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The P53-Induced Lincrna-P21 Derails Somatic Cell Reprogramming by Sustaining H3k9me3 and Cpg Methylation at Pluripotency Gene Promoters

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The P53-Induced Lincrna-P21 Derails Somatic Cell Reprogramming by Sustaining H3k9me3 and Cpg Methylation at Pluripotency Gene Promoters Cell Research (2015) 25:80-92. npg © 2015 IBCB, SIBS, CAS All rights reserved 1001-0602/15 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters Xichen Bao1, 2, *, Haitao Wu1, 2, 3, *, Xihua Zhu1, 2, 3, *, Xiangpeng Guo 1, 2, Andrew P Hutchins2, Zhiwei Luo1, 2, Hong Song2, Yongqiang Chen2, Keyu Lai2, Menghui Yin4, Lingxiao Xu5, Liang Zhou6, Jiekai Chen2, Dongye Wang1, 2, 7, Baoming Qin2, 8, 9, Jon Frampton10, Hung-Fat Tse9, 11, 12, Duanqing Pei2, 9, Huating Wang13, Biliang Zhang4, Miguel A Esteban1, 2, 9 1Laboratory of Chromatin and Human Disease, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China; 2Key Lab- oratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China; 3University of Chinese Academy of Sciences, Beijing 100049, China; 4Laboratory of RNA Chemical Biology, State Key Laboratory of Respiratory Diseases, Guang- zhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China; 5School of Life Sciences, Shandong University, Jinan, Shandong 250100, China; 6Department of Radiation Medicine, School of Public Health and Tropic Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China; 7Drug Discovery Pipeline Group, Guang- zhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China; 8Laboratory of Metabolism and Cell Fate, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guang- dong 510530, China; 9Hong Kong - Guangdong Joint Laboratory of Stem Cells and Regenerative Medicine, the University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong 510530, China; 10School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK; 11Cardiology Divi- sion, Department of medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China; 12Shenzhen Institutes of Research and Innovation, The University of Hong Kong, Hong Kong SAR, China; 13Li Ka Shing Institute of Health Sciences, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong SAR, China Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological pro- cesses, but few have examined their roles in somatic cell reprogramming. Through expression profiling and function- al screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. In- stead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltrans- ferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results pro- vide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation. Keywords: somatic cell reprogramming; long noncoding RNAs; p53; lincRNA-p21; heterochromatin; H3K9 methylation; DNA methylation Cell Research (2015) 25:80-92. doi:10.1038/cr.2014.165; published online 16 December 2014 *These three authors contributed equally to this work. Correspondence: Xichen Baoa, Miguel A Estebanb aTel: +86-20-3229-0481; Fax: +86-20-3206-8110 Introduction E-mail: [email protected] bTel: +86-20-3202-2920; Fax: +86-20-3206-8110 Induced pluripotent stem cells (iPSCs) have major E-mail: [email protected] implications for regenerative medicine, in vitro disease Received 29 May 2014; revised 20 September 2014; accepted 14 Novem- modeling and toxicology screening [1]. Yet, to fulfill ber 2014; published online 16 December 2014 Xichen Bao et al. npg 81 these expectations, it is necessary to understand how the speeds [3], preiPSCs provide a relatively well-defined reprogramming machinery erases and rewrites the somat- model for dissecting the chain of events eventually lead- ic epigenetic code. This is important because, among oth- ing to the full acquisition of pluripotency [10, 11]. Using er reasons, reprogramming is prone to errors that could preiPSCs, we thus sought to identify lncRNAs that con- hamper biomedical applications of the derived cells [2]. trol the late stage of mouse somatic cell reprogramming. Reprogramming by exogenous factors is nowadays We selected 51 lincRNAs previously reported to reg- considered as a multistep process in which cells must ulate embryonic stem cell (ESC) pluripotency (26) or overcome a series of roadblocks to ultimately acquire differentiation (25) [22] and 16 p53-regulated lincRNAs pluripotency [3, 4], though under certain conditions it [18] (Figure 1A). The rationale for choosing the latter can also be deterministic [5-7]. Two fundamental repro- group is that p53 is an important regulator of ESC differ- gramming roadblocks are the activation of a p53-mediat- entiation and a major barrier for reprogramming [8]. We ed response that promotes apoptosis and cell senescence, performed quantitative PCR (qPCR) analysis of these and the inefficient removal of preexisting somatic-like 67 lincRNAs in mouse embryonic fibroblasts (MEFs), 4 epigenetic marks. Accordingly, depletion of p53 [8] or independent preiPSC clones (generated from Oct4-GFP the H3K9 methyltransferases Setdb1, Suv39h1 and Su- transgenic MEFs [21] using Oct4, Sox2, Klf4 and c-Myc; v39h2 [9-11], and inhibition of the maintenance DNA OSKM), iPSCs and ESCs. The analysis showed distinct methyltransferase DNMT1 [12] improve reprogramming patterns of lincRNA expression in the 4 cell types that efficiency. were grouped into 7 classes (Figure 1B, 1C and Supple- Noncoding RNAs have important roles in establishing mentary information, Table S1A). Classes I and II were or removing different reprogramming roadblocks. How- the largest and contained 17 and 26 lincRNAs, respec- ever, despite the extensive characterization of microRNA tively, while class VI included only 1 lincRNA (p53-reg- (miRNA) networks in reprogramming [13], only 2 long ulated) (Supplementary information, Table S1A). In con- noncoding RNAs (lncRNAs) are known to modulate this trast to classes I-VI, class VII lincRNAs did not show a process. LncRNAs are noncoding RNA species longer consistent pattern among the different cell types. Of note, than 200 nucleotides and act as decoys, guides or scaf- pluripotency- and differentiation-related lincRNAs were folds [14]. LincRNA-RoR was the first lncRNA impli- distributed evenly among classes I-IV and VII, but only cated in reprogramming [15] and partly functions as a 1 differentiation-related lincRNA was included in class ‘sponge’ that titrates down miRNAs targeting pluripoten- V. Conversely, p53-regulated lincRNAs were mostly in cy regulators (e.g., miR-145) [16]. Conversely, lincRNA classes II (8) and V (comprising 4 out of 5 constituents). TUNA forms a complex with several RNA-binding pro- We also performed qPCR analysis for all 67 lincRNAs teins at the promoters of Nanog, Sox2 and Fgf4, where in MEFs reprogrammed with OSKM (Supplementary it facilitates the deposition of the active histone mark information, Figure S1 and Table S1B), and observed H3K4me3 [17]. that 58 out of the 67 lincRNAs increase by at least 2-fold In this report, we identify that the p53-induced large during reprogramming (Supplementary information, Ta- intergenic noncoding RNA p21 (lincRNA-p21; also ble S1B). termed Trp53cor1) [18-20] acts as a barrier for repro- We then selected 15 lincRNAs from classes I-VI for gramming by sustaining heterochromatic state at pluripo- knockdown and functional analysis in a validated pre- tency gene promoters. iPSC to iPSC conversion model [10]. We generated 2 individual short hairpin RNA (shRNA) viral vectors for Results each of the 15 lincRNAs, and identified at least 1 effec- tive shRNA (> 50% depletion) for 11 out of the 15 lin- Functional screening reveals roles of lincRNAs in the cRNAs (Figure 1D, upper panel). Activation of the Oct4- preiPSC to iPSC conversion GFP reporter was employed as readout for detecting the Somatic cells undergoing reprogramming can be full acquisition of pluripotency. After infection with the trapped in a partially reprogrammed state termed the shRNAs, the preiPSCs were passaged onto feeders and preiPSC state [21]. These cells have overcome the senes- treated with vitamin C (Vc) for 8 days before GFP+ col- cence barrier and downregulated multiple somatic mark- ony counting [10]. Notably, knockdown of 3 lincRNAs ers, but fail to fully activate the pluripotency network. (-1463, -1526, and -p21) with the 2 independent shRNAs Importantly, preiPSCs are stable and can be readily con- showed enhanced GFP+ colony formation compared to verted to fully reprogrammed iPSCs through a variety of the control, while lincRNA-1307 knockdown had the op- approaches [10, 21]. Because reprogramming cells con- posite effects (Figure 1D, lower panel and 1E). In sum- sist of heterogeneous populations progressing at different mary, our screening successfully identified 4 lincRNAs www.cell-research.com | Cell Research npg LincRNA-p21 is an epigenetic barrier for reprogramming 82 Cell Research | Vol 25 No 1 | January
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