How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.
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The RNA-Seq and UMI-4C data generated have been deposited in the Gene Expression Omnibus under accession number GSE126618. The accession numbers of the published datasets used are as follows: HiC in ESCs: GSE96611 (ref. 61); H3K27ac ChIP-Seq in Flk1+ mesendodermal progenitors: GSE47082 (ref. 62); H3K27ac ChIP-Seq in Eomes + mesendodermal progenitors: GSE103262 (ref. 63); H3K27ac ChIP-Seq in activin A-induced mesendodermal precursors: GSE38596 (ref. 64); and H3K27ac ChIP-Seq in neural progenitor cells: GSE35496 (ref. 65). Source data for all figures have been provided as Supplementary Table 4. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
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We thank A. Smith for providing the Rex1GFPd2 cells, K. Nimura for the Whsc1 ΔSET cells, E. J. Robertson for the Eomes-GFP cells, A. Surani for the XGFP EpiSCs, J. E. Bradner for the JQ1 compound, L. Batlle and C. Berenguer for technical help, the CRG core facilities of flow cytometry, advanced light microscopy and tissue engineering, and L. Di Croce, B. Payer, M. Behringer, J. Licht, K. M. Loh, K. Kaji and R. Stadhouders for advice and discussions. T.V.T. and J.L.S. were supported by Juan de la Cierva postdoctoral fellowships (MINECO; FJCI-2014-22946 and IJCI-2014-21872), B.D.S. by an EMBO long-term fellowship (number ALTF 1143-2015), G.S. by a Marie Sklodowska-Curie fellowship (H2020-MSCA-IF-2016, miRStem), A.D. by a Severo Ochoa fellowship and J.G. by a Boehringer Ingelheim Graduate Student Fellowship. R.J. was supported by NIH grants R01 NS088538-01 and 2R01MH104610-15. This work was supported by the EU-FP7 project BLUEPRINT, the Spanish Ministry of Economy, Industry and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa 2013–2017 and the CERCA Program Generalitat de Catalunya. T.V.T., J.L.S. and B.D.S. were supported by a CRG award for junior collaborative projects.
R.J. is an advisor/co-founder of Fate Therapeutics, Fulcrum Therapeutics, Omega Therapeutics and Dewpoint Therapeutics.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 An in silico screen reveals a role of Whsc1 in the exit from pluripotency of ESCs.
a, Heatmap showing gene expression dynamics of Chromatin-related Factors (CRFs) during embryoid body (EB) differentiation of R1 (GSE2972) (left panel) and J1 ESCs (GSE3749) (right panel) (BMC Genomics 8, 85, 2007). Selected candidate genes, Cbx4, L3mbtl3 and Whsc1, are indicated. b, Expression of pluripotency, mesendoderm and neuroectoderm markers during exit from pluripotency induced by N2B27, Activin A and FBS. Relative expression (fold changes relative to 0 hr) is indicated as numbers which represent mean from n=3 independent experiments. c, Left panel: Evaluation of knockdown efficiency by RT-qPCR after shRNA transductions in Rex1GFPd2 cells. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. Right panel: Flow cytometry analysis of GFP (Rex1) expression in Rex1GFPd2 cells depleted of Cbx4 or L3mbtl3 at 0 hr and 72 hrs after induction. Three independent experiments were performed with similar results. d, Evaluation of serial induction of exit from pluripotency. Left-top panel: schematic representation of experimental procedure; left-bottom panel: quantification of AP+ colonies. Right panel: representative images of control (shScr) and Whsc1 depleted Rex1GFPd2 cells subjected to two rounds of exit from pluripotency. Differentiated cells (cells lost GFP/Rex1 expression) were eliminated by Blasticidin and pluripotent cells were stained by AP. Scale bar: 500 μm. Two independent experiments were performed with similar results. e, Cell proliferation assay of control (shScr) and Whsc1 depleted (shWhsc1.2 and shWhsc1.4) Rex1GFPd2 cells. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. f, Evaluation of pluripotency gene expression in control (shScr) and Whsc1 depleted (shWhsc1.2 and shWhsc1.4) Rex1GFPd2 cells by RT-qPCR. Data represent mean±s.d. from n=3 independent experiments. g, Western blot analysis of pluripotency factors, Nanog, Sox2 and Oct4 in control (shScr) and Whsc1-depleted (shWhsc1.2 and shWhsc1.4) Rex1GFPd2 cells. Tubulin was used as loading control. Two independent experiments were performed with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.
Supplementary Figure 2 Whsc1 depletion results in reduced mesendoderm differentiation in EpiSCs and in teratomas in vivo.
a, Expression of Whsc1, mesendoderm and ectoderm markers and Pou5f1 was quantified by RT-qPCR in control (shScr) and Whsc1 depleted (shWhsc1.2 and shWhsc1.4) EBs generated from EpiSCs (XGFP EpiSCs). Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. b, HE staining of teratomas from control (shScr) and Whsc1 depleted (Whsc1.2 and Whsc1.4) cells. Endodermal (gut), mesodermal (cartilage) and ectodermal (neuroepithelia) were indicated on the figures. Scale Bar: 200 μm. Ten teratomas from each group were examined, and representative images are shown. c, Expression of Whsc1, mesendoderm, ectoderm and pluripotency makers was quantified by RT-qPCR in control (shScr) and Whsc1 depleted teratomas (shWhsc1.2 and shWhsc1.4). Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test.
Supplementary Figure 3 Whsc1 is required for efficient generation of cardiac progenitors and definitive endoderm but not of neural progenitor cells.
a, Percentage of EBs with beating cells from control and Whsc1-depleted ESCs counted from Day 6 to 10 after induction of differentiation. Data represent mean from n=2 independent experiments with similar results. b-c, RT-qPCR quantification of the expression of cardiac progenitor and pluripotency markers 10 days after induction of differentiation. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. d-e, Definitive endoderm differentiation using Eomes-GFP ESCs. (d) FACS profiles of ESCs 0 and 7 days after induction of differentiation and (e) quantification of Eomes-GFP positive cells in control and Whsc1-depleted cells 7 days after induction of differentiation. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. f-g, Expression levels by RT-qPCR of definitive endoderm (f) and pluripotency markers (g) during induction of definitive endoderm in shScr and shWhsc1.4 cells. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. h, Expression levels by RT-qPCR of neural markers at several time points after neural progenitor induction and quantification of pluripotency gene expression by RT-qPCR 8 days after induction of differentiation. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test.
Supplementary Figure 4 The SET-domain is dispensable for the functions of Whsc1 in pluripotency exit and mesendodermal differentiation.
a, Schematics of wild type (WT) and the SET-domain deleted (ΔSET) Whsc1 proteins (ΔSET). b, Expression levels by RT-qPCR of Whsc1 and pluripotency genes in WT and ΔSET cells, as well as in ΔSET cells expressing shScr or shWhsc1.4 constructs at 48 and 72hrs after induction of exit from pluripotency by transferring cells into N2B27 medium containing Activin A and FBS. Data represent mean±s.d. from n=3 independent experiments. c, Expression levels by RT-qPCR of Whsc1, mesendoderm, ectoderm markers and pluripotency markers in Day 6 EBs derived from WT and ΔSET ESCs, as well as in ΔSET cells with shScr or shWhsc1.4 constructs. Data represent mean±s.d. from n=6 independent experiments. d, Western blots of Whsc1 expression in WT and ΔSET cells, as well as ΔSET cells with control or Whsc1 shRNA (shWhsc1.4). Three independent experiments were performed with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8. e, Western blots of H3K36me2, H3K36me3 and H3K27me3 levels in Whsc1 +/+ and Whsc1 −/− ESCs. Total H3 was used as loading control. Three independent experiments were performed with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.
a, Top panel: HiC contact maps at 10kb resolution from ESCs around Foxa2, Gata4, Sox1 and Pax6 loci (GSE96611). TADs are marked by solid black lines and subTAD are indicated by dashed black lines. The regions analysed using H3K27ac ChIP-seq data are indicated as black boxes; Bottom panel: H3K27ac ChIP-seq profiles on the loci of Foxa2, Gata4, Sox1 and Pax6 loci in neural progenitor cells (NPC) (GSE35496), Eomes+ mesendodermal progenitors (GSE103262), Activin A-induced mesendodermal precursors (MP) (GSE38596) and Flk1+ mesendodermal progenitors (GSE47082). The black arrows below each panel correspond to putative regulatory regions up or downstream of the respective gene. b, ChIP-qPCR quantification of Whsc1 occupancy on the same enhancers as shown in Fig. 5a (above) in D6 EBs from Whsc1 +/+ and Whsc1 −/− cells with numbers indicating distance to the TSS in kb. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. c, Replicated UMI-4C profiles for baits located on the Foxa2 (left) and Gata4 (right) promoters assayed in Whsc1 +/+ and Whsc1 −/− ESCs and D6 EBs. Top panel, contact profiles generated from the average of two independent biological replicates; bottom panel: average contact fold change D6 EBs versus D0 ESCs from two independent biological replicates. d, H3K27ac enrichments on putative regulatory regions of Foxa2, Gata4, Sox1 and Pax6 were quantified by ChIP-qPCR in Day 6 Whsc1 +/+ and Whsc1 −/− EBs. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. e, ChIP-qPCR quantification of H3K4me2 occupancies on putative regulatory regions of T, Gata6, Foxa2, Gata4, Sox1 and Pax6 in D6 Whsc1 +/+ and Whsc1 −/− EBs. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. f, ChIP-qPCR quantification of Whsc1 (left) and H3K27ac (right) occupancies on putative regulatory regions of T, Gata6, Gata4 and Foxa2 in Whsc1 +/+ and Whsc1 −/− ESCs. These regulatory regions were bound by Whsc1 in D6 with increase H3K27ac enrichments (Fig. 5b, d, Supplementary Fig. 5b and d). Data represent mean±s.d. from n=3 independent experiments.
a, siRNA knockdown efficiency was evaluated by RT-qPCR in Rex1GFPd2 cells 72 hrs after induction of exit from pluripotency. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. b, Kinetics of GFP expression determined by FACS in Rex1GFPd2 ESCs transfected with siRNAs against T, Gata6, Gata4, Foxa2 at different times after differentiation induction. Data represent mean±s.d. from n=3 independent experiments.
Gating strategy for FACS analysis.
Scans of unprocessed Western Blots.
Supplementary Figs. 1–8, and titles and footnotes of Supplementary Tables 1–4.
List of CRFs
Differentially expressed genes in Whsc1−/− ESCs
Table of primers, antibodies, single guide RNAs and siRNAs
Statistical source data
Representative day 10 beating EB derived from shScrambled ESCs under cardiac differentiation conditions
Representative day 10 EB derived from shWhsc1.4 ESCs under cardiac differentiation conditions