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BMP4 resets mouse epiblast stem cells to naive pluripotency through ZBTB7A/B-mediated chromatin remodelling

Abstract

BMP4 regulates a plethora of developmental processes, including the dorsal–ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.

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Fig. 1: Identification of BMP4 as a driver for mouse PNT.
Fig. 2: Enhancement of BMP4-driven PNT by chemicals.
Fig. 3: BMP4- and factor-induced PNT.
Fig. 4: Chromatin dynamics during BiPNT.
Fig. 5: BMP4 drives PNT by reorganizing chromatin accessibility.
Fig. 6: Zbtb7a/b is required and sufficient for PNT.
Fig. 7: Zbtb7a/b is required to open naive chromatin.
Fig. 8: ZBTB7A occupies loci undergoing both activation and silencing.

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Data availability

ATAC-seq, RNA-seq and ChIP-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE122935, GSE122936 and GSE146653, respectively. A super-series of all datasets is provided at GSE122937. Previously published Smad1/5 ChIP-seq data that were reanalysed here are available under accession number GSE70581 (ref. 33). Source data for Figs. 1, 2 and 4–7 and Extended Data Figs. 1–8 are provided with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank A. Smith and G. Guo for discussion and suggestions. This research was supported by grants from The National Key Research and Development Program of China (2017YFA0504100, 2016YFA0101800 and 2018YFE0204800); National Natural Science Foundation of China (numbers 31421004, 31530038, 31830060, 31970681 and 21907095; Frontier Science Research Program of the Chinese Academy of Sciences (QYZDJ-SSW-SMC009); Science and Technology Planning Project of Guangdong Province (numbers 2017B030314056 and 2019A1515011024); Key Research & Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110104003). The research is partly supported by Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation.

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Authors and Affiliations

Authors

Contributions

J.L. and S.Y. designed the project. S.Y., C.Z. and S.Cao performed the experiments. J.H., X.W., G.Z., X.H. and S.X. analysed the data. Y.Q., J.Y., W.X. and M.Z. isolated the primary EpiSC cell lines. B.C., S.Chu, W.P. and R.L. performed the blastocyst injection. K.W., J.G., H.L., L.G., B.W., L.W., L.X. and C.L. performed the ATAC-seq and RNA-seq experiments. J.K. performed the ChIP-seq experiments. J.C. supervised the data analysis. J.L. and D.P. supervised the whole study. D.P. conceived the whole study, wrote the manuscript and approved the final version.

Corresponding authors

Correspondence to Jing Liu or Duanqing Pei.

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Extended data

Extended Data Fig. 1 BMP4 induces mouse primed-to-naive transition (PNT).

a, q-PCR analysis for the expression of Oct4, Sox2 and Klf4 in ESCs and EpiSCs. Data are mean ± s.d., n = 3 independent experiments. b, Representative images for EpiSCs cultured in ActivinA/bFGF, iCD1 and iCD1+BMP4 medium for 6 days. Scale bars, 250 μm. c, Colonies of passaged cultures from reset pluripotent cells (rESCs). Scale bar, 250 μm. d, q-PCR analysis for the expression of naïve and primed pluripotent markers in ESCs and rESCs. Data shown are the mean of 3 technical replicates and are from 1 of 2 independent experiments. The experiments in b-c were repeated independently three times with similar results. Statistics source data are shown in Statistical Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 The screening of small molecules to enhance PNT.

a, Schematic for the drug screening to promote PNT. b, The dosage effect of two hit small molecules. Data shown are the mean of 3 technical replicates and are from 1 of 2 independent experiments. c, Whole well fluorescent images of resetting cultures with the protocol in a with DMSO as control, EPZ5676 and EPZ6438 alone or together (2EPZ). Scale bars, 5mm. The experiments were repeated independently three times with similar results. d, Quantification for c. Data are mean ± s.d., two-tailed, unpaired t-test; n=3 biological replicates, + EPZ5676 vs. control ***P < 0.001, +EPZ6438 vs. control ***P = 0.0001, + 2EPZ vs. control ***P < 0.001, + 2EPZ vs. + EPZ5676 ***P < 0.001, + 2EPZ vs. + EPZ6438 **P = 0.0029. e, Summary of chemical induced PNT21,29. Statistics source data are shown in Statistical Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Characterization of the rESCs derived from the enhanced PNT protocol.

a, Representative images for primary and passaged (P7) rESCs. Scale bars, 250 μm. b, q-PCR analysis for the expression of naïve and primed pluripotent markers in ESCs and newly derived rESCs by the enhanced PNT protocol. Data are mean ± s.d., n = 3 biological replicates. c, Immuno-staining of NANOG and REX1 in EpiSCs and newly derived rESCs by the enhanced protocol. Scale bars, 250 μm. d, Normal karyotype for the newly derived rESCs (rESC-2#, male). e, Chimera with germline transmission from rESC-2#. f, Normal karyotype for a female EpiSCs cell line (EpiSC-2#, female). g, Whole well fluorescent images for the rESCs from the female EpiSCs (EpiSC-2#) at day8. Scale bars, 5 mm. h, FACS analysis for the proportion of Oct4-GFP positive cells in g, with EpiSCs and ESCs as negative control and positive control, respectively. The experiments in a, c-h were performed three times with similar results. Statistics source data are shown in Statistical Source Data Extended Data Fig. 3.

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Extended Data Fig. 4 Comparison of BiPNT and EiPNT, KiPNT, NiPNT.

a, Numbers of Oct4-GFP positive colonies for PNT induced with BMP4 (BiPNT) or exogenous Esrrb (EiPNT), Klf2 (KiPNT) and Nr5a2 (NiPNT). Data shown are the mean of 3 technical replicates and are from 1 of 2 independent experiments. b, FACS analysis for the proportion of Oct4-GFP positive cells in a. c, Numbers of Oct4-GFP positive colonies for BiPNT, EiPNT, KiPNT and NiPNT with or without LDN193189 (100 nM) treatment. Data shown are the mean of 3 technical replicates and are from 1 of 2 independent experiments. d, Whole well fluorescent images of the colonies for c. Scale bars, 5 mm. The experiments were performed twice with similar results. Statistics source data are shown in Statistical Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 Chromatin accessibility dynamics for PNT.

a, Peaks numbers for the OC, CO or PO groups or subgroups in the two-stage chromatin accessibility dynamics (CAD) analysis, respectively. Stage 1, left panel; stage 2, right panel. b, Heatmap and pileup of ATAC-seq signals for PNT stage1 and stage 2, respectively. The heatmap and pileups are centered on the ATAC-seq peaks (upstream 5 kb and downstream 5 kb of the peaks). c, Gene ontology (GO) analysis for genes with a transcriptional state site within 20 kb at ATAC-seq peaks for CO/OC in stage 1. For CO group, genes up-regulated for more than 2-fold compared with Day0 were selected for GO analysis. For OC group, genes downregulated more than 2-fold compare with Day0 were selected. d, Gene ontology (GO) analysis for genes with a transcriptional state site within 20 kb at ATAC-seq peaks for each CO/OC group in stage 2. For CO group, genes upregulated for more than 2-fold compared with Day0 were selected for GO analysis. For OC group, genes downregulated more than 2-fold compare with Day0 were selected. e, Expression patterns for Tfap2a and Tfap2c by RNA-seq data. f, q-PCR analysis for the expression of Tfap2a and Tfap2c. Data are mean ± s.d., n = 3 biological replicates. Statistics source data are shown in Statistical Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 Function of BMP4 during PNT.

a, The effect of different BMPs in PNT. BMP2, 10 ng/ml; BMP4, 10 ng/ml, BMP6, 10 ng/ml; BMP7, 10 ng/ml. Data shown are the mean of 3 technical replicates and are from 1 of 2 independent experiments. b, The inhibition of BMP signaling by LDN193189 can totally block the PNT. Data are mean ± s.d., n = 3 biological replicates. c, Immunoblotting for the activation of SMAD1/5/8 by BMP4. The experiments were performed three times with similar results. d, Quantification of the efficiency for the treatment of BMP4 in stage 2 medium. Data are mean ± s.d., n = 3 biological replicates. e, ChIP-seq tracks showing binding of Smad1/5 to the gene loci of Zbtb7b, Zbtb7c, Klf2 and Nr5a233 Statistics source data are shown in Statistical Source Data Extended Data Fig. 6. Unprocessed blots are shown in Unprocessed Blots Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Zbtb7a and Zbtb7b are required for PNT.

a, Representative images of Oct4-GFP positive colonies obtained with over-expression of mCherry, Zbtb7a and Zbtb7b at day8 of PNT without BMP4. Scale bar, 250 μm. b, Representative images of Oct4-GFP positive colonies generated by Zbtb7a or Zbtb7b in Oct4 and Sox2 mediated MEF reprogramming. Scale bar, 250 μm. c, Both Zbtb7a and Zbtb7b can promote Oct4 and Sox2 mediated MEF reprogramming. Data are mean ± s.d., n = 3 biological replicates. d, Western blot for ZBTB7A and ZBTB7B at day3 in Zbtb7a-/-, Zbtb7b-/- and double knockout (DKO)EpiSCs. e, Numbers of Oct4-GFP positive colonies for PNT (upper panel) and whole cell fluorescent images of the colonies (lower panel) induced with wildtype (WT), Zbtb7a-/-, Zbtb7b-/- EpiSCs and rescue of Zbtb7a or Zbtb7b knockout with exogenous Zbtb7a and Zbtb7b. Data are mean ± s.d., n = 3 biological replicates. Scale bars, 5 mm. f, Western blot for ZBTB7A and ZBTB7B at day3 in WT and DKO-ESC. g, q-PCR analysis for the expression of core pluripotent factors in WT and DKO-ESC. Data are mean ± s.d., n = 3 biological replicates. h, Representative images of WT and DKO ESCs maintained with BMP4/LIF medium for 2 passages. Scale bars, 250 μm. i, q-PCR analysis for the expression of core pluripotent genes Oct4, Sox2 and Nanog in WT and DKO ESCs maintained with BMP4/LIF medium at different passages. Data are mean ± s.d., n = 3 biological replicates. The experiments in a-b, d, f, h were performed three times with similar results. Statistics source data are shown in Statistical Source Data Extended Data Fig. 7. Unprocessed blots are shown in Unprocessed Blots Extended Data Fig. 7.

Source data

Extended Data Fig. 8 Zbtb7a and Zbtb7b are essential for the activation of naïve program.

a, Scatterplot showing the difference of the transcriptomic profiles at day 3 of PNT between WT and the indicated knockout. b, The rescue experiment for PNT induced with DKO EpiSCs with the transgene of Klf2, Nr5a2 and Esrrb. Data are mean ± s.d., two-tailed, unpaired t-test; n = 3 biological replicates, Klf2 vs. DKO *P = 0.0232, Nr5a2 vs. DKO *P = 0.0282, Esrrb vs. DKO **P = 0.0018. Statistics source data are shown in Statistical Source Data Extended Data Fig. 8.

Source data

Supplementary information

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1: the growth factors used for screening. Supplementary Table 2: the compounds used for screening. Supplementary Table 3: the primers used for qRT-PCR.

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Yu, S., Zhou, C., Cao, S. et al. BMP4 resets mouse epiblast stem cells to naive pluripotency through ZBTB7A/B-mediated chromatin remodelling. Nat Cell Biol 22, 651–662 (2020). https://doi.org/10.1038/s41556-020-0516-x

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