Chromatin-modifying enzymes as modulators of reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling1. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address Correspondence and requests for materials should be addressed to: George.Daley@childrens.harvard.edu. S.A.A. is a consultant for Epizyme Inc.GQD is a member of the scientific advisory boards and holds stock in or receives consulting fees from the following companies: Johnson & Johnson, Verastem, Epizyme, iPierian, Solasia KK, and MPM Capital, LLP. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author contributions T.T.O. performed project planning, experimental work, data interpretation and preparation of the manuscript. N.K., A.C, N.Z., J.U. and O.B.M. performed experimental work. P.C. and A.U.S. participated in data analysis. K.M.B. and S.A.A. provided critical materials and participated in the preparation of the manuscript. P.B.G. and E.S.L., participated in data acquisition, data interpretation and preparation of the manuscript. G.Q.D supervised research and participated in project planning, data interpretation and preparation of the manuscript. NIH Public Access Author Manuscript Nature. Author manuscript; available in PMC 2012 November 19. Published in final edited form as: Nature. ; 483(7391): 598–602. doi:10.1038/nature10953. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors. To examine the influence of chromatin modifiers on somatic cell reprogramming, we employed a loss-of-function approach to interrogate the role of 22 select genes in DNA and histone methylation pathways. We tested a pool of 3 hairpins for each of 22 target genes and observed knockdown efficiencies of >60% for 21 out of 22 targets (Supplementary Fig. 1). We infected fibroblasts differentiated from the H1 human embryonic stem cell (ESC) line (dH1fs) with shRNA pools, transduced them with reprogramming vectors expressing Oct4, Sox2, Klf4 and c-Myc (OSKM), and identified the resulting iPSCs by Tra-1-60 staining (Fig. 1a)4. Eight shRNA pools reduced reprogramming efficiency (Fig. 1b). Among the target genes were Pou5F1/Oct4 (included as a control), and Ehmt1 and SetDB1, two H3K9 methyltransferases whose histone mark is associated with transcriptional repression. The remaining five shRNA pools targeted components of polycomb repressive complexes (PRC), major mediators of gene silencing and heterochromatin formation5. Inhibition of PRC1 (Bmi1, Ring1) and PRC2 components (Ezh2, Eed, Suz12) significantly decreased reprogramming efficiency while having negligible effects on cell proliferation (Fig. 1c, Supplementary Fig. 2). This finding is of particular significance given that Ezh2 is necessary for fusion-based reprogramming6 and highlights the importance of transcriptional silencing of the somatic cell gene expression program during generation of iPSCs. In contrast to genes whose functions appear to be required for reprogramming, inhibition of three genes enhanced reprogramming: YY1, Suv39H1, and Dot1L (Fig. 1b, 1d). YY1 is a context-dependent transcriptional activator or repressor7, whereas Suv39H1 is a histone H3K9 methyltransferase implicated in heterochromatin formation8. Interestingly, enzymes that modify H3K9 were associated with both inhibition and enhancement of reprogramming, which suggested that unraveling the mechanisms for their effects might be challenging. Thus, we focused on Dot1L, a histone H3 Lysine 79 methyltransferase that has not previously been studied in the context of reprogramming9. We utilized two hairpin vectors that resulted in the most significant downregulation of Dot1L and concomitant decrease in global H3K79 levels (Supplementary Fig. 3a, b). Fibroblasts expressing Dot1L shRNAs formed significantly more iPSC colonies when tested separately or in a context where they were fluorescently labeled and co-mixed with control cells (Fig 2a, Supplementary Fig. 4). This enhanced reprogramming phenotype could be reversed by overexpressing an shRNAresistant wildtype Dot1L, but not a catalytically-inactive Dot1L, suggesting that inhibition of catalytic activity of Dot1L is key to enhance reprogramming10 (Fig. 2a). Our findings with dH1fs were applicable to other human fibroblasts, as IMR-90 and MRC-5 cells also exhibited 3-fold and 6-fold increases in reprogramming efficiency, respectively, upon Dot1L Onder et al. Page 2 Nature. Author manuscript; available in PMC 2012 November 19. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript suppression (Supplementary Fig. 5). To validate our findings independently of shRNAmediated knockdown, we utilized a recently discovered small molecule inhibitor of Dot1L catalytic activity. EPZ00477711 (referred to as iDot1L) abrogated H3K79 methylation at concentrations ranging from 1uM to 10uM and increased reprogramming efficiency 3–4 fold (Fig. 2b, Supplementary Fig. 6a, b). Combination of inhibitor treatment with Dot1L knockdown did not further increase reprogramming efficiency, reinforcing our previous observation that inhibition of Dot1L’s catalytic activity is key to reprogramming (Supplementary Fig. 6c). iPSCs generated through Dot1L inhibition exhibited characteristic ESC morphology, immunoreactivity for SSEA4, SSEA3, Tra-1-81, Oct4 and Nanog, and differentiated into all three embryonic germ layers in vitro and in teratomas (Supplementary Fig. 7 a–c). Therefore, iPSCs generated following Dot1L inhibition display all of the hallmarks of pluripotency. We next assessed Dot1L inhibition in murine reprogramming. iDot1L treatment led to 3-fold enhancement of reprogramming of Oct4-GFP MEFs (Fig. 2c). Reprogramming of tail-tip fibroblasts (TTFs) derived from a conditional knockout Dot1L mouse strain yielded significantly more iPSC colonies upon deletion of Dot1L12 (Supplementary Fig. 8a). Cremediated excision of both floxed Dot1L alleles in iPSC clones derived from homozygous TTFs was confirmed by genomic PCR (Supplementary Fig. 8b). Dot1L inhibition also increased reprogramming efficiency of MEFs and peripheral blood cells derived from an inducible secondary iPSC mouse strain13 (Supplementary Fig. 8c, d). Taken together, these results demonstrate that Dot1L inhibition enhances reprogramming of both mouse and human cells. We next examined the cellular mechanisms by which Dot1L inhibition promotes reprogramming. Dot1L inhibition affected neither retroviral transgene expression nor cellular proliferation (Supplementary Fig. 9 a–c). Although previous studies indicated that Dot1L null cells have increased apoptosis and accumulation of cells in G2 phase9, we failed to observe a significant increase in apoptosis or change in the cell cycle profile of Dot1Linhibited fibroblasts (Supplementary Fig. 9d, e). In human iPSC clones derived from shDot1L fibroblasts, Dot1L inhibition was no longer evident, reflecting the known silencing of retroviruses that occurs during reprogramming (Supplementary Fig. 10a). qPCR analysis revealed that the silencing occurred by day 15 after OSKM transduction (Supplementary Fig. 10b, c). To define the crucial time window for Dot1L inhibition, we treated fibroblasts with iDot1L at 1-week intervals during reprogramming. iDot1L treatment in either the first or second week was sufficient to enhance reprogramming, whereas treatment in the third week or a 5-day pretreatment had no effect (Supplementary Fig. 10d, e). Immunofluorescence analysis revealed significantly greater numbers of Tra-1-60-positive cell clusters on day 10 and day 14 in shDot1L cultures (Supplementary Fig. 11a, b), indicating that the emergence of iPSCs is accelerated upon Dot1L inhibition. When we extended the reprogramming experiments by 10 more days, shDot1L cells still yielded more iPSC colonies than controls (Supplementary Fig. 11c). Taken together, these findings indicate that Dot1L inhibition acts in early to middle stages to accelerate and increase the efficiency of the reprogramming process. To assess whether Dot1L inhibition could replace any of the reprogramming factors, we infected control and Dot1L-inhibited fibroblasts with 3 factors, omitting one factor at a time. In the absence of Oct4 or Sox2 no iPSC colonies emerged (Fig. 2d). When we omitted either Klf4 or c-Myc, Dot1L-inhibited fibroblasts gave r