Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2 Soroush Tahmasebia,b,1, Seyed Mehdi Jafarnejada,b,1, Ingrid S. Tama,b, Thomas Gonatopoulos-Pournatzisc, Edna Matta-Camachoa,b, Yoshinori Tsukumoa,b,2, Akiko Yanagiyaa,b, Wencheng Lid, Yaser Atlasie, Maxime Caronf,g, Ulrich Braunschweigc, Dana Pearla,b, Arkady Khoutorskyh, Christos G. Gkogkasi, Robert Nadonf,g, Guillaume Bourquef,g, Xiang-Jiao Yanga,b,j, Bin Tiand, Hendrik G. Stunnenberge, Yojiro Yamanakaa,f, Benjamin J. Blencowec,k, Vincent Giguèrea,b,j, and Nahum Sonenbergb,c,3 aGoodman Cancer Research Center, McGill University, Montreal, QC H3A 1A3, Canada; bDepartment of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; cDonnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; dDepartment of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ 07103; eDepartment of Molecular Biology, Faculty of Science, Radboud University, Nijmegen, 6525GA, The Netherlands; fDepartment of Human Genetics, McGill University, Montreal, QC H3A 1A3, Canada; gMcGill University and Genome Quebec Innovation Centre, Montreal, QC H3A 0G1, Canada; hDepartment of Anesthesia, McGill University, Montreal, QC, H3G 1Y6, Canada; iPatrick Wild Centre, Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, United Kingdom; jDepartment of Medicine, McGill University Health Center, Montreal, QC H3A 1A3, Canada; and kDepartment of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2015. Contributed by Nahum Sonenberg, September 22, 2016 (sent for review August 15, 2016; reviewed by George Q. Daley and Maurice S. Swanson) Translational control of gene expression plays a key role during family of small molecular weight (15–20 kDa) translational inhibitors the early phases of embryonic development. Here we describe a (4E-BP1, -2, and -3 in mammals), that, when dephosphorylated, av- transcriptional regulator of mouse embryonic stem cells (mESCs), idly bind eIF4E and block its association with eIF4G to form the Yin-yang 2 (YY2), that is controlled by the translation inhibitors, eIF4F complex. Following phosphorylation by mTORC1, 4E-BPs Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays dissociate from eIF4E, allowing the formation of the eIF4F complex a critical role in regulating mESC functions through control of key and activation of translation (8, 10–12). 4E-BPs inhibit cap-dependent Oct4 pluripotency factors, including Octamer-binding protein 4 ( ) translation in embryonic and somatic stem cells (3, 4, 13, 14). Al- and Estrogen-related receptor-β (Esrrb). Importantly, overexpres- though eIF4E promotes cap-dependent translation of all cellular sion of YY2 directs the differentiation of mESCs into cardiovascular mRNAs, the translation of a subsetofmRNAs,whichgenerally lineages. We show that the splicing regulator Polypyrimidine tract- ′ binding protein 1 (PTBP1) promotes the retention of an intron in the contain a long and highly structured 5 -UTR, is strongly dependent “ ” 5′-UTR of Yy2 mRNA that confers sensitivity to 4E-BP–mediated on eIF4E (9, 15). These mRNAs are known as eIF4E-sensitive and translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and docu- Significance ment a multilayer regulatory mechanism that controls its expression. Embryonic stem cells (ESCs) maintain a low translation rate; mRNA translation | 4E-BPs | PTBP | embryonic stem cell | YY2 therefore control of mRNA translation is critical for preserving their stemness. We identified a hitherto unstudied transcription tringent control of mRNA translation is critical during early factor, Yin-yang 2 (YY2), which is translationaly regulated and Sembryonic development, because relatively small changes in controls self-renewal and differentiation of mouse ESCs (mESCs). the expression of development-related genes can dramatically Although YY2 is essential for mESC self-renewal, increased YY2 affect the self-renewal and differentiation of stem cells. In fact, a expression directs differentiation of mESCs toward cardiovas- modest (twofold or less) increase or decrease in Octamer-binding cular lineages. Examination of the Yy2 5′-UTR revealed a multi- protein 4 (OCT4) or Sex-determining region Y (SRY)-box 2 layer regulatory mechanism through which YY2 expression is (SOX2) protein levels impairs ESC self-renewal and triggers dif- dictated by the combined actions of the splicing regulator, ferentiation (1, 2). mRNA translation, which is low in undiffer- Polypyrimidine tract-binding protein 1 (PTBP1), and the trans- entiated embryonic stem cells (ESCs) and multipotent somatic lation inhibitors, Eukaryotic initiation factor 4E-binding pro- stem cells (e.g., hematopoietic stem cells and skin stem cells), teins (4E-BPs). YY2 directly controls the expression of several increases significantly during differentiation (3–5). Importantly, pluripotency and development-related genes. This study de- genome-wide analysis of the transcriptome vs. proteome of ESCs scribes a synchronized network of alternative splicing and mRNA during the early stages of differentiation demonstrated that pro- translation in controlling self-renewal and differentiation. tein levels correlate poorly with mRNA levels (Pearson’s R < 0.4), underscoring the importance of posttranscriptional regulation in Author contributions: S.T., S.M.J., and N.S. designed research; S.T., S.M.J., I.S.T., T.G.-P., E.M.-C., Y.T., A.Y., W.L., D.P., A.K., X.-J.Y., and Y.Y. performed research; C.G.G. contrib- ESC differentiation (6). uted new reagents/analytic tools; S.T., S.M.J., I.S.T., T.G.-P., E.M.-C., Y.T., A.Y., W.L., Y.A., mRNA translation can be divided into three steps: initiation, M.C., U.B., R.N., G.B., B.T., H.G.S., Y.Y., B.J.B., and V.G. analyzed data; and S.T., S.M.J., and elongation, and termination. Translational control has been docu- N.S. wrote the paper. mented most extensively at the initiation step, at which ribosomes Reviewers: G.Q.D., Children’s Hospital Boston; and M.S.S., University of Florida. are recruited to the mRNA by the concerted action of Eukaryotic The authors declare no conflict of interest. translation initiation factors (eIFs) (7). Control of translation is 1S.T. and S.M.J. contributed equally to this article. – exerted mainly by two key protein complexes: eIF4F (eIF4E 2Present address: Division of Molecular Target and Gene Therapy Products, National In- Met eIF4G–eIF4A) and the ternary complex (eIF2–GTP–Met-tRNA i) stitute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan. (7). The mammalian target of rapamycin complex 1 (mTORC1) 3To whom correspondence should be addressed. Email: [email protected]. controls the assembly of eIF4F through the phosphorylation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. eIF4E-binding proteins (4E-BPs) (8, 9). The 4E-BPs consist of a 1073/pnas.1615540113/-/DCSupplemental. 12360–12367 | PNAS | November 1, 2016 | vol. 113 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1615540113 Downloaded by guest on September 30, 2021 encode proteins that control fundamental cellular processes such as cell proliferation and survival (16). INAUGURAL ARTICLE We showed that 4E-BPs are required for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs) (17). In the current study, we describe a tightly coordi- nated network in mESCs whereby the expression of the Yin-yang 2 (YY2) transcription factor is controlled by the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) and the 4E-BP translation inhibitors. Our data reveal that stringent regulation of YY2 expression by this network is critical for mESC self-renewal and lineage commitment. Results Transcriptome and Translatome Profiling of WT and 4E-BP1/2–Null mESCs. To investigate the role of 4E-BPs in mESCs, we first derived mESCs from WT and Eukaryotic translation initiation factor 4E- binding protein 1 (Eif4ebp1) and Eukaryotic translation initiation Fig. 1. The lack of 4E-BPs deregulates the expression of pluripotency factors factor 4E-binding protein 2 (Eif4ebp2) double-knockout (DKO) in mESCs. (A and B) The log2 abundanceofRFPs(A) and mRNA (RNA-Seq) (B) mice and then examined the eIF4F complex using a 7-methyl-GTP of transcripts that were included in Babel analysis are plotted for WT and 7 (m GTP)-agarose pull-down assay. The eIF4F amount was ele- Eif4ebp1 and Eif4ebp2 DKO mESCs. (C) Babel analysis of transcripts with a vated, as demonstrated by increased (∼10.6-fold) eIF4G1 pull-down significant change in RFPs independent of the corresponding change in mRNA in DKO mESCs (Fig. S1A). However, polysome profiling (Fig. S1B) abundance (black dots; FDR < 0.1). Triml2 and Trmt61b, respectively, are and [35S]methionine/cysteine-labeling assays (Fig. S1C) did not mRNAs with the highest and the lowest RFP ratios in DKO compared with WT detect a substantial difference in global mRNA translation between mESCs; FC, fold change. (D) Western blot analysis of NANOG expression in a WT and DKO mESCs. These data are consistent with previous WT mESC and in two independent DKO mESC clones (C1 and C2). Numbers indicate the ratio of NANOG expression in each clone to that in the WT mESC findings that the lack of 4E-BPs affects the