Regulation of embryonic haematopoietic multipotency by EZH1

Abstract

All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.

Extended Data 1. (related to Figure 1). EZH1 knockdown activates lymphoid potential from PSCs

(a) List of all candidate epigenetic modifiers in loss-of-function shRNA screen. (c) CD34⁺ cells were isolated after 9 days of EB differentiation (top left), transduced with shLUC or shEZH1 and cultured under endothelial-to-haematopoietic (EHT)-promoting conditions. After 6 days, rounded haematopoietic cells (top right) were collected and co-cultured on OP9-DL1 stroma. Flow cytometric analysis of T cell potential in shLUC and shEZH1 cells without 5F is shown for two independent iPS lines in one experiment (n=2 biological replicates). (b) Representative flow plots of CD4+CD8+ T cell potential across top 6 candidates from 4 independent hairpins in two independent experiments (n=8). (d) Expansion and differentiation potential of 5F+shEZH1 cells after long-term in vitro culture. 5F+shEZH1 cells were maintained in +Dox cultures for 14 days respecification (~10²-fold expansion), plus an additional 6 weeks (~10³-fold expansion) and then plated into OP9-DL1 for T cell differentiation. Representative flow cytometric analyses of T cell potential of 5F+shLUC and 5F+shEZH1 cells after 13 weeks of expansion and differentiation in n=2 biological replicates. (e) Flow cytometric analysis (left) and quantification (right) of the proportion of CD34⁺ and CD34⁻ hematopoietic progenitors in +Dox suspension culture at day 25 in n=2 biological replicates.

Extended Data 2. (related to Figure 2). Ezh1, but not Ezh2, suppresses T cell potential and requires its catalytic domain

Ezh1, but not Ezh2, suppresses T cell potential and requires its catalytic domain(a) Quantitative PCR of PRC2 expression during the course of differentiation from hPSC-CD34+, respecification, expansion, OP9-DL1 co-culture and CD4⁺CD8⁺ T cells in n=2 biological replicates in one experiment. (b) Quantitative PCR of mRNA knockdown efficiency of individual shRNAs for PRC2 genes for n=2 replicates. (c) Western blot for EZH1 and GAPDH protein levels. (d) Scheme for rescue experiments. GFP⁺ 5F cells were transduced with shRNAs and selected with puromycin. 5F+shRNA cells were then transduced with full-length murine Ezh1 ORF (mEzh1) or mEzh1 with the catalytic SET domain deleted (mEzh1ΔSET), marked by mCherry fluorescence. Triple-transduced (GFP⁺, puro-resistant, mCherry⁺) cells were sorted and seeded onto OP9-DL1. T cells were analyzed by flow cytometry after 5 weeks of differentiation. (e) Expression of full-length murine Ezh1 (mEzh1), catalytic-deleted mEzh1ΔSET, or full-length Ezh2 in shLUC and shEZH1 cells by quantitative PCR. (f) Western blot validation of expression of mEzh1 or mEzh1ΔSET in shLUC and shEZH1 cells. (g) Representative flow cytometry plots of T cell potential for 5F+shLUC cells for rescue experiments detailed in (d) and from n=3 biological replicates (top). CD4+CD8+ T cells were verified for mCherry+ (bottom). See also Fig. 2c. All plots are gated on CD45+. (h) 5F+shRNA cells were transduced with full-length murine Ezh2 ORF (mEzh2) marked by mCherry fluorescence. Triple-transduced (GFP+, puro-resistant, mCherry+) cells were sorted and seeded onto OP9-DL1. T cells were analyzed by flow cytometry after 5 weeks of differentiation. Representative flow plots for n=2 biological replicates in one experiment. Quantitation for (h) is shown in (i) as mean ± SEM.

Extended Data 3. (related Figure 3). Ezh1 regulates hematopoietic and lymphoid programs in vitro and in vivo

(a) Representative images of E10.5 embryo (top), YS (middle) and AGM (bottom) from n>30 embryos. Lin⁻cKit⁺VE-Cadherin⁺CD45⁺CD41⁺ cells from E10.5 YS and AGM were FACS-sorted followed by RNA-seq analysis. (b) Genes upregulated and downregulated by >2-fold in Ezh1^+/− or Ezh1^−/− YS and AGM compared to WT. (c) and (d) GO term annotations of upregulated genes in Ezh1^+/− and Ezh1^−/− YS and AGM compared to WT. (e) GO analysis of enriched pathways of 1033 nearest neighbor genes associated with upregulated ATAC-seq peaks (top) GO analysis of nearest 1012 neighbor genes associated with downregulated ATAC peaks (bottom). (f) Comparison of upregulated ATAC-seq peaks in 5F+shEZH1 cells with HSPC, B,T cell networks and (d) HSPC hierarchy signatures. (g) Box plot of expression of genes associated with upregulated (ATAC UP) and downregulated (ATAC DOWN) ATAC-seq peaks. *p< 0.05 via one-way ANOVA (h) ATAC-seq density map of cKit⁺VE-cadherin⁺CD45⁺ HSPCs sorted from ~30 embryos of E10.5 WT and Ezh1^−/− AGM (top) from one experiment. Significantly upregulated ATAC-seq peaks were compared to HSPC, T, B cell networks and signatures of the human HSPC hierarchy (bottom). (i) GO terms of enriched pathways of regions associated with significantly upregulated ATAC-seq peaks annotated by GREAT analysis in Ezh1^+/− AGM (top) and Ezh1^−/− AGM (bottom) compared to WT. (j) GO terms of enriched pathways of regions associated with significantly downregulated ATAC-seq peaks annotated by GREAT analysis in Ezh1^+/− AGM (top) and Ezh1^−/− AGM (bottom) compared to WT. (k) TF binding to genes with upregulated ATAC peaks in Ezh1^+/− (left) and Ezh1^−/− AGM (right) from (i) compared to WT AGM.

Extended Data 4. (related to Figure 3). Genome-wide chromatin occupancy reveals EZH1 enrichment at bivalent HSC genes and non-canonical active lymphoid genes

(a) Breakdown of EZH1 binding at promoter regions and associated histone marks. GO term analysis of EZH1-bound active (b), bivalent (c) and repressed (d) genes. (e) Distribution of EZH1-bound genes across the hematopoietic hierarchy (left) and their associated histone marks (right). R = repressed (H3K27me3-marked), A = active (H3K4me3-marked), B = bivalent (H3K4me3 and H3K27me3-marked). (f) GSEA analysis of EZH1-bound genes correlated with RNA-seq data upon EZH1 knockdown. (g) Sankey diagram showing genome-wide changes in histone methylation status upon EZH1 knockdown. (h) Upregulated genes exhibit reciprocal decreases in H3K27me3 levels, as quantified by EpiChIP software. (i) Activated (formerly bivalent) HSC genes exhibit increased gene expression upon EZH1 knockdown and loss of H3K27me3. (j) Correlations between changes in H3K27me3 and gene expression levels upon EZH1 knockdown, subdivided by subgroups corresponding to methylation changes. (k) breakdown of bivalent-bivalent (left), bivalent-repressed (center), and bivalent-null (right) genes upon EZH1 knockdown across the hematopoietic hierarchy. (l) Overlap of EZH1 and EZH2 enriched peaks and the distribution of all EZH1 enriched, EZH2 enriched or common genes across the hierarchy (left), or specifically bivalent genes that become activated upon EZH1 knockdown (middle) and active genes, marked by H3K4me3 in shLUC (right). (m) SUZ12 binding (from the ChEA database) across the hematopoietic hierarchy. (n) Canonical and non-canonical targets, previously identified by Xu et al. Mol Cell (2015) across the hematopoietic hierarchy. (o) Breakdown of histone marks on non-canonical ProB genes and (p) the genome-wide distribution from CEAS analysis. (q) Changes in actively marked, non-canonical ProB genes (green bar, panel o), upon EZH1 knockdown. (r) SUZ12 and EZH2 binding (ChEA database) at ATAC peaks in Ezh1^+/− and Ezh1^−/− AGM. *p< 0.05 via one-way ANOVA.

Extended Data 5. (related to Figure 4). Ezh1 deficiency enhances embryonic HSPC engraftment

(a) Whole E10.5 AGM and YS were transplanted intravenously into sublethally irradiated NSG adult females. Chimaerism was monitored via retroorbital bleeding every 4 weeks. Representative flow plots are shown for analysis after 4 weeks in n≥3 mice. (b) Whole E9.5 PSP was transplanted intravenously into sublethally irradiated NSG adult females. Chimaerism was monitored via retroorbital bleeding every 4 weeks. Representative flow plots are shown for analysis after 8 weeks in n≥3 mice. (c) Representative flow plots of lineage analysis in E10.5 AGM Ezh1^+/− and Ezh1^−/− primary transplant recipients after 24 weeks, and in E9.5 PSP Ezh1^+/− primary transplant recipient after 16 weeks (n≥3 mice per group). (d) Primary recipients in (a) were sacrificed after 24 weeks post-transplantation and 4 ×10⁶ whole bone marrow was transplanted into sublethally irradiated adult NSG females intravenously. Chimaerism was monitored via retroorbital bleeding. Representative flow plots of E10.5 AGM and YS secondary transplants after 4 weeks in n≥3 mice. (e) Secondary transplantation of E10.5 YS primary recipients in (Fig. 4f). (Right) Lineage distribution of E10.5 YS secondary recipients. Data is pooled across three independent experiments. *p<0.05, ** p<0.01 by unpaired two-sided t-test; see supplementary information for exact p values per time point. N.E. = not engrafted.

Extended Data 6. (related to Figure 4). Ezh1-deficient embryonic HSPCs contribute to adult-type lymphopoiesis in vivo

(b) Flow analysis of B1 and B2 progenitors in the peritoneal cavity of engrafted primary recipients (n=1 mouse per group). (b) Flow analysis of TCRβ and TCRγδ frequencies of donor-derived peripheral CD3⁺ T cells from engrafted primary recipients (n=1 mouse per group).

Figure 1. In vitro screen for epigenetic modifiers that restrict definitive lymphoid potential

(a) Scheme for human PSC differentiation into haematopoietic progenitors. CD34⁺ cells were transduced with HOXA9, ERG, RORA, SOX4, and MYB (5F). 5F cells were then transduced with individual shRNAs (×4 each) targeting each epigenetic modifier and seeded onto OP9-DL1 stroma to induce T cell differentiation. (b) Strictly standardized mean difference (SSMD) of CD4⁺CD8⁺ T cell frequencies across all 4 shRNAs targeting each epigenetic modifier in 5F cells in n=2 independent experiments using two different iPSC lines, CD45-iPS and MSC-iPS1. (c) Prospective analysis of T cell and B cell frequencies from 5F+shRNA targeting top candidates (n=2 biological replicates). (d) Flow analysis of CD4⁺CD8⁺ T cell development of 5F cells with shRNAs targeting luciferase (shLUC) or EZH1 (shEZH1) after 5 weeks differentiation on OP9-DL1. (e) Flow analysis of CD19⁺ B cell potential. (f) Quantitation (mean ± SEM) of T cell potential of 5F+shEZH1 cells compared to 5F+shLUC cells pooled across 2 hairpins and 5 independent experiments (n=10) using multiple iPSC lines (CD34-iPS, CD45-iPS, MSC-iPS1). Source data files show individual values obtained for each hairpin. ***p=0.001 by unpaired two-tailed t-test (g) Quantitation of colony-forming potential in n=3 independent experiments. (h) Flow analysis of myeloid (CD11b⁺) and (i) erythroid (CD71⁺GLYA⁺) potential. Experiments replicated at least twice.

Figure 2. Repression of canonical PRC2 subunits does not activate lymphoid potential

(a) Representative flow plots of T cell potential of 5F cells with shRNAs targeting individual components of PRC2. (b) Quantitation (mean ± SEM) of T cell potential of 5F+shRNA targeting the indicated subunit in (a) shown as using two hairpins across two independent experiments (n=4) *p=0.0457, **p=0.0061 by unpaired two-tailed t-test. (c) Representative flow analysis of T cell potential in 5F+shEZH1 cells with mEzh1 or mEzh1ΔSET co-expressed. (d) Quantitation of flow analysis in (c) shown as mean ± SEM of n=3 biological replicates. *p=0.0146, **p=0.0011 by one-way ANOVA. All plots are gated on CD45⁺. Data is pooled across two independent experiments.

Figure 3. EZH1 directly binds to and modulates expression and chromatin accessibility of HSC and lymphoid genes

(a) Heatmap of upregulated (104) and downregulated (49) genes (>2-fold; Benjamini-Hochberg corrected t-test, p<0.1) from RNA seq analysis of CD34⁺CD38⁻ HSPCs 5F+shEZH1 (n=10 biological replicates) compared to 5F+shLUC cells (n=8 biological replicates). (b) GO analysis of biological processes associated with significantly upregulated genes in (a), subdivided by GO hierarchical categories and p-values labeled along radius. (c) Enrichment of human HSC and progenitor signatures by GSEA in 5F+shEZH1 compared with 5F+shLUC cells, overlaid on the map of human HSPC hierarchy. (d) Density map of upregulated and downregulated ATAC peaks by MAnorm in 5F+shEZH1 cells compared to 5F+shLUC from n=2 biological replicates. (e) GO terms of enriched biological processes of ATAC-seq peaks in (d) by GREAT analysis. (f) Tracks of representative genes that acquire a significant ATAC peak upon EZH1 knockdown. (g) ChIP-seq density map of EZH1 peaks within bivalent (B), repressed (R), active (A) or null (N) promoter groups from n=2 biological replicates. K4 = H3K4me4, K27 = H3K27me3 (h) Waterfall plot of CellNet predicted regulators of EZH1-bound bivalent gene networks. (i) Sitepro quantitative analysis of H3K27me3 levels at all upregulated genes around the transcription start site upon EZH1 knockdown, relative to shLUC in n=2 biological replicates. (j) Sankey diagram illustrating histone methylation changes of all bivalent genes in shLUC cells and after EZH1 knockdown n=2 biological replicates (left). Genes that lose H3K27me3 (become activated) are specifically enriched in HSC signature, whereas bivalent genes that are unchanged or repressed are enriched in ProB signature (right) by Fisher’s exact test. (k) ChIP-seq tracks of EZH1, H3K4me3 and H3K27me3 at representative HSC promoter regions in shLUC and shEZH1 cells. Experiments replicated at least twice.

Figure 4. Ezh 1 deficiency increases lymphoid potential and engraftment of embryonic HPSCs

(a) Representative images of E9.5 and E10.5 embryos (n>50 embryos). (b) Quantitative PCR of each PRC2 subunit in E10.5 WT YS and AGM as mean ± SEM of n=3 biological replicates. *p=0.0439,****p<0.0001 by unpaired two-tailed t-test (c) ATAC-seq density map of cKit⁺VE-cadherin⁺CD45⁺ HSPCs sorted from 30 pooled embryos of E10.5 WT and Ezh1^+/− AGM. (d) Significantly upregulated ATAC-seq peaks were compared to HSPC, T, B cell networks and signatures of the human HSPC hierarchy. *p<0.05 by Fisher’s exact test. (e) Engraftment of E10.5 AGM (3.5ee) in sublethally-irradiated adult NSG females. Donor chimerism marked by CD45.2⁺ was measured in peripheral blood every 4 weeks up to 16 weeks post-transplantation. Each dot represents a single transplant recipient. (Right) Lineage distribution of engrafted mice showing T cell (T), B cell (B), and myeloid (M) contribution. (f) Engraftment of E10.5 YS (5ee). (Right) Lineage distribution of engrafted mice. (g) Engraftment of E9.5 PSP (10ee). (Right) Lineage distribution of engrafted mice. (h) Serial transplantation of whole BM from primary recipients of E10.5 AGM cells in (e). Secondary transplant was carried out after 24 weeks of primary transplant. (Right) Lineage distribution of engrafted mice. n≥3 mice per group; *p<0.05, ** p<0.01, ***p<0.0001 by unpaired two-tailed t-test. See supplemental information for exact p values per time point. Data is pooled across four independent experiments in (e, f), four independent experiments in (g), three independent experiments in (h); experiment in (c) performed once.