/3 deficiency impedes reprogram- mediated RNA decay in the early stage of reprogramming. ming via accelerating EMT in a Hippo signaling-dependent manner. Ythdf2/3 Knockdown Inhibits Reprogramming by Promoting the EMT Process through the Hippo Signaling YTHDF2 and YTHDF3 Are Required for MEF-Related Pathway Chromatin Loci Closed during Reprogramming To elucidate the corresponding downstream mechanisms of Somatic transcription factors that could inhibit the MEF-related YTHDF2/3-TEAD2 in reprogramming, we performed chromatin chromatin loci closing are barriers to reprogramming (Li et al., immunoprecipitation sequencing (ChIP-seq) of TEAD2 at day 1 2017b). Therefore, we wondered whether the mRNA decay of of reprogramming. De novo motif analysis (Heinz et al., 2010) MEF-related transcription factors mediated by YTHDF2/3 would identified the classical TEAD2 binding motif as the most signifi- affect this process. To answer this question, we harvested cells cant one (Figure S4A). After peak annotation, we found that in our reprogramming system following treatment with Ythdf2, TEAD2 bound to the promoter region of many EMT-related Ythdf3, or control shRNAs for Assay for Transposase-Accessible genes in reprogramming, such as Zyx, Pdgfc, Amotl2, Cyr61, Chromatin with high-throughput sequencing (ATAC-seq) (Buen- and Ctgf (Figure 4A). The expression of these genes was in- rostro et al., 2013, 2015). For data processing, we first down- hibited by knockdown of Tead2 at day 1 of reprogramming (Fig- loaded and analyzed previously reported ATAC-seq data from ure S4B), demonstrated that these genes were directly regulated MEFs and ESCs as controls (Li et al., 2017b). We divided the by TEAD2. Gene Ontology analysis of TEAD2 binding genes peaks into two groups: the CO group (peaks indicating closed

Cell Reports 32, 108120, September 8, 2020 5 ll OPEN ACCESS Report

F G

Figure 3. Tead2 mRNA Is a Major Target of YTHDF2/3-Mediated Deadenylation in the Early Stage of Reprogramming (A) Effects of Ythdf2/3 knockdown on reprogramming at speciﬁc time frames. Ythdf2/3 was knocked down beginning on the indicated day and proceeding throughout the remainder of the reprogramming time course. GFP-positive colonies were counted at day 7. (B) mRNA lifetime of selected somatic genes at day 1 of reprogramming in Ythdf2/3-knockdown cells or control cells measured by RNA-seq. Data of cells treated with actinomycin D (ActD) were normalized to DMSO treatment. (C) Western blot analysis of TEAD2 protein level with knockdown of Ythdf2/3 on MEFs, reprogramming cells at day 1 and day 3. (D) YTHDF1-3 and GFP RIP-qPCR data of the indicated transcripts at day 1 of OKS-induced reprogramming. RIP data were shown as the percentage of input RNA and normalized to GFP. (E) m6A RIP-qPCR data of the indicated transcripts at day 1 of reprogramming. RIP data were shown as the percentage of input RNA. (F) qRT-PCR analysis of Tead2 mRNA lifetime at day 1 of Cnot1-orPan3-defective reprogramming with overexpression of DsRed, YTHDF2, or YTHDF3. Data of cells treated with ActD were normalized to DMSO treatment. (G) Effects of TEAD2, YAP1, TAZ, TGFB1, or HOXA9 overexpression in OKS-induced reprogramming. Colonies were counted at day 7. Data are presented as the mean ± SD of three (A, F, and G) or two (B, D, and E) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; NS, no signiﬁcance; two-tailed Student’s t test.

6 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS

DEF

Figure 4. Ythdf2/3 Loss of Function Impedes Reprogramming through the Hippo Pathway by Upregulating EMT Genes (A) Genome browser track of TEAD2 ChIP-seq peaks for selected genes at day 1 of reprogramming. (B) Gene Ontology analysis of the top 500 TEAD2-targeted genes of ChIP-seq data obtained from day 1 of reprogramming. (C) Heatmap of selected TEAD2-targeted EMT-related gene expression with Ythdf2/3 knockdown on different days during reprogramming. (D) Images of wound healing assay of Ythdf2/3-defective reprogramming from day 0 to day 2. Scale bar, 200 mm. (E) Quantitatively analysis of wound healing assay in (D) by ImageJ. Data are presented as the mean ± SD (n = 5). (F) Effects of the knockdown of selected transcription factors on Ythdf2/3-defective reprogramming. Data are presented as the mean ± SD of three independent experiments. (G) Fluorescent images from 24-well plates at day 7 of OKS-driven reprogramming related to (F). Scale bar, 5 mm. (H) Model of somatic cell reprogramming regulated by YTHDF2- and YTHDF3-mediated mRNA decay via the Hippo signaling pathway. **p < 0.01, ***p < 0.001, NS, no signiﬁcance, two-tailed Student’s t test.

Cell Reports 32, 108120, September 8, 2020 7 ll OPEN ACCESS Report chromatin in MEFs but open chromatin in ESCs), and the OC ramming system (Figure 3G), in which reprogramming processes group (peaks indicating open chromatin in MEFs but closed with fast kinetics and high efficiency (Chen et al., 2011) and might chromatin in ESCs); we then compared the peaks at each chro- have overcome most reprogramming barriers reported previ- matin locus in MEFs and ESCs as described previously (Li et al., ously. To conclude, our findings provide mechanistic insight 2017b). We compared the peaks between shYthdf2-or into how YTHDF2 and YTHDF3 modulate somatic cell reprog- shYthdf3-treated cells and control cells at days 1 and 3 of re- ramming at the posttranscriptional level. programming and divided the CO and OC groups of reprogramming data into two subgroups: the FO (failed to open) STAR+METHODS group and the FC (failed to close) group (Figure S4F). We focused on the peaks of the FC group, which far outnumbered Detailed methods are provided in the online version of this paper peaks of the FO group (Figure S4G). Among these peaks, we and include the following: identified TEAD2 binding genes such as Ctgf and Pdgfc and found that they were specific to the FC group (Figure S4H). d KEY RESOURCES TABLE We also analyzed the transcription factor motifs relevant to d RESOURCE AVAILABILITY FO and FC peaks. As predicted, the motifs of the reprogram- B Lead Contact ming factors OCT4, SOX2, and KLF4 were enriched in the B Materials Availability FO peaks, indicating that the reprogramming process was B Data and Code Availability impaired by the knockdown of Ythdf2/3, whereas the FC peaks d EXPERIMENTAL MODEL AND SUBJECT DETAILS were governed by the somatic transcription factors TEAD, B Mice AP-1, and the RUNX family (Figure S4I). Overall, these analyses B Cell lines and culture conditions reveal that the deadenylation of somatic transcripts regulated d METHOD DETAILS by YTHDF2/3 is essential for the closure of MEF-related chro- B Retrovirus infection and iPSCs induction matin loci in the initial stage of reprogramming. Somatic cell B Plasmid construction reprogramming is a highly heterogeneous process, but for B Lentiviral shRNAs technical reasons, the cells harvested for experiments were a B Reprogramming rescued assay bulk population consisting of reprogramming cells, non-reprog- B Western blot ramming cells, and cells in diverse reprogramming trajectories B RNA extraction and real-time quantitative PCR anal- without sorting in this study. Therefore, some less obvious ysis events may be masked that would be solved with single-cell B Co-immunoprecipitation (coIP) assays technologies. B mRNA lifetime profiling B RIP (RNA-immunoprecipitation)-qPCR DISCUSSION B m6A RIP-qPCR B Wound-healing assay 6 In this study, we investigated the function of m A through its B ChIP-seq readers and demonstrated that YTHDF2 and YTHDF3 are B RNA-seq required for the initial stage of somatic cell reprogramming in B ATAC-seq 6 an m A-dependent manner. We found that YTHDF3-mediated B RNA-seq data processing recruitment of the PAN2-PAN3 deadenylase complex has func- B ChIP-seq and ATAC-seq data processing tions in reprogramming synergistic with those of the YTHDF2- B Gene ontology analysis CCR4-NOT complex. Furthermore, we identified that Tead2 B Motif analysis mRNA was a major target of YTHDF2/3. Accumulation of d QUANTIFICATION AND STATISTICAL ANALYSIS Tead2 mRNA upon Ythdf2/3 knockdown promoted EMT and repressed the generation of iPSCs. Knockdown of the Hippo signaling effectors Yap1, Taz,orTead2 or the EMT-induced fac- SUPPLEMENTAL INFORMATION tor Tgfb1 could rescue this reduced reprogramming efficiency. Supplemental Information can be found online at https://doi.org/10.1016/j. In conclusion, YTHDF2 and YTHDF3 recruit different deadeny- celrep.2020.108120. lase complexes to facilitate the degradation of somatic mRNAs, such as Tead2, and help MEFs discard their somatic identity dur- ACKNOWLEDGMENTS ing reprogramming (Figure 4H). The impaired reprogramming of Ythdf2 deficiency could not We are grateful to Chih-Hung Hsu and Yang Yu for constructive suggestions. be recovered by Ythdf3 overexpression, and vice versa (Fig- This work was supported by the National Key R&D Program of China ure 1E), suggesting the mechanisms of YTHDF2 and YTHDF3 (2019YFA0110200 and 2016YFA0100400), the Frontier Science Research Pro- in mRNA decay are distinct. Further functional studies are gram of the CAS (ZDBS-LY-SM007), the Key Research and Development Pro- needed to reveal the detailed mechanisms of these proteins in gram of Guangzhou Regenerative Medicine and Health Guangdong Labora- tory (2018GZR110104003), the Science and Technology Planning Project of cell fate transition. In addition, YAP was described as a pluripo- Guangdong Province (2017B030314056), the China Postdoctoral Science tent factor involved in ESC maintenance that accelerates so- Foundation (2019M662859), the National Postdoctoral Program for Innovative matic cell reprogramming (Lian et al., 2010; Tamm et al., 2011; Talent (BX20190089), and the National Natural Science Foundation of China Zhao et al., 2017b). YAP had no obvious influence in our reprog- (31771424).

8 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS

AUTHOR CONTRIBUTIONS Fu, Y., Dominissini, D., Rechavi, G., and He, C. (2014). Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, J.L. and M.G. carried out most experiments and assembled the ﬁgures. J.L. 293–306. and S.X. performed the bioinformatics analysis. Y.C. and K.W. were involved Geula, S., Moshitch-Moshkovitz, S., Dominissini, D., Mansour, A.A., Kol, N., in plasmid constructions and high-throughput sequencing library construc- Salmon-Divon, M., Hershkovitz, V., Peer, E., Mor, N., Manor, Y.S., et al. tions. H.L., X.Y., and Junwei Wang conducted high-throughput sequencing. (2015). Stem cells. m6A mRNA methylation facilitates resolution of naı¨ve plu- X.B. and W.L. provided advice and suggestions about the experimental de- ripotency toward differentiation. Science 347, 1002–1006. signs. J.L., M.G., and J.C. wrote the manuscript, and Jie Wang helped to improve it. J.C. conceived and supervised the project. Gu, J., Wang, M., Yang, Y., Qiu, D., Zhang, Y., Ma, J., Zhou, Y., Hannon, G.J., and Yu, Y. (2018). GoldCLIP: Gel-omitted Ligation-dependent CLIP. Genomics Proteomics Bioinformatics 16, 136–143. DECLARATION OF INTERESTS Guo, L., Lin, L., Wang, X., Gao, M., Cao, S., Mai, Y., Wu, F., Kuang, J., Liu, H., Yang, J., et al. (2019). Resolving Cell Fate Decisions during Somatic Cell Re- The authors declare no competing interests. programming by Single-Cell RNA-Seq. Mol. Cell 73, 815–829.e7.

Received: January 17, 2020 Halder, G., and Johnson, R.L. (2011). Hippo signaling: growth control and Revised: July 12, 2020 beyond. Development 138, 9–22. Accepted: August 17, 2020 Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Published: September 8, 2020 Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage- determining transcription factors prime cis-regulatory elements required for REFERENCES macrophage and B cell identities. Mol. Cell 38, 576–589. Hsu, P.J., Zhu, Y., Ma, H., Guo, Y., Shi, X., Liu, Y., Qi, M., Lu, Z., Shi, H., Wang, Adams, J.M., and Cory, S. (1975). Modified nucleosides and bizarre 50-termini J., et al. (2017). Ythdc2 is an N6-methyladenosine binding protein that regu- in mouse myeloma mRNA. Nature 255, 28–33. lates mammalian spermatogenesis. Cell Res. 27, 1115–1127. Aguilo, F., Zhang, F., Sancho, A., Fidalgo, M., Di Cecilia, S., Vashisht, A., Lee, Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., D.F., Chen, C.H., Rengasamy, M., Andino, B., et al. (2015). Coordination of Yang, Y.G., and He, C. (2011). N6-methyladenosine in nuclear RNA is a major m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Plurip- substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887. otency and Reprogramming. Cell Stem Cell 17, 689–704. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Batista, P.J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D.M., Lu- Bowtie 2. Nat. Methods 9, 357–359. jan, E., Haddad, B., Daneshvar, K., et al. (2014). m(6)A RNA modification con- Lau, N.C., Kolkman, A., van Schaik, F.M., Mulder, K.W., Pijnappel, W.W., trols cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, Heck, A.J., and Timmers, H.T. (2009). Human Ccr4-Not complexes contain 707–719. variable deadenylase subunits. Biochem. J. 422, 443–453. Bokar, J.A., Shambaugh, M.E., Polayes, D., Matera, A.G., and Rottman, F.M. Li, B., and Dewey, C.N. (2011). RSEM: accurate transcript quantification from (1997). Purification and cDNA cloning of the AdoMet-binding subunit of the hu- RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, man mRNA (N6-adenosine)-methyltransferase. RNA 3, 1233–1247. 323. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., (2013). Transposition of native chromatin for fast and sensitive epigenomic Abecasis, G., and Durbin, R.; 1000 Genome Project Data Processing Sub- profiling of open chromatin, DNA-binding proteins and nucleosome position. group (2009). The Sequence Alignment/Map format and SAMtools. Bioinfor- Nat. Methods 10, 1213–1218. matics 25, 2078–2079. Buenrostro, J.D., Wu, B., Chang, H.Y., and Greenleaf, W.J. (2015). ATAC-seq: Li, R., Liang, J., Ni, S., Zhou, T., Qing, X., Li, H., He, W., Chen, J., Li, F., Zhuang, A Method for Assaying Chromatin Accessibility Genome-Wide. Curr. Protoc. Q., et al. (2010). A mesenchymal-to-epithelial transition initiates and is required Mol. Biol. 109, 21.29.1–21.29.9. for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7, 51–63. Chen, J., Liu, J., Chen, Y., Yang, J., Chen, J., Liu, H., Zhao, X., Mo, K., Song, H., Li, F., Zhao, D., Wu, J., and Shi, Y. (2014). Structure of the YTH domain of hu- Guo, L., et al. (2011). Rational optimization of reprogramming culture condi- man YTHDF2 in complex with an m(6)A mononucleotide reveals an aromatic tions for the generation of induced pluripotent stem cells with ultra-high effi- cage for m(6)A recognition. Cell Res. 24, 1490–1492. ciency and fast kinetics. Cell Res. 21, 884–894. Li, A., Chen, Y.S., Ping, X.L., Yang, X., Xiao, W., Yang, Y., Sun, H.Y., Zhu, Q., Chen, T., Hao, Y.J., Zhang, Y., Li, M.M., Wang, M., Han, W., Wu, Y., Lv, Y., Baidya, P., Wang, X., et al. (2017a). Cytoplasmic m6A reader YTHDF3 pro- Hao, J., Wang, L., et al. (2015). m(6)A RNA methylation is regulated by micro- motes mRNA translation. Cell Res. 27, 444–447. RNAs and promotes reprogramming to pluripotency. Cell Stem Cell 16, 289–301. Li, D., Liu, J., Yang, X., Zhou, C., Guo, J., Wu, C., Qin, Y., Guo, L., He, J., Yu, S., et al. (2017b). Chromatin Accessibility Dynamics during iPSC Reprogramming. Desrosiers, R., Friderici, K., and Rottman, F. (1974). Identification of methyl- Cell Stem Cell 21, 819–833.e6. ated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc. Natl. Acad. Sci. USA 71, 3971–3975. Li, Z., Qian, P., Shao, W., Shi, H., He, X.C., Gogol, M., Yu, Z., Wang, Y., Qi, M., Zhu, Y., et al. (2018). Suppression of m6A reader Ythdf2 promotes hematopoi- Diepenbruck, M., Waldmeier, L., Ivanek, R., Berninger, P., Arnold, P., van Nim- etic stem cell expansion. Cell Res. 28, 904–917. wegen, E., and Christofori, G. (2014). Tead2 expression levels control the sub- cellular distribution of Yap and Taz, zyxin expression and epithelial-mesen- Lian, I., Kim, J., Okazawa, H., Zhao, J., Zhao, B., Yu, J., Chinnaiyan, A., Israel, chymal transition. J. Cell Sci. 127, 1523–1536. M.A., Goldstein, L.S., Abujarour, R., et al. (2010). The role of YAP transcription Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch, J., Amariglio, N., Kupiec, 24, 1106–1118. M., et al. (2012). Topology of the human and mouse m6A RNA methylomes re- Liu, J., Yue, Y., Han, D., Wang, X., Fu, Y., Zhang, L., Jia, G., Yu, M., Lu, Z., vealed by m6A-seq. Nature 485, 201–206. Deng, X., et al. (2014). A METTL3-METTL14 complex mediates mammalian nu- Du, H., Zhao, Y., He, J., Zhang, Y., Xi, H., Liu, M., Ma, J., and Wu, L. (2016). clear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95. YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold the CCR4-NOT deadenylase complex. Nat. Commun. 7, 12626. change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.

Cell Reports 32, 108120, September 8, 2020 9 ll OPEN ACCESS Report

Mahoney, W.M., Jr., Hong, J.H., Yaffe, M.B., and Farrance, I.K. (2005). The Src/Yes-associated protein localized in the cytoplasm. Genes Dev. 15, transcriptional co-activator TAZ interacts differentially with transcriptional 1229–1241. enhancer factor-1 (TEF-1) family members. Biochem. J. 388, 217–225. Wang, X., Lu, Z., Gomez, A., Hon, G.C., Yue, Y., Han, D., Fu, Y., Parisien, M., Mangus, D.A., Evans, M.C., Agrin, N.S., Smith, M., Gongidi, P., and Jacobson, Dai, Q., Jia, G., et al. (2014a). N6-methyladenosine-dependent regulation of A. (2004). Positive and negative regulation of poly(A) nuclease. Mol. Cell. Biol. messenger RNA stability. Nature 505, 117–120. 24, 5521–5533. Wang, Y., Li, Y., Toth, J.I., Petroski, M.D., Zhang, Z., and Zhao, J.C. (2014b). Meyer, K.D., and Jaffrey, S.R. (2014). The dynamic epitranscriptome: N6- N6-methyladenosine modification destabilizes developmental regulators in methyladenosine and gene expression control. Nat. Rev. Mol. Cell Biol. 15, embryonic stem cells. Nat. Cell Biol. 16, 191–198. 313–326. Wang, X., Zhao, B.S., Roundtree, I.A., Lu, Z., Han, D., Ma, H., Weng, X., Chen, Meyer, K.D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C.E., and Jaffrey, K., Shi, H., and He, C. (2015). N(6)-methyladenosine Modulates Messenger S.R. (2012). Comprehensive analysis of mRNA methylation reveals enrichment RNA Translation Efficiency. Cell 161, 1388–1399. in 30 UTRs and near stop codons. Cell 149, 1635–1646. Ota, M., and Sasaki, H. (2008). Mammalian Tead proteins regulate cell prolif- Wang, H., Zuo, H., Liu, J., Wen, F., Gao, Y., Zhu, X., Liu, B., Xiao, F., Wang, W., 6 eration and contact inhibition as transcriptional mediators of Hippo signaling. Huang, G., et al. (2018). Loss of YTHDF2-mediated m A-dependent mRNA Development 135, 4059–4069. clearance facilitates hematopoietic stem cell regeneration. Cell Res. 28, 1035–1038. Pan, D. (2010). The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505. Wu, M., Reuter, M., Lilie, H., Liu, Y., Wahle, E., and Song, H. (2005). Structural Patil, D.P., Chen, C.K., Pickering, B.F., Chow, A., Jackson, C., Guttman, M., insight into poly(A) binding and catalytic mechanism of human PARN. EMBO J. and Jaffrey, S.R. (2016). m(6)A RNA methylation promotes XIST-mediated 24, 4082–4093. transcriptional repression. Nature 537, 369–373. Xiao, W., Adhikari, S., Dahal, U., Chen, Y.S., Hao, Y.J., Sun, B.F., Sun, H.Y., Li, Ping, X.L., Sun, B.F., Wang, L., Xiao, W., Yang, X., Wang, W.J., Adhikari, S., A., Ping, X.L., Lai, W.Y., et al. (2016). Nuclear m(6)A Reader YTHDC1 Regulates Shi, Y., Lv, Y., Chen, Y.S., et al. (2014). Mammalian WTAP is a regulatory sub- mRNA Splicing. Mol. Cell 61, 507–519. unit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, Xu, C., Wang, X., Liu, K., Roundtree, I.A., Tempel, W., Li, Y., Lu, Z., He, C., and 177–189. Min, J. (2014). Structural basis for selective binding of m6A RNA by the Ramı´rez, F., Dundar,€ F., Diehl, S., Gruning,€ B.A., and Manke, T. (2014). deep- YTHDC1 YTH domain. Nat. Chem. Biol. 10, 927–929. Tools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Yu, G., Wang, L.G., Han, Y., and He, Q.Y. (2012). clusterProfiler: an R package Res. 42, W187–W191. for comparing biological themes among gene clusters. OMICS 16, 284–287. Ramos, A., and Camargo, F.D. (2012). The Hippo signaling pathway and stem Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., cell biology. Trends Cell Biol. 22, 339–346. Nusbaum, C., Myers, R.M., Brown, M., Li, W., and Liu, X.S. (2008). Model- Risso, D., Schwartz, K., Sherlock, G., and Dudoit, S. (2011). GC-content based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137. normalization for RNA-Seq data. BMC Bioinformatics 12, 480. Zhang, C., Chen, Y., Sun, B., Wang, L., Yang, Y., Ma, D., Lv, J., Heng, J., Ding, Roundtree, I.A., Luo, G.Z., Zhang, Z., Wang, X., Zhou, T., Cui, Y., Sha, J., Y., Xue, Y., et al. (2017). m6A modulates haematopoietic stem and progenitor Huang, X., Guerrero, L., Xie, P., et al. (2017). YTHDC1 mediates nuclear export cell specification. Nature 549, 273–276. of N6-methyladenosine methylated mRNAs. eLife 6, e31311. Shi, H., Wang, X., Lu, Z., Zhao, B.S., Ma, H., Hsu, P.J., Liu, C., and He, C. Zhao, B.S., Roundtree, I.A., and He, C. (2017a). Post-transcriptional gene (2017). YTHDF3 facilitates translation and decay of N6-methyladenosine- regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42. modified RNA. Cell Res. 27, 315–328. Zhao, Y., Fei, X., Guo, J., Zou, G., Pan, W., Zhang, J., Huang, Y., Liu, T., and Stewart, S.A., Dykxhoorn, D.M., Palliser, D., Mizuno, H., Yu, E.Y., An, D.S., Sa- Cheng, W. (2017b). Induction of reprogramming of human amniotic epithelial batini, D.M., Chen, I.S., Hahn, W.C., Sharp, P.A., et al. (2003). Lentivirus-deliv- cells into iPS cells by overexpression of Yap, Oct4, and Sox2 through the acti- ered stable gene silencing by RNAi in primary cells. RNA 9, 493–501. vation of the Hippo-Yap pathway. Exp. Ther. Med. 14, 199–206. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells Zheng, G., Dahl, J.A., Niu, Y., Fedorcsak, P., Huang, C.M., Li, C.J., Va˚ gbø, from mouse embryonic and adult fibroblast cultures by defined factors. Cell C.B., Shi, Y., Wang, W.L., Song, S.H., et al. (2013). ALKBH5 is a mammalian 126, 663–676. RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Tamm, C., Bo¨ wer, N., and Annere´ n, C. (2011). Regulation of mouse embryonic Cell 49, 18–29. stem cell self-renewal by a Yes-YAP-TEAD2 signaling pathway downstream of Zhu, T., Roundtree, I.A., Wang, P., Wang, X., Wang, L., Sun, C., Tian, Y., Li, J., LIF. J. Cell Sci. 124, 1136–1144. He, C., and Xu, Y. (2014). Crystal structure of the YTH domain of YTHDF2 re- Vassilev, A., Kaneko, K.J., Shu, H., Zhao, Y., and DePamphilis, M.L. (2001). veals mechanism for recognition of N6-methyladenosine. Cell Res. 24, 1493– TEAD/TEF transcription factors utilize the activation domain of YAP65, a 1496.

10 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Monoclonal anti-GAPDH-HRP KangCheng Bio-tech Cat# KC-5G5; RRID: AB_2631280 Rabbit Polyclonal anti-YTHDF1 Proteintech Cat# 17479-1-AP; RRID: AB_2217473 Rabbit Polyclonal anti-YTHDF2 Aviva systems biology Cat# ARP67917_P050; RRID: AB_2861185 Rabbit Polyclonal anti-YTHDF3 Abcam Cat# ab103328; RRID:AB_10710895 Rabbit Polyclonal IgG Abcam Cat# ab37415; RRID:AB_2631996 Rabbit Polyclonal anti-CNOT1 Proteintech Cat# 14276-1-AP; RRID:AB_10888627 Rabbit Monoclonal anti-CCR4A Abcam Cat# ab221151; RRID: AB_2861188 Rabbit Polyclonal anti-PAN2 Proteintech Cat# 16427-1-AP; RRID:AB_1851434 Rabbit Polyclonal anti-PAN3 Abcam Cat# ab88642; RRID:AB_10714404 Rabbit Polyclonal anti-PARN Proteintech Cat# 13799-1-AP; RRID:AB_2160430 Rabbit Polyclonal anti-TEAD2 Proteintech Cat# 21159-1-AP; RRID: AB_2861186 Rabbit Polyclonal anti-TAZ Proteintech Cat# 23306-1-AP; RRID:AB_2721185 Rabbit Polyclonal anti-YAP1 Proteintech Cat# 13584-1-AP; RRID:AB_2218915 Rabbit Polyclonal anti-TGF-Beta 1 Proteintech Cat# 21898-1-AP; RRID:AB_2811115 Rabbit Polyclonal anti-HOXA9 Proteintech Cat# 18501-1-AP; RRID: AB_2861187 Mouse Monoclonal anti-FLAG Sigma Cat# F1804; RRID:AB_262044 Mouse Monoclonal anti-Halo Promega Cat# G9211; RRID:AB_2688011 Chemicals, Peptides, and Recombinant Proteins Leukemia Inhibitory Factor (LIF) Millipore Cat# ESGE107 CHIR99021 Sigma Cat# SML1046-5MG PD0325901 Sigma Cat# PZ0162-5MG Fetal Bovine Serum Natocor Cat# SFBE Fetal Bovine Serum Front Biomedical Cat# OPT500 DMEM-Dulbecco’s Modiﬁed Eagle Medium, High HyClone Cat# SH30022.01 Glucose GlutaMAX GIBCO Cat# 35050079 Non-Essential Amino Acids Solution GIBCO Cat# 11140076 2-Mercaptoethoethanol GIBCO Cat# 21985-023 Sodium Pyruvate Solution GIBCO Cat# 11360070 Penicillin-Streptomycin Solution Hyclone Cat# SV30010 Phosphate-Buffer Saline (PBS) GIBCO Cat# C14190500BT DNase/RNase Free Deionized Water TIANGEN Cat# RT121 Lipofectamine 3000 Reagent Thermo Fisher Cat# L3000001 Trypsin-EDTA (0.25%), phenol red GIBCO Cat# 25200114 0.1% Gelatin Solution Millipore Cat# ES-006-B RRI Promega Cat# N2511 TRI Reagent MRC Cat# TR118-200 Oligo-dT TaKaRa Cat# 3806 ChamQ Universal SYBR qPCR master mix Vazyme Cat# Q711-02 ReverTra Ace Toyobo Cat# TRT-101 Polybrene Sigma Cat# 40804ES76 Puromycin GIBCO Cat# A1113802 iCD1 medium DeliCell Cat# 820250 (Continued on next page)

Cell Reports 32, 108120, September 8, 2020 e1 ll OPEN ACCESS Report

Continued REAGENT or RESOURCE SOURCE IDENTIFIER Actinomycin D Sigma Cat# A1410 DMSO Sigma Cat# D1435 Halo-tagged beads Promega Cat# G7282 PK enzyme NEB Cat# P8107S ECL solution Millipore Cat# WBULS0500 RNase A Thermo Scientific Cat# EN0531 EDTA-free protease inhibitor cocktail Roche Cat# 4693132001 Protease Inhibitor Cocktail Promega Cat# G6521 DTT Sigma Cat# 43816 Triton X-100 Sigma Cat# T8787 IGEPAL CA-630 Sigma Cat# 198596 Tween20 Sigma Cat# P1379 Dynabeads Protein A Invitrogen Cat# 10008D Dynabeads Protein G Invitrogen Cat# 10009D ERCC ExFold RNA spike-in control Invitrogen Cat# 4456739 Critical Commercial Assays VAHTS Universal DNA Library Prep Kit for Illumina V3 Vazyme Cat# ND607 VAHTS mRNA-seq V3 Library Prep Kit for Illumina Vazyme Cat# NR611 MinElute PCR Purification Kit QIAGEN Cat# 28006 EpiMark N6-Methyladenosine Enrichment Kit NEB Cat# E1610S VAHTS DNA Clean Beads Vazyme Cat# NR411 TruePrep DNA Library Prep Kit V2 for Illumina Vazyme Cat# TD501 VAHTS Library Quantification Kit for Illumina Vazyme Cat# NQ101 NextSeq 500 using a NextSeq 500 High Output Kit v2 Illumina Cat# FC-404-2002 (150 cycles) Deposited Data RNA-seq data, ChIP-seq data and ATAC-seq data This study GEO: GSE156437 ATAC-seq data of MEFs and ESCs (Li et al., 2017b) GEO: GSE93029 Experimental Models: Cell Lines OG2 mouse embryonic stem cells (mESCs) This study N/A Mouse induced pluripotent stem cell lines (iPSCs) This study N/A Primary mouse embryonic fibroblast (MEFs) This study N/A Platinum-E (Plat-E) A gift from The Fourth Military N/A Medical University HEK293T ATCC Cat# CRL-1126 Experimental Models: Organisms/Strains OG2 transgenic mice: CBA/CaJ x C57BL/6J The Jackson Laboratory Mouse strain datasheet: 004654 129 mice Beijing Vital River Laboratory Stock No.: 217 Animal Technology Oligonucleotides Oligonucleotides are summarized in Tables S1 and S2 This study N/A Recombinant DNA pMX-Oct3/4 (Takahashi and Yamanaka, 2006) Addgene Cat# 13366 pMX-Sox2 (Takahashi and Yamanaka, 2006) Addgene Cat# 13367 pMX-Klf4 (Takahashi and Yamanaka, 2006) Addgene Cat# 13370 pMX-cMyc (Takahashi and Yamanaka, 2006) Addgene Cat# 13375 PLKO.1-puro (Stewart et al., 2003) Addgene Cat# 8453 (Continued on next page)

e2 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS

Continued REAGENT or RESOURCE SOURCE IDENTIFIER psPAX2 a gift from Didier Trono Addgene Cat# 12260 pMD2.G a gift from Didier Trono Addgene Cat# 12259 pSUPER-puro OligoEngine Cat# VEC-pBS-0008 pMX-DsRed This study N/A pMX-YTHDF1 This study N/A pMX-YTHDF2 This study N/A pMX-YTHDF3 This study N/A pMX-YTHDF2-W432A This study N/A pMX-YTHDF3-W438A This study N/A pMX-Tead2 This study N/A pMX-Yap1 This study N/A pMX-Taz This study N/A pMX-Tgfb1 This study N/A pMX-Hoxa9 This study N/A pMX-Halo-Ythdf1 This study N/A pMX-Halo-Ythdf2 This study N/A pMX-Halo-Ythdf3 This study N/A pMX-Halo-Ythdf2 W432A This study N/A pMX-Halo-Ythdf3 W438A This study N/A pMX-Halo-GFP This study N/A Software and Algorithms ZEN Zeiss https://www.zeiss.com/microscopy/int/ products/microscope-cameras.html Bio-RAD CFX Manager BIO-RAD http://www.bio-rad.com/en-us/product/ cfx-manager-software?tab=Download Prism 6 GraphPad https://www.graphpad.com/ scientificsoftware/prism/ ImageJ NIH, USA https://imagej.nih.gov/ij Bowtie2 (Langmead and Salzberg, 2012) http://bowtie-bio.sourceforge.net/bowtie2/ manual.shtml MACS2 (Zhang et al., 2008) https://pypi.org/project/MACS2/ Samtools (Li et al., 2009) http://www.htslib.org/ Deeptools (Ramı´rez et al., 2014) https://deeptools.readthedocs.io/en/ develop/ Homer (Heinz et al., 2010) http://homer.ucsd.edu/homer/ EDAseq (Risso et al., 2011) https://bioconductor.org/packages/devel/ bioc/html/EDASeq.html DESeq2 (Love et al., 2014) R package DESeq2 clusterProfile (Yu et al., 2012) R package clusterProfiler Other Bioruptor Plus sonicator Diagenode Cat# B01020001

RESOURCE AVAILABILITY

Lead Contact Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Jiekai Chen ([email protected])

Cell Reports 32, 108120, September 8, 2020 e3 ll OPEN ACCESS Report

Materials Availability All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and Code Availability The accession number for the RNA-seq, mRNA lifetime proﬁling, TEAD2 ChIP-seq and ATAC-seq datasets reported in this paper is GEO: GSE156437.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice All animal procedures were performed according to NIH guidelines. Animals were housed in individually ventilated cages containing sterile quarter-inch corncob bedding for environmental enrichment. Mice were maintained on 12 h light/dark cycle and provided with food and water in individually ventilated units in a temperature controlled room (22 - 23C). Animal care and experimental protocols were approved by the Guangzhou Institutes of Biomedicine and Health Ethical Committee. OG2 (Oct4-GFP reporter) transgenic mice (CBA/CaJ 3 C57BL/6J) were original from Jackson Laboratory (Mouse strain datasheet: 004654). 129 (Stock No.: 217) mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Female and male mice were used between 8 and 12 weeks of age. Male OG2 mice and female 129 mice were crossed to generate Mouse embryonic ﬁbroblasts (MEFs), and all embryos included both females and males.

Cell lines and culture conditions MEFs were derived from E13.5 embryos from female 129 mice crossing with male OG2 mice. HEK293T cells were purchased from ATCC (CRL-1126), and Plat-E cell line was a gift from The Fourth Military Medical University. OG2-MEFs, HEK293T and Plat-E cells were maintained in high-glucose Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Natocor, Cat# SFBE), 1 3 GlutaMAX, and nonessential amino acids (1 3 NEAAs). Male OG2 mESCs were derived from E3.5 embryos ICM from female 129 mice crossing male OG2 mice. iPS cell lines were derived from OCT4/SOX2/KLF4 retrovirus induced MEFs with the same genetic background as the OG2 mESCs. mESCs and iPSCs were cultured on gelatin coated plate with high-glucose DMEM supplemented with 15% FBS (Front Biomedical, Cat# OPT500), 1 3 GlutaMAX, 1 3 NEAAs, 1 3 Penicillin-Streptomycin, 1 3 Sodium Pyruvate, 0.1 mM b-Mercaptoethanol, 2i (1 mM PD0325901, 3 mM CHIR99021) and leukemia inhibitory factor (LIF). All the cells were cultured at 37 C with 5% CO2.

METHOD DETAILS

Retrovirus infection and iPSCs induction iPSCs were generated as previously described (Chen et al., 2011; Guo et al., 2019). In brief, 8 3 106 Plat-E cells were plated onto 10 cm dish and cultured overnight, and then retrovirus were made by transfection of 25 mg of pMXs vector DNA containing Oct4, Sox2, and Klf4 or pSUPER-puro vector DNA containing shRNAs into Plat-E cells using the calcium phosphate method. The cells were incubated with DNA-calcium phosphate mixture overnight, and 12 hours after transfection, the medium was replaced with fresh 10% FBS medium. The viral supernatants were collected 48 hours and 72 hours posttransfection and filtered through a 0.45 mm filter (Millipore). MEFs were plated at density of 1 3 104 per well (24-well plate) 12 hours before infection with retrovirus and a final concentration of 4 mg/ml polybrene was added during the infection. The infection was performed repeatedly the following day and the shRNA retrovirus infected cells were selected with 1 mg/ml puromycin for two days (from reprogramming D 1 to D1). Day 0 was defined as the day that viral supernatants were removed and the specific reprogramming medium were added. OKS iPSCs were induced in iCD1 medium (Chen et al., 2011), and OKSM iPSCs were generated in serum/LIF medium. iPSC colonies were picked at day 7 and cultured in mESC medium supplemented with 2i and LIF. Whole-well fluorescent scans were taken at day 7 using a living cell workstation (Nikon, BioStation CT).

Plasmid construction Human YTHDF1, YTHDF2, YTHDF3, YTHDF2-W432A, and YTHDF3-W438A and mouse Tead2, Yap1, Taz, Tgdf1, and Hoxa9 were generated and cloned into the pMXs-3 3 Flag vector. Halo-tagged mouse Ythdf1, Ythdf2 and Ythdf3 and GFP were also cloned into the pMXs-3 3 Flag vector. The expression of these genes was checked by western blot analysis. The shRNAs used for reprogramming in this study were cloned into the pSUPER-puro vector. Five shRNA sequences were designed for each gene and the two with the highest knockdown efficiency confirmed by qPCR and western blotting were selected for the next functional studies. The sequences of the shRNAs used to efficiently target genes are listed in Table S1.

Lentiviral shRNAs shRNA targeting sequences against Ythdf2, Ythdf3 and control were designed, synthesized and subloned into the PLKO.1-puro vector (not use for reprogramming) (Stewart et al., 2003). 1.5 3 106 293T cells were plated onto 6-well plate and cultured overnight, and e4 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS then lentivirus was made by co-transfection of each pLKO.1 shRNA vector with psPAX2 and pMD2.G in a 5:3:2 ratio into 293T cells using the calcium phosphate method. The viral supernatants were collected 48 hours post-transfection and ﬁltered through a 0.45 mm ﬁlter (Millipore). 2 3 105 MEFs, mESCs or iPCs were seeded in a 6-well plate coated by 0.1% gelatin and infected with each lentivirus supernatant in the presence of 4 mg/ml polybrene. Medium was replaced by fresh media with 2 mg/ml puromycin for MEFs and 1 mg/ml puromycin for mESCs and iPSCs at 12 hours post-infection. After continuous puromycin selection for 3 days, the survived cells were collected for RNA-seq.

Reprogramming rescued assay Retroviral production and infection was performed as described above. To knock-down Ythdf1/2/3, Cnot1, Ccr4a or Pan2/3 during reprogramming, each shRNA retrovirus against these genes were co-infected with reprogramming factor retrovirus into MEFs. To perform the rescued assay, these MEFs were also infected with retrovirus that overexpressing human YTHDF1, YTHDF2, YTHDF3, YTHDF2-W432A or YTHDF3-W438A, respectively. The cells were selected with 1 mg/ml puromycin for two days (from reprogramming D 1 to D1). The shRNA knockdown efﬁciency and protein expression level were measured by western blotting at day 1 and the Oct4-GFP colonies were counted at day 7 of reprogramming.

Western blot Cells were collected, lysed in RIPA buffer supplemented with protease inhibitor cocktail on ice for 15 min followed by boiled at 100C for 10 min. After centrifugation, the supernatants were loaded onto SDS-polyacrylamide gel and electrophoresed at 100 V for 120 min, and the proteins were transferred to PVDF membranes at 100 V for 60 min. After blocked with 5% skinny fat milk diluted in TBS-T (10 mM Tris-Hcl, pH 8.0, 150 mM NaCl, 0.1% Tween20) for 60 min at room temperature, the membranes were incubated with primary antibodies in blocking solution at 4C overnight with rocking. On the next day, the membrane was washed four times for 5 min in 1 3 TBS-T, and then incubated with HRP secondary antibodies at room temperature for 1 hour. After washing with 1 3 TBS-T for ﬁve times, proteins were detected using ECL solution and a chemiluminescent imaging and analysis system (MiniChemi). The antibodies used in this project are listed in Key Resources Table.

RNA extraction and real-time quantitative PCR analysis Cells were washed once with PBS and lysed by TRIzol reagent, following which total RNAs were extracted by the chloroform-iso- propanol method and cDNA was synthesized with ReverTra Ace, RRI and oligo-dT. Real-time quantitative PCR was performed using ChamQ Universal SYBR qPCR master mix and CFX96 real-time system (Bio-Rad). Gene expression levels were normalized to those of Gapdh. Sequences of the primers used for RT-qPCR are listed in Table S2.

Co-immunoprecipitation (coIP) assays Reprogramming cells (1 3 107 per sample) were collected by trypsinisation at day 1 and washed twice with cold PBS once and lysed with 1 mL of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 5 mg/ml of RNase A and EDTA-free protease inhibitor cocktail) on ice for 10 min. After centrifuged at 14,000 3 g for 10 min at 4C, a total of 80 mL of the supernatant was used as the input, and the rest of the supernatant was divided equally for IP and IgG. Then, the IP sample was mixed with 2 mg of anti-YTHDF2 or anti-YTHDF3 antibody, and the IgG sample was mixed with 2 mg of rabbit IgG. Then, 50 mL of a mixture of Dynabeads protein A and protein G magnetic beads was added into tubes containing the IP and IgG samples. Both the IP and IgG samples were incubated overnight with gentle rotation at 4C. On the next day, the beads were washed with 1 mL of washing buffer (same as the IP buffer) and gently rotated at 4C for 5 minutes. This wash step was repeated five times. The immunocomplex was eluted with 100 mLof13 SDS-PAGE loading buffer and analyzed by electrophoresis and immunoblotting. mRNA lifetime profiling Control, Ythdf2- and Ythdf3-defective cells at day 1 of reprogramming were treated with 10 mg/ml actinomycin D or DMSO for 4 hours and 8 hours, respectively. Total RNAs were then extracted, and before library construction for RNA-seq or reverse transcription for qPCR, 0.1 mL ERCC ExFold RNA spike-in control was added to each 1 mg RNA sample. For RNA-seq, data analysis was performed as previously described (Wang et al., 2014a). In brief, the raw reads were mapped to the mm10 transcriptome and ERCC RNA sequence, and TPM was converted to attomole by linear fitting of the RNA spike-in. The degradation rate of RNA, k, and the mRNA lifetime were calculated.

RIP (RNA-immunoprecipitation)-qPCR RIP-qPCR was performed based on GoldCLIP (Gu et al., 2018) with some modiﬁcations. In brief, Halo-tagged GFP or YTHDF1/2/3 was overexpressed during reprogramming. At day 1, the cells were washed once with PBS and cross-linked at UVC (254 nm, 400 mJ/ cm2). Crosslinked cells (1 3 107 per sample) were then scraped off the plates and lysed with 1 mL of RIP lysis buffer (50 mM Tris-Hcl, pH 7.5, 100 mM NaCl, 1 mM DTT, 2 mM CaCl2, 0.5% Triton X-100, 10% Glycerol and Protease Inhibitor Cocktail). After centrifugation, Halo-tagged beads was incubated with the supernatant with rotation at 4C for about 10-16 h. Beads associated with Halo- tagged protein complexes were washed under denatured conditions as described previously (Gu et al., 2018), except that no nuclease was added to the mixture, and RNA was not fragment throughout the whole process. After denaturing wash, the RNAs

Cell Reports 32, 108120, September 8, 2020 e5 ll OPEN ACCESS Report were released from the proteins by PK enzyme digestion. Then, the RNAs were reverse transcribed using random hexamers and analyzed for enrichment by qPCR. The data were calculated as the percent of input RNA and normalized to GFP RIP. m6A RIP-qPCR Total RNA was isolated from reprogramming cells at day 1, and performed m6A RIP using EpiMark N6-Methyladenosine Enrichment Kit according to the manufacturer’s instructions. In brief, 1 mL 1:1000 diluted m6A spike-in RNA from the kit was added to 100 mg total RNA, followed by fragmentation according to previously published protocols (Dominissini et al., 2012). In brief, RNA samples were chemically fragmented into 100-300 nucleotide-long fragments by 5 min incubation at 94C in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7.0). The fragmentation reaction was stopped with 0.05 M EDTA, followed by standard ethanol precipitation. 6 Samples were resuspended in H2Oat1 mg/ul concentration. 2 mLm A antibody was attached to protein G Magnetic Beads and incubated with fragmented RNA in Reaction Buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40) for 1 hour at 4C with orbital rotation. Then washed the beads and eluted RNA following the manufacturer’s protocols. The RNAs were reverse transcribed using random hexamers and analyzed for enrichment by qPCR. The enrichment were calculated as the percent of input RNA

Wound-healing assay At day 0 of reprogramming after OKS and shRNA retrovirus infection, cells were scratched by a sterile pipette tip and cultured in iCD1 medium supplement with 1 mg/ml puromycin. Cell migration distances into the scratched area were measured with ImageJ in 5 randomly chosen ﬁelds under a microscope.

ChIP-seq ChIP was performed as described previously with some modifications (Li et al., 2017b). Briefly, reprogramming cells were harvested by trypsinisation at day 1 and chemically crosslinked with 1% formaldehyde for 10 min at room temperature with gentle rotation. The reaction was quenched by 0.125M glycine for 5 min at room temperature and cells were washed twice with cold PBS. 1 3 107 cells were lysed in ChIP lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitor cocktail) for 10 min at 4C. The DNA was fragmented to 150-500 bp by sonication, and then centrifuged at 14,000 g for 10 min at 4C. The supernatant was diluted with ChIP IP buffer (0.01% SDS, 1% Triton X-100, 2mM EDTA, 50mM Tris-HCl, pH 8.0, 150 mM NaCl and protease inhibitor cocktail). Immunoprecipitation was performed with 5 mg rabbit anti-TEAD2 antibody coupled to 50 mL protein A/G mix overnight at 4C with gently rotation. Beads were washed at 4C for 5 min in the following order: twice low salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), twice high salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), twice LiCl buffer (500mM LiCl, 50 mM Tris-HCl pH 8.0, 2 mM EDTA, 1% NP-40, 0.5% Sodium Deox- ycholate), once TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA) with final elution in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS buffer. The eluted chromatin and input samples were reverse crosslinked by overnight incubation at 65C. After treated with protein- ase K at 56C for 30 min, the DNA was purified using the MinElute PCR Purification Kit. The libraries were constructed by a VAHTS Universal DNA Library Prep Kit for Illumina V3 according to the manufacturer’s instructions.

RNA-seq Total RNA was extracted as described above. The VAHTS mRNA-seq V3 Library Prep Kit for Illumina was used for RNA library prep- aration. In brief, mRNA was enriched from 1 mg of total RNA using oligo-dT Dynabeads, followed by fragmentation at 94C 8min. Sec- ond, First-strand and second-strand cDNA were synthesis successively using corresponding reagents. After purified by VAHTS DNA Clean Beads, the cDNA was end repaired and added with a dA base, followed by adaptor ligation, two-round purification, PCR amplification and purification. Finally, sequencing was performed on an Illumina NextSeq 500 (Illumina). Libraries were single-end reads with a 75 bp read length or paired-end reads with a 150 bp read length.

ATAC-seq ATAC-seq was performed as previously described (Buenrostro et al., 2013, 2015; Li et al., 2017b). In brief, 50,000 cells were washed in PBS and lysed using 50 mL cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Nuclei were centrifuged at 500 3 g for 5 min and resuspended with 50 mL transposition reaction mix from a TruePrep DNA Library Prep Kit V2 for Illumina. The samples were incubated at 37C for 30 min, and DNA was purified using a MinElute kit. Libraries were first preamplified for 5 cycles, and then assessed by quantitative PCR as described (Buenrostro et al., 2015) to determine the suitable PCR cycles. After appropriate amplification, libraries were purified with the MinElute kit and quantified using a VAHTS Library Quantification Kit for Il- lumina. Gel electrophoresis was used for examining the library integrity. Finally, the sequencing was carried out on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles).

RNA-seq data processing The reference transcriptome index was produced with RSEM (Li and Dewey, 2011) using the reference genome mm10 (ensemble gene annotation track v79) and ERCC RNA spike-in sequence. The reads were mapped to the reference transcriptome using RSEM and bowtie 2 (with options ‘rsem-calculate-expression -p 12–bowtie2–no-bamout’) (Langmead and Salzberg, 2012), and normalized using EDASeq (Risso et al., 2011). We chose the Trans per Million (TPM) value to normalize and evaluate gene expression e6 Cell Reports 32, 108120, September 8, 2020 ll Report OPEN ACCESS levels. RNA-seq data were expressed as GC-normalized tag counts for DESeq2 (Love et al., 2014) to analyze differential gene expression. Genes were considered as differentially expressed if gene expression was more than 1.5-fold change and p < 0.05. Data analysis and visualization was performed by package in R.

ChIP-seq and ATAC-seq data processing The MEF and ESC ATAC-seq data were from GEO: GSE93029 and ATAC-seq analysis was performed as described previously (Li et al., 2017b). All ChIP-seq and ATAC-seq data were aligned to the mm10 genome using bowtie2 (with the options ‘-p 20–very-sensitive–end-to-end–nounal–no-mixed -X 2000’). Reads mapping to mitochondrial DNA or unassigned sequences were discarded. SAMtools (Li et al., 2009) (view –q 30) was used for removing the low-quality mapped reads, and uniquely mapped reads were only retained after removed PCR duplicates. Alignment bam ﬁles were transformed into bigwig ﬁles using deepTools (Ramı´rez et al., 2014) with the RPKM normalization method. Peaks were called using MACS2 (-g mm -f BAMPE) (Zhang et al., 2008) and divided the peaks into evenly spaced 50 bp bins, then the bin with the maximum signal extracted by deeptools was used as the observed value of peaks. For ATAC-seq, random genomic regions of the same number and size as all the ATAC peaks were taken and measured the average coverage signal as background value, and the peaks with value larger than the background value were annotated as ‘open’, or else ‘closed’. Peaks were annotated by using the annotatePeaks.pl script from the Homer package (Heinz et al., 2010).

Gene ontology analysis For RNA-seq data, differentially expressed gene lists were generated from RNA-seq data from shYthdf2 and shYthdf3 conditions using shLuc as reference and genes were ﬁltered with more than 1.5-fold change and p < 0.05. For ChIP-seq data, the genes bound by TEAD2 were sorted according to the log2 fold-change (RPKM (TEAD2-ChIP) / RPKM (Input)) from the highest to the lowest, then the top 500 genes were chosen for GO analysis. The enriched GO terms were determined with R package clusterProﬁler (Yu et al., 2012).

Motif analysis The ﬁndMotifsGenome.pl function of Homer (Heinz et al., 2010) was used for de novo motif discovery with default settings. Motifs were only kept if the P value was < 0.01 and (< percent of target > / < percent of background > ) was > 1.5.

QUANTIFICATION AND STATISTICAL ANALYSIS

All the somatic cell reprogramming experiments were repeated three times independently with duplicate samples. Other experiments in this study were performed with three biological repeats unless otherwise specified. Data are presented as the mean ± s.d. that calculated by GraphPad Prism 6.0 and used two-tailed unpaired Student’s t test to analyze statistical differences in Microsoft Excel. Differences for which P-values < 0.05 were considered as statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, NS, no significant).

Cell Reports 32, 108120, September 8, 2020 e7