Abstract
Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.
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Data availability
Microarray, RNA-seq and ChIP-seq data have been deposited in the GEO under accession nos. GSE95608, GSE93706 and GSE149017. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank M. Sinn, I. Gelker, H.W. Choi and M. Haustein for technical assistance. We also thank J. Müller-Keuker for creating the graphical abstract. This work was supported by the Max Planck Society.
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K.K. conceived the study, performed the experiments, interpreted the data and wrote the manuscript. J.Y. and B.S. contributed to the plasmid construction and western blotting. J.B. contributed to the chemical screening. Jonghun K. and D.W.H. contributed to the karyotyping. M.J.A. contributed to the microarray. Johnny K. interpreted the data and wrote the manuscript. G.W. and D.H. contributed to the teratoma and chimera assays. J.C. and P.C. contributed to the ChIP-seq. H.R.S. supervised this study, interpreted the data and wrote the manuscript.
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Extended data
Extended Data Fig. 1 Reprogramming competence of OCT factors in humans.
a, A schematic representation of eight OCT proteins. The numbers indicate the length of amino acids. N-TAD, N-terminal transactivation domain; DBD, DNA-binding domain; C-TAD, C-terminal transactivation domain. The DBD has a bipartite structure with two subdomains, POU-specific domain (red) and POU-homeodomain (blue), which are connected by a linker (green). b, Among eight OCT family members, only OCT4 and OCT6 were reprogramming-competent. P value was 0.0002. Data are presented as mean values ± s.d. (b, n = 3 biologically independent samples). Differences between samples were compared using a two-tailed Student’s t-test (***P < 0.001).
Extended Data Fig. 2 shRNA-mediated DOT1L inhibition enables reprogramming with OCT7, OCT8 and OCT9.
a, Knockdown efficiency of two shRNA targeting DOT1L mRNA was determined by qPCR. The shLuc construct was used as a control. P values were 0.0006, 0.0024 and 0.0009 from left to right. b, shRNA-mediated DOT1L inhibition resulted in a dramatic reduction of H3K79me1/2 levels. c, shRNA-mediated DOT1L inhibition not only dramatically enhanced OCT4- and OCT6-based reprogramming but also enabled iPSC generation in O7SKM-, O8SKM- and O9SKM-transduced cells. P values were 0.0005, 0.007, 0.0046, 0.0011, 0.0037, 0.0038, 0.0267, 0.004, 0.0037, 0.0081, 0.0004, 0.0023, 0.0026, 0.0003, 0.0011 from left to right. Data are presented as mean values ± s.d. (a,c, n = 3 biologically independent samples). Differences between samples were compared using a two-tailed Student’s t-test (***P < 0.001, **P < 0.01, *P < 0.05). The experiments were repeated independently three times with similar results and representative images are shown (b). See Source Data for uncropped blot images.
Extended Data Fig. 3 SGC0946-mediated DOT1L inhibition facilitates reprogramming process.
a, SGC0946-mediated DOT1L inhibition did not alter transactivation activities of TADs of OCT factors. b, SGC0946-mediated DOT1L inhibition resulted in the activation of LIN28A, NANOG, SALL4 and SOX2 on day 6 of OCT7-, OCT8- and OCT9-based reprogramming (n = 2 biologically independent samples). c, Coverage plots of OCT4FLAG, OCT7FLAG and OCT7FLAG + SGC0946 ChIP-seqs around + /-500bp from the center of OCT4FLAG peaks. Rows represent OCT4FLAG binding sites. d, SGC0946-mediated DOT1L inhibition did not alter levels of the indicated histone marks. e, SGC0946-mediated DOT1L inhibition did not change expression levels of EED, EZH2 and SUZ12. Data are presented as mean values ± s.d. (a, n = 3 biologically independent samples). The experiments were repeated independently three times with similar results and representative images are shown (d,e). See Source Data for uncropped blot images.
Extended Data Fig. 4 Inhibiting multiple epigenetic pathways enhances human and mouse reprogramming.
a, Inhibition of multiple epigenetic pathways dramatically enhanced reprogramming efficiencies in humans. P values were <0.0001, 0.0003, <0.0001, 0.0004, <0.0001, <0.0001, <0.0001, 0.0035, <0.0001, <0.0001, <0.0001, <0.0001, 0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0004, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0004, <0.0001, <0.0001, <0.0001, 0.0003 from left to right. b, Inhibition of multiple epigenetic pathways dramatically enhanced reprogramming efficiencies in mice. P values were 0.0008, 0.0014, 0.0003, <0.0001, 0.0002, 0.0009, 0.0004, <0.0001, 0.0006, <0.0001, <0.0001, 0.0002, <0.0001, <0.0001, <0.0001, <0.0001 from left to right. Data are presented as mean values ± s.d. (a,b, n = 3 biologically independent samples). Differences between samples were compared using a two-tailed Student’s t-test (***P < 0.001, **P < 0.01, *P < 0.05). S, SGC0946; RG, RG-108; SB, SB431542; C, CI-994; RN, RN-1.
Extended Data Fig. 5 Characterization of human iPSC lines that were generated with OCT7, OCT8 and OCT9 under S/SB/RN treatment.
a, Expression of pluripotency makers in iPSC lines. The O7-1 and O7-2 lines were generated with O7SKM; the O8-1 and O8-2 lines were generated with O8SKM; and the O9-1 and O9-2 iPSC lines were generated with O9SKM. DAPI was used as a nuclear counterstain. Scale bars, 500 μm. b, Karyotype analysis demonstrated correct chromosome content and no obvious deletions or duplications in all iPSC lines. c, No OCT4 transgene was detected in iPSC lines generated with other OCT factors. Instead, only transgenes that were used for the generation of the respective iPSC lines were detected in corresponding iPSC lines. d, All iPSC lines displayed gene expression profiles similar to H1 hESCs but distinct from fibroblasts. e, OCT4 and NANOG promoters were methylated in fibroblasts but largely unmethylated in the iPSC lines and H1 hESCs. f, iPSC lines formed teratomas consisting of all three embryonic germ layers, providing evidence for in vivo differentiation potential. Scale bars, 100 μm. The experiments were repeated independently three times with similar results and representative images are shown (a,c,f). See Source Data for uncropped blot images.
Extended Data Fig. 6 Characterization of mouse iPSC lines that were generated with Oct6, Oct7, Oct8, Oct9 and Oct11 under S/CI/RN treatment.
a, All iPSC lines were stained positive for Oct4-GFP, NANOG and SSEA1. The mO6-1 and mO6-2 lines were generated with O6SKM; the mO7-1 and mO7-2 lines were generated with O7SKM; the mO8-1 and mO8-2 lines were generated with O8SKM; the mO9-1 and mO9-2 lines were generated with O9SKM; and the mO4 line was generated with O4SKM. b, iPSC lines produced chimeras as determined by PCR. C, corresponding iPSC lines were used as positive controls. H2O was used as a negative control. L, DNA ladder. c, Karyotype analysis demonstrated correct chromosome content and no obvious deletions or duplications in the iPSC lines. d, iPSC lines formed teratomas consisting of all three embryonic germ layers, providing evidence for in vivo differentiation potential. e, mO11-1 and mO11-2 lines, which were generated with O11SKM, were stained positive for Oct4-GFP, NANOG and SSEA1. f, mO11-1 and mO11-2 lines formed teratomas. g, All iPSC lines displayed gene expression profiles similar to mESCs but distinct from MEFs. h, No Oct4 transgene was detected in mO11-1 and mO11-2 lines. Instead, Oct11 transgene was integrated in these iPSC lines. i, mO11-1 and mO11-2 lines produced chimeras as determined by PCR. C, the mO11-1 iPSC line was used as a positive control. H2O was used as a negative control. L, DNA ladder. Scale bar, 250 μm (a,e), 100 μm (d,f). The experiments were repeated independently three times with similar results and representative images are shown (a,b,d,e,f,h,i). See Source Data for uncropped blot images.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8 and Table 1.
Supplementary Dataset 1
The list of epigenetic compounds.
Supplementary Dataset 2
The list of candidate genes.
Supplementary Dataset 3
The list of primers.
Supplementary Dataset 4
Compound ID.
Supplementary Data 1
Unprocessed blots for Supplementary Fig. 4a.
Supplementary Data 2
Unprocessed blots for Supplementary Fig. 4b.
Supplementary Data 3
Unprocessed blots for Supplementary Fig. 4c.
Supplementary Data 4
Unprocessed blots for Supplementary Fig. 4d.
Source data
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Source Data Extended Fig. 6i
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Kim, KP., Choi, J., Yoon, J. et al. Permissive epigenomes endow reprogramming competence to transcriptional regulators. Nat Chem Biol 17, 47–56 (2021). https://doi.org/10.1038/s41589-020-0618-6
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DOI: https://doi.org/10.1038/s41589-020-0618-6