Cell-extrinsic hematopoietic impact of Ezh2 inactivation in fetal liver endothelial cells

Abstract

Despite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating Ezh2, a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (Vav-iCre) Ezh2-inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis. In contrast, endothelium (Tie2-Cre) targeted Ezh2-inactivation resulted in embryonic lethality with severe anemia at E13.5 despite normal emergence of functional HSCs. Ezh2-deficient FL endothelium overexpressed Mmp9 which cell-extrinsically depleted the membrane-bound form of Kit ligand (mKitL), an essential hematopoietic cytokine, in FL. Furthermore, Mmp9 inhibition in vitro restored mKitL expression along with the erythropoiesis supporting capacity of FL endothelial cells. These data establish that Ezh2 is intrinsically dispensable for FL HSCs and provides proof of principle that modulation of epigenetic regulators in niche components can exert a marked cell-extrinsic impact.

Introduction

Ezh2 is a histone methyltransferase and core component of the polycomb repressive complex 2 (PRC2) which mediates tri-methylation of H3K27, a mark of transcriptionally silent chromatin.¹ PRC2 is essential for early mouse embryonic development, as embryos that carry a null mutation of any of its core components die prior to gastrulation.^2–4 Studies of conditional knockout models have provided fundamental insights into the role of Ezh2 in the regulation of hematopoiesis, revealing an essential role for PRC2 in lymphocyte differentiation.^{5, 6} Subsequently, several groups have described the role of PRC2 in hematopoietic stem cell (HSC) function.^7–11 Loss of Ezh2 has minimal impact on adult HSCs, likely due to redundancy with its homolog, Ezh1, which is crucial for preventing the senescence of adult HSCs.^8–10 In contrast, the role of Ezh2 and PRC2 in fetal hematopoiesis remains less clear,^8–12 with studies using different targeting approaches to inactivate Ezh2 implicating either no role⁸ or a major cell-intrinsic role¹⁰ of Ezh2 in the regulation of fetal HSCs and erythropoiesis. Importantly, deletion of PCR2 core components Suz12¹¹ or Eed⁸ with a hematopoietic-specific Cre line (Vav-Cre) led to minor or no impact on fetal hematopoiesis respectively.

As HSCs emerge from hemogenic endothelium,¹³ endothelium-specific Cre lines such as Tie2-Cre^{10, 14} induce deletion in both endothelial and hematopoietic cells. Furthermore, endothelial and other vascular cells have a well-characterized role in the regulation of the adult bone marrow (BM) HSC niche¹⁵ and a vascular HSC niche has also recently been identified in FL.¹⁶ We therefore reasoned that loss of Ezh2 function in FL vascular cells might impact FL hematopoiesis, either through disruption of HSC emergence from the hemogenic endothelium, or by a cell-extrinsic microenvironmental impact of Ezh2-inactivated vascular cells on hematopoietic cells by regulating the expression of important hematopoietic niche factors. To explore this possibility, we inactivated Ezh2 in hematopoietic cells with (Tie2-Cre; Tie2-Ezh2-KO) or without (Vav-iCre; Vav-Ezh2-KO) simultaneous deletion in endothelial cells, allowing us to distinguish the hematopoietic impact of Ezh2 deletion in the FL vascular HSC niche from deletion in HSCs alone.

Methods

Mice

Ezh2^fl/fl,⁵ mKitL^fl/fl,¹⁷ Tie2-Cre,¹⁴ Vav-iCre,¹⁸ Gata1-EGFP,¹⁹ Rosa26-LSL-tdTomato²⁰ and Mmp9^-/-21 mice have been described, and had all been backcrossed to a C57BL/6 background. All mouse experiments were performed in accordance with UK Home Office project licence 30/3103. Embryonic development was estimated considering the day of vaginal plug formation as 0.5 d post-coitum (dpc) and somite pairs (sp). E10.5 embryos have sp range from 31 to 36.

In vivo experiments

For transplantation experiments, C57BL/6 (CD45.1) recipient mice were lethally irradiated (10 Gy, split dose) before intra-venous transplantation of 500,000 E12.5 total FL cells of indicated genotypes, together with 1,000,000 CD45.1 unfractionated BM competitor cells. CD45.2 and CD45.1 contribution to blood cell lineages were monitored every 4 weeks post-transplantation for 16 weeks. Analysis of hematological parameters was performed using a Sysmex KX-21N analyser.

FACS analysis and sorting

FACS experiments were performed using LSRII, LSRFortessa, LSRFortessa X20, AriaII, AriaIII and Aria Fusion cytometers (BD Biosciences). Data was analyzed using FlowJo software (TreeStar). Cells were incubated in Fc-block before being stained with antibodies. Gates were set using a combination of fluorescence-minus-one controls and also populations that are known to be negative for the antigen. Antibodies used are detailed in the Supplemental Information.

FL tissue preparation

Dissected FL at indicated fetal stage were digested in a mix of Collagenases IV (2 mgml^-1, Worthington) and DNase I (200 Uml^-1, Calbiochem) at 37 ºC for 15 min. The dissociated cells were centrifuged at 300 g for 6 min and resuspended in 10% FBS/PBS.

Whole-mount immunofluorescence staining and analysis

The yolk sac, head, limb buds and lateral body wall were removed from the E10.5 embryos and fixed for 45 min in 4% PFA/PBS on ice. Whole-mount immunofluorescence staining was performed as described previously.²² Images were acquired with a Zeiss 780 confocal upright microscope and analyzed using Fiji and Imaris software. Antibodies used are detailed in the Supplemental Information.

Immunofluorescence staining and imaging

Freshly isolated E12.5 FL were included in OCT for snap freezing, sliced at 7 µm thickness and processed as described previously.¹⁷ Cytospin preparations were performed using Cytospin 4 (Thermo Shandon) by centrifuging at 350 r.p.m. for 10 min. Slides were air-dried, fixed with acetone for 5 min at 4 ºC and processed as described previously.¹⁷ Images were acquired with a Zeiss 780 confocal inverted microscope and analyzed using Fiji software. Antibodies used are detailed in the Supplemental Information.

Hematoxylin and Eosin staining

Embryos were dissected and fixed in 4% PFA for 2 h at 4°C and processed as described previously.²³

Chromatin immunoprecipitation (ChIP)

C166 (ATCC) and MS-5 (DSMZ) were cultured in DMEM (Gibco) and IMDM (Gibco) respectively, supplemented with 10% FCS (Hyclone), 1% penicillin/streptomycin (P/S, PAA Laboratories), 1% L-Glutamine (Gibco). For H3K27ac and H3K27me3 ChIP, cells were fixed with 1% formaldehyde for 10 min. For Ezh2 ChIP, cells were fixed with 2 mM DSG (Disuccinimidyl Glutarate, Sigma) and 1% formaldehyde for 1 h each. Fixed chromatin samples were fragmented using an S220 Focused-ultrasonicator (Covaris) to an average size of 200-500 bp. Immunoprecipitation was then performed using Protein A/G Dynabeads (Life Technologies). Antibodies and primers used are detailed in the Supplemental Information.

Quantitative real-time PCR analysis

Total FL RNA was purified using TRIzol (Invitrogen) as recommended. Reverse transcription was performed using SuperScript III (Invitrogen) with random hexamer. cDNA was amplified and analyzed with specific Taqman probes (listed in Supplemental Information) on an ABI 7500 Fast real-time PCR instrument. Data were normalized to Hprt.

In vitro co-culture and methylcellulose colony-forming assay

6,000-10,000 E12.5 FL endothelial or stromal cells were FACS sorted and co-cultured with 150 FACS sorted E12.5 FL LSK cells for 5 d at 37 ºC in 200 µl of StemSpan SFEM (StemCell Technologies) supplemented with 1% P/S, 1% L-glutamine and cytokines mIL3 (5 ngml⁻¹, PeproTech), and hTHPO (10 ngml⁻¹, PeproTech). Media were half-changed at day 3. Non-adherent hematopoietic cells were harvested at day 5 and cultured with Methocult M3434 (Stem Cell Technologies) for 7 d at 37 ºC. Burst forming unit-erythroid (BFU-E) colony formation was determined by 2,7-diaminofluorene (DAF, Sigma) staining. For Mmp9 inhibitor ²⁴ (10 nM, CAS 1177749-58-4, Millipore) treatment, the inhibitor or DMSO (control) were added to the co-culture from day 1 and replenished at day 3 while changing media.

5,000 MS-5 cells were cultured in 10% FCS/IMDM for 24 h at 37 ºC for prior treatment with 10mM GSK126, 10mM Mmp9 inhibitor or DMSO for 24 h. 200 FACS sorted BM LSK cells were then added to the co-culture with or without drug treatment for 72 h at 37 ºC in 200 µl of StemSpan SFEM complete medium as indicated above. Non-adherent hematopoietic cells were harvested at day 3 and cultured with Methocult M3436 (Stem Cell Technologies) for 7 d at 37 ºC. BFU-E colony formation was determined by DAF staining.

Mmp9 ELISA

Supernatant of co-culture was collected by centrifuging for 5 min at 500 g to remove any non-adherent cells. Samples were assessed by murine Mmp9 ELISA kits (R&D systems) according to the manufacturer's instructions. Optical density measurements were determined using a microplate reader (Molecular Devices-SpectraMaxM2e) set to 450 nm with wavelength correction set to 570 nm.

Statistical evaluation

Statistical analyses was performed using GraphPad Prism 7.0. All results are presented in graphs are the mean ± SEM of at least three independent experiments, unless otherwise noted in the figure legends. Each exact n value is indicated in the corresponding figure legend. A two-tailed unpaired t-test was used for all analysis. FACS and immunofluorescence data presented are representative of at least three independent experiments that yielded similar results, unless otherwise noted in the figure legends.

Results

Loss of Ezh2 cell-intrinsically promotes fetal HSC expansion

Comparison of Tie2-Ezh2-KO and Vav-Ezh2-KO embryos revealed a striking difference in survival, with embryonic lethality at E13.5 in Tie2-Ezh2-KO embryos in contrast to Vav-Ezh2-KO embryos which survived at expected Mendelian ratios to E18.5 (P < 0.0001; Table 1). Tie2-Ezh2-KO embryos showed marked anemia in comparison with Vav-Ezh2-KO despite close to 100% deletion efficiency in all hematopoietic cells using both Cre lines at E12.5 (Figure 1A-B). As a previous study had implicated loss of HSCs as the cause of the anemia in Tie2-Ezh2-KO FL hematopoiesis,¹⁰ we first set out to compare numbers of hematopoietic stem/progenitor cells in Tie2-Ezh2-KO and Vav-Ezh2-KO embryos (supplemental Figure 1). E12.5 FL cellularity was reduced in Tie2-Ezh2-KO embryos (2.3-fold reduction; P = 0.0003), in contrast to normal cellularity in Vav-Ezh2-KO embryos (Figure 1C). Numbers of Lin^-Sca1⁺c-Kit⁺ (LSK) cells, which encompass a heterogeneous mix of progenitor cells and infrequent HSCs,^{25, 26} were reduced in Tie2-Ezh2-KO FL (1.8-fold reduction; P = 0.0014) but were not significantly affected in Vav-Ezh2-KO FL (Figure 1C). The reduction of LSK cells in Tie2-Ezh2-KO embryos was primarily accounted for by a reduction in LSKCD150^-CD48⁺ multipotent progenitor cells (Figure 1C). In contrast, using a more stringent definition of HSCs,²⁶ the number of LSKCD150⁺CD48^- phenotypic HSCs was similar to wildtype (WT; Cre-negative Ezh2^fl/fl or Ezh2^fl/+) embryos in both Tie2-Ezh2-KO and Vav-Ezh2-KO FL (Figure 1C). Importantly, numbers of phenotypically defined HSCs were much closer to the number of repopulating units per E12.5 FL as compared to LSK cell numbers.²⁷ HSCs emerge from the aorta-gonad-mesonephros (AGM) region, and Tie2-Cre but not Vav-iCre will delete in the hemogenic endothelium.²⁸ However, numbers of intra-aortic c-Kit⁺ cells and clusters were similar to WT embryos for both Tie2-Ezh2-KO and Vav-Ezh2-KO (supplemental Figure 2), supporting that Tie2-Cre mediated Ezh2-inactivation does not affect HSCs emergence.

Table 1. Viability of Tie2-Ezh2-KO and Vav-Ezh2-KO embryos.

	Tie2-Ezh2-KO		Vav-Ezh2-KO
Stage	Total live embryos	Percentage of live KO	Total live embryos	Percentage of live KO
E8.5	8	25	20	25
E10.5	66	32	66	21
E12.5	300	23	244	23
E13.5	24	8	34	27
E14.5	10	0	33	12
E15.5	8	0	17	18
E16.5	-	-	9	44
E18.5	-	-	44	27

The expected Mendelian frequency of KO embryo is 25%.

Figure 1. Ezh2 is dispensable for fetal HSC.

(A) Representative image of WT (n = 21; 5 independent experiments), Tie2-Ezh2-KO (n = 4; 2 independent experiments) and Vav-Ezh2-KO (n = 7; 3 independent experiments) embryos at E13.5. Scale bars, 1 mm. (B) Representative FACS histogram plots showing the recombination efficiency of Tie2-Cre (n = 6; 1 experiment) and Vav-iCre (n = 3; 1 experiment) in E12.5 FL LSK (Lin^-Sca1⁺c-Kit⁺) cells using Rosa26-LSL-tdTomato reporter. (C) Absolute numbers of Tie2-Ezh2-WT (n = 37), Tie2-Ezh2-KO (n = 9), Vav-Ezh2-WT (n = 16) and Vav-Ezh2-KO (n = 7) total mononuclear cells, LSK, MPP (LSKCD150^-CD48⁺) and HSC (LSKCD150⁺CD48^-) per FL at E12.5. Data pooled from 5 (Tie2-Cre) and 4 (Vav-iCre) independent experiments. (D) Absolute numbers of Vav-Ezh2-WT (E12.5, n = 16; E18.5, n = 13) and Vav-Ezh2-KO (E12.5 and E18.5, n = 7) LSK and HSC per FL at E12.5 (4 independent experiments) and E18.5 (3 independent experiments). (E) Absolute numbers of Vav-Ezh2-WT (n = 7) and Vav-Ezh2-KO (n = 4) LSK and HSC per femur and tibia at E18.5 (2 independent experiments). (F) Peripheral myeloid cell (Mac1⁺) reconstitution kinetics in CD45.1 mice transplanted with 500,000 CD45.2 Tie2-Ezh2-WT (n = 5), Vav-Ezh2-WT (n = 4), Tie2-Ezh2-KO (n = 10) or Vav-Ezh2-KO (n = 6) total E12.5 FL cells; Vav-Ezh2-WT (n = 11) or Vav-Ezh2-KO (n = 13) E18.5 FL cells, in all cases together with 1,000,000 CD45.1 competitor BM cells. Data are pooled from 2 (E12.5) or 3 (E18.5) independent experiments. (G) Contribution of CD45.2 E12.5 FL cells to HSC compartment at 16 weeks following transplantations shown in (F). Two-tailed t-test were used to assess statistical significance. Error bars represent SEM.

To determine whether Ezh2-inactivated HSCs expand normally during fetal development, we next assessed Vav-Ezh2-KO embryos at E18.5, at which stage LSK cell and HSC numbers were increased relative to WT embryos (2.2-fold increase for LSK cells, P < 0.0001 and 2.0-fold increase for HSCs, P = 0.0012; Figure 1D), supporting that Ezh2 KO FL HSCs expand more rapidly than WT counterparts. Analysis of Vav-Ezh2-KO fetal BM at E18.5 revealed a similar increase in number of LSK cells (1.9-fold; P = 0.0681) and HSC (5.3-fold; P = 0.0261), indicating that the migration from FL and colonization of HSCs in fetal BM was not compromised by the loss of Ezh2 (Figure 1E). To further examine any potential cell-intrinsic defect of HSC function caused by Ezh2-inactivation, we performed in vivo competitive repopulation assay using Tie2-Ezh2-KO and Vav-Ezh2-KO FL donor cells at E12.5 and Vav-Ezh2-KO FL donor cells at E18.5. We found that Ezh2 KO FL was functionally comparable to WT FL (Figure 1F-G) for long-term myeloid cell production. In line with the previously reported role of Ezh2 in early lymphoid development,^{5, 6} where loss of Ezh2 blocks the maturation of lymphocytes and leads to accumulation of immature pro-B and DN3 T cells, the engraftment of the mature lymphoid lineage was severely compromised in recipients of Tie2-Ezh2-KO and Vav-Ezh2-KO FL cells (supplemental Figure 3A). However, both Tie2-Ezh2-KO and Vav-Ezh2-KO FL cells were capable of sustaining long-term engraftment of the myeloid lineage (Figure 1F), including presence of phenotypic HSCs at week 16 following transplantation (Figure 1G; supplemental Figure 3B), indicating that Ezh2 KO FL HSCs are capable of long-term self-renewal. Furthermore, in line with the increased numbers of phenotypic HSCs in E18.5 Vav-Ezh2-KO FL (Figure 1D), myeloid engraftment was higher in recipients of equal numbers of FL cells from Vav-Ezh2-KO (Figure 1F). Taken together, these data provide definitive evidence that Ezh2 is dispensable for the emergence and expansion of phenotypic and functional HSCs during fetal hematopoiesis.

Ezh2-inactivation in endothelial cells leads to cell-extrinsically mediated anemia

As Tie2-Ezh2-KO but not Vav-Ezh2-KO E12.5 embryos were severely anemic, we next focused our analysis on erythroid development. Erythroid staging revealed a marked loss (>24-fold) of CD71⁺Ter119⁺ erythroid precursors in Tie2-Ezh2-KO but not Vav-Ezh2-KO embryos (Figure 2A-B), indicating a Tie2-Ezh2-KO specific failure of late stages of fetal erythropoiesis. To determine whether this disruption in erythropoiesis was cell-intrinsically mediated, we traced the erythroid-lineage chimerism post-transplantation with a recently generated Gata1-EGFP reporter mouse.¹⁹ This analysis demonstrated that Ezh2-inactivation did not cell-intrinsically affect the erythroid reconstitution following transplantation of FL (Figure 2C). Collectively, these data do not support that the anemia phenotype of Tie2-Ezh2-KO embryos is due to a cell-intrinsic impact of Ezh2 deletion on FL HSCs or progenitor cells. Therefore, we hypothesised that depletion of Ezh2 in FL endothelium was causing a cell-extrinsic suppression of FL progenitors, particularly those of erythropoietic lineage.

Figure 2. Absence of Ezh2 leads to cell-extrinsically mediated anemia.

(A) Representative FACS plots showing distinct block in E12.5 FL erythropoiesis in Tie2-Ezh2-KO but not Vav-Ezh2-KO embryos. Shown are the mean values with SEM for the frequencies of the indicated population across all experiments. (B) Absolute numbers of E12.5 FL erythroid progenitor populations (Tie2-Ezh2-WT, n = 26, 5 independent experiments; Tie2-Ezh2-KO, n = 7, 4 independent experiments; Vav-Ezh2-WT, n = 6, 2 independent experiments; Vav-Ezh2-KO, n = 5, 2 independent experiments). Two-way ANOVA were used to assess statistical significance. Error bars represent SEM. (C) Peripheral Gata1-EGFP^- erythroid (Ter119⁺) reconstitution kinetics in CD45.1 Gata1-EGFP recipient mice transplanted with 500,000 CD45.2 Tie2-Ezh2-WT (n = 7), Vav-Ezh2-WT (n = 2), Tie2-Ezh2-KO (n = 12) or Vav-Ezh2-KO (n = 3) total E12.5 FL cells; Vav-Ezh2-WT (n = 5) or Vav-Ezh2-KO (n = 7) E18.5 FL cells, together with 1,000,000 CD45.1 Gata1-EGFP competitor BM cells. Data are pooled from 1 (Vav-iCre; E12.5) or 2 (Tie2-Cre; E12.5 and Vav-iCre; E18.5) independent experiments. Two-tailed t-test were used to assess statistical significance. Error bars represent SEM.

Endothelial Ezh2-inactivation causes global loss of FL mKitL

We next assessed levels of erythropoietin (Epo) and KitL expression in Tie2-Ezh2-KO as these are two crucial cytokines for cell-extrinsic regulation of normal fetal erythropoiesis.^{29, 30} However, both Epo (9.8-fold; P < 0.0001) and KitL (3.6-fold; P < 0.0001) mRNA levels were upregulated in Tie2-Ezh2-KO E12.5 FL (Figure 3A), likely due to the increased proportion of hepatoblasts (3-fold; P < 0.0001; Figure 3B-D), which are the major producer of these cytokines in the FL,³¹ as a consequence of the marked reduction of erythroid precursors in Tie2-Ezh2-KO FL (Figure 2B). KitL is expressed as two major isoforms, the membrane-bound (mKitL) and the soluble (sKitL) isoforms.³² The mKitL isoform is important for supporting erythropoiesis.³³ Therefore, we further examined the expression of mKitL by immunofluorescence staining using an intracellular KitL-specific antibody that is specific for mKitL.³⁴ Strikingly, despite the increased total KitL mRNA and protein expression (Figure 3A,D), there was an almost complete loss of mKitL in Tie2-Ezh2-KO E12.5 FL (Figure 3E). Importantly, the loss of mKitL was not endothelium-specific, but also affected hepatoblasts (Figure 3D-E; supplemental Figure S4), which express KitL but are not targeted by Tie2-Cre (Figure 3F), suggesting the presence of Ezh2 KO endothelium-derived cell-extrinsic factor(s) causing a loss of mKitL expression.

Figure 3. Tie2-Cre mediated deletion of Ezh2 leads to a specific loss of mKitL expression.

(A) KitL and Epo mRNA expression levels in E12.5 FL cells (WT, n = 6; KO, n = 4; 2 independent experiments). (B) Gating strategy used to define hepatoblasts (Ter119^-CD45^-CD31^-Dlk-1⁺) population within the total FL live (7AAD^-) singlet population. (C) Proportion of hepatoblasts in E12.5 FL (WT, n = 10; KO, n = 11; 4 independent experiments). Two-tailed t-test were used to assess statistical significance. Error bars represent SEM. (D and E) Immunofluorescence staining of E12.5 FL (n = 3 each) using antibodies against KitL (D, red), Alb (D, green, hepatoblast marker), c-Kit (E, magenta, hematopoietic cell marker), CD31 (E, red, endothelial marker), mKitL (E, green). Nuclei was stained with DAPI (D and E, blue). Scale bars, 20 µm. (F) Representative FACS histogram plots showing the recombination efficiency of Tie2-Cre in E12.5 FL hepatoblasts (n = 5, 2 independent experiments) and endothelial cells (n = 6, 1 experiment; Ter119^-CD45^-CD31⁺Lyve1⁺) using Rosa26-LSL-tdTomato reporter. Shown are the mean values with SEM for the frequencies of the indicated population across all experiments.

Loss of Ezh2 impairs endothelium erythropoietic supporting ability via overexpression of Mmp9

As various FL HSC niche populations play an essential role in supporting fetal hematopoiesis, we further examined the Tie2-Ezh2-KO E12.5 FL HSC niche components and found that in addition to hepatoblasts, the proportion of both stromal (1.8-fold; P = 0.0037) and endothelial cells (1.5-fold; P = 0.0056) were also increased in the FL (Figure 3E, 4A-B), likely due to relative loss of the erythroid lineage cells (Figure 2A-B) with overall normal numbers of stromal and endothelial cells (Figure 4C). Therefore, the impaired fetal erythropoiesis was due to defective niche populations rather than a reduction of their cell numbers.

Figure 4. Reduced erythropoietic supporting ability of Ezh2-deficient endothelium.

(A) Gating strategy used to define stromal cell (SC; Ter119^-CD45^-CD31^-) and endothelial cell (EC; Ter119^-CD45^-CD31⁺Lyve1⁺) populations within the total FL live (7AAD^-) singlet population. (B and C) Proportion (B) and absolute numbers (C) of SC and EC (WT, n = 8; KO, n = 7; 2 independent experiments) per FL at E12.5. (D) Experimental design of the co-culture assay. (E) Measurement of WT or Tie2-Ezh2-KO E12.5 FL SC erythropoietic supporting ability with co-culture assay (2 independent experiments). LSK indicates LSK cells only. SC indicates SC only. (F) Measurement of WT, Tie2-Ezh2-KO or mKitL-KO E12.5 FL EC (Ter119^-CD45^-CD31⁺) erythropoietic supporting ability with co-culture assay (7 independent experiments). LSK indicates LSK cells only. EC indicates EC only. (G) Measurement of WT or Tie2-Ezh2-KO E12.5 FL EC myeloid supporting ability with co-culture assay (2 independent experiments). Numbers of replicates are indicated at the bottom of each column. Two-tailed t-test were used to assess statistical significance. Error bars represent SEM.

To gain a deeper mechanistic understanding of Ezh2 KO stromal and endothelial cells’ erythropoietic supporting ability, we established an in vitro co-culture assay with WT E12.5 FL LSK cells and Tie2-Ezh2-KO E12.5 FL-derived stromal or endothelial cells (Figure 4D). In agreement with previous studies³⁵, the presence of stromal or endothelial cells was crucial for maintaining progenitors in vitro as LSK cells that were cultured alone lost their erythroid colony forming potential (Figure 4E-F). Stromal cells (in the absence of endothelial cells) from Tie2-Ezh2-KO FL showed a normal erythropoietic supporting ability in vitro (Figure 4E). In contrast, erythropoietic supporting ability of Tie2-Ezh2-KO FL-derived endothelial cells was markedly reduced (3.2-fold reduction; P < 0.0001; Figure 4F). Moreover, E12.5 FL endothelium derived from a mKitL-specific KO mouse line¹⁷ also showed a similar defect as for Tie2-Ezh2-KO endothelium (2.2-fold reduction; P = 0.001; Figure 4F). Interestingly, the myeloid supporting ability of Tie2-Ezh2-KO endothelium remained normal, in keeping with a key role of Kit signaling in erythropoiesis^{29, 36} (Figure 4G). Together, these data support that a major cause of the defective FL erythropoiesis associated with Tie2-Ezh2-KO was the loss of mKitL expression in FL niche populations.

As mKitL is a known target of Mmp9³⁷, a metallopeptidase that is capable of cleaving the transmembrane domain of mKitL protein, causing release of sKitL and loss of mKitL expression, we next explored the possibility that Ezh2-inactivation might lead to the overexpression of Mmp9 in FL endothelial cells. We found that Mmp9 gene expression was markedly upregulated in Tie2-Ezh2-KO FLs (11.3-fold; P < 0.0001; Figure 5A). This result was further supported by the significantly higher Mmp9 protein level (18.4-fold; P < 0.0001; Figure 5B) that was present in the supernatant of co-cultured Tie2-Ezh2-KO endothelium. To determine whether Ezh2 directly regulated Mmp9 expression, an embryonic-derived endothelial cell line (C166) was used. Similarly to the impact of Ezh2-KO in FL endothelial cells, treatment of C166 cells with an Ezh2 specific inhibitor (GSK126) led to a marked increase in Mmp9 expression (6.3-fold; P = 0.004; Figure 5C). ChIP-qPCR revealed an enriched binding of Ezh2 at the Mmp9 promoter (5-fold; P < 0.0001), associated with reduced H3K27me3 (2.3-fold reduction; P < 0.0001) and increased H3K27ac levels (6.3-fold; P < 0.0001) upon GSK126-mediated Ezh2 inhibition (Figure 5D). Taken together, these data support a direct role of Ezh2 on the regulation of Mmp9 in FL endothelial cells.

Figure 5. EZH2 directly regulates Mmp9 expression.

(A) Mmp9 mRNA expression level in E12.5 FL cells (2 independent experiments). (B) Mmp9 protein level in co-culture supernatant in (G) quantified by ELISA. (C) Mmp9 mRNA expression level in DMSO and 10mM GSK126 (Ezh2 inhibitor) treated C166 cells (1 experiment). (D) ChIP-qPCR for Ezh2, H3K27me3 and H3K27ac at Mmp9 locus in DMSO and GSK126 treated C166 cells (3 independent experiments). (E) Representative immunofluorescence staining (2 independent experiments) of E12.5 FL endothelial cells collected post-in vitro treatment with DMSO or 10 mM Mmp9 inhibitor using antibodies against CD31 (red, endothelial marker) and mKitL (green). Nuclei was stained with DAPI (blue). Scale bars, 20 µm. (F) Quantification of mKitL mean fluorescence intensity (MFI) in (E). (G) In vitro co-culture assay with DMSO or Mmp9 inhibitor treatment (5 independent experiments). (H) Hematoxylin and Eosin-stained E13.5 FL sections from indicated genotypes, representative of three embryos per genotype. Scale bars, 50 µm. Numbers of replicates are indicated at the bottom of each column. Two-tailed t-test were used to assess statistical significance. Error bars represent SEM.

We therefore assessed whether inhibition of Mmp9 could restore expression of mKitL and the erythropoiesis supporting capacity of FL endothelial cells. Indeed, Mmp9-specific inhibitor (CAS 1177749-58-4) treatment of Tie2-Ezh2-KO endothelial cells in vitro rescued the cell-surface expression of mKitL (Figure 5E-F) and almost completely restored their erythropoietic supporting capability (Figure 5G). We next sought to determine whether knockout of Mmp9 in the Tie2-Ezh2-KO embryo also rescue the FL anemia phenotype. In agreement with our in vitro findings, genetic knockout of Mmp9 led to a restoration of erythropoiesis in Tie2-Ezh2-KO embryos as demonstrated by histology (Figure 5H) and also in line with previously reported rescue of anemia associated with Tie2-Ezh2-KO by Mmp9 genetic knockout as observed macroscopically.²³ Collectively, our data support that Ezh2-inactivation in FL endothelial cells leads to a cell-extrinsic disruption of erythropoiesis, mediated by overexpression of Mmp9 with consequent loss of mKitL expression.

Finally, to determine whether inactivation of Ezh2 in niche cells might also cause a cell-extrinsic disruption of adult hematopoiesis, we examined the impact of Ezh2 inactivation in MS-5 cells, an adult murine BM derived stromal cell line. In keeping with our findings in fetal hematopoiesis, GSK126-mediated Ezh2-inhibition led to an upregulation of Mmp9 expression in MS-5 cells (2.2-fold; P = 0.003; Figure 6A). ChIP-qPCR analysis also showed enriched binding of Ezh2 at the Mmp9 promoter (4.9-fold; P = 0.0001; Figure 6B). Furthermore, Ezh2 inhibition with GSK126 was accompanied by reduced H3K27me3 (1.7-fold reduction; P = 0.0003) and increased H3K27ac (4.1-fold; P = 0.0316) levels at the Mmp9 promoter (Figure 6B), once again supporting a direct role of Ezh2 in the regulation of Mmp9 expression. Using an in vitro co-culture assay, we determined that erythropoietic supporting ability of MS-5 was severely impaired by GSK126-mediated Ezh2 inhibition, which could be rescued by Mmp9 inhibitor treatment (Figure 6C). Moreover, cell-surface expression mKitL in MS-5 cells was lost upon Ezh2 inhibition and could be restored by concurrent inhibition of Mmp9 (Figure 6D). These results support that Ezh2 also has an important role in the regulation of Mmp9 in adult BM derived stromal cells.

Figure 6. Reduced erythropoietic supporting ability of Ezh2-inactivated adult BM stromal cells.

(A) Mmp9 mRNA expression level in DMSO and 10mM GSK126 (Ezh2 inhibitor) treated MS-5 cells (1 experiment). (B) ChIP-qPCR for Ezh2, H3K27me3 and H3K27ac at Mmp9 locus in DMSO and GSK126 treated MS-5 cells (3 independent experiments). (C) In vitro co-culture assay of MS-5 cells treated with DMSO, GSK126 and/or Mmp9 inhibitor (2 independent experiments). Numbers of replicates are indicated at the bottom of each column. Two-tailed t-test were used to assess statistical significance. Error bars represent SEM. (D) Representative immunofluorescence staining (n ≥ 30, 2 independent experiments) of MS-5 cells collected post-in vitro treatment with DMSO, GSK126 and/or 10 mM Mmp9 inhibitor using antibodies against mKitL (green). Nuclei were stained with DAPI (blue). Scale bars, 20 µm.

Discussion

Epigenetic factors have been shown to be important cell-intrinsic regulators of normal and malignant hematopoiesis.³⁸ Ezh2 loss-of-function mouse models have demonstrated a key role for Ezh2 during early lymphoid development.^{5, 6} Furthermore, somatic inactivating mutations of Ezh2 occur not only in patients with chronic myeloid malignancies³⁹ but also fetal origin juvenile myelomonocytic leukemia,⁴⁰ supporting the notion that hematopoietic cell specific Ezh2-inactivation might confer a clonal advantage to fetal HSCs. Moreover, loss-of-function of Ezh2 is associated with re-activation of a fetal-like gene expression program in adult HSCs.⁴¹ However, a previous study using Tie2-Cre to induce Ezh2-inactivation, suggested an essential role of Ezh2 in fetal but not adult HSC.¹⁰ This finding has not been supported by other studies using different approaches to inactivate PRC2.^{8, 9} Therefore, we directly compared the phenotype of Tie2-Ezh2-KO and Vav-Ezh2-KO embryos. In keeping with previous work,¹⁰ we observed a reduced number of FL LSK cells, disrupted erythropoiesis and severe anemia, specifically in Tie2-Ezh2-KO embryos. However, it is well established that LSK is a highly heterogeneous population containing a small minority of bona fide HSCs.⁴² By incorporating SLAM markers,²⁶ we established that numbers of phenotypic FL HSCs were not affected by the loss of Ezh2. Moreover, in functional (transplantation) assays, Tie2-Ezh2-KO FL could sustain long-term myeloid and erythroid cell reconstitution, with a defect in lymphoid reconstitution as expected from previous studies.^{5, 6} We also observed altered early myeloid reconstitution kinetics following transplantation of both Tie2-Ezh2-KO and Vav-Ezh2-KO FL, with reduced myeloid cell engraftment at 4 weeks following transplantation that was fully recovered by 8 weeks. As this defect was present following transplantation of both Tie2-Ezh2-KO and Vav-Ezh2-KO FL (Figure 1F) this would support a cell-intrinsic cause for this observation, that does not help to explain the anemia phenotype specifically associated with Tie2-Ezh2-KO, and may relate to numbers, or differentiation kinetics, of Ezh2-deficient FL progenitor cells. Furthermore, Vav-Ezh2-KO FLs showed increased numbers of HSCs at later stages of gestation, suggesting that Ezh2-inactivation cell-intrinsically promotes the expansion of FL HSCs; a finding in line with the presence of Ezh2 inactivating mutations in hematological malignancies of fetal origin, or those showing fetal-like gene expression programs.⁴⁰

In contrast to the severe anemia phenotype of Tie2-Ezh2-KO embryos, hematopoietic specific inactivation of Ezh2 with Vav-iCre did not result in anemia at E12.5. As Tie2-Cre also targets endothelial cells,¹⁴ our data strongly suggested an endothelial cell mediated mechanism of defective fetal hematopoiesis in the Tie2-Ezh2-KO model. One possibility we considered is that Ezh2 function is critical for emergence of HSCs from the hemogenic endothelium but is dispensable for HSC function following emergence, similar to findings with the transcription factor Runx1.²⁸ However, we showed that Ezh2 is dispensable for HSC emergence through demonstration of morphologically normal numbers of hematopoietic clusters in the AGM at E10.5 and as Tie2-Ezh2-KO FL contained normal numbers of phenotypic and functional HSCs at E12.5. We therefore concluded that the impact of Ezh2-inactivation in endothelial cells was likely to be cell-extrinsically mediated.

As the major hematopoietic defect in Tie2-Ezh2-KO embryos was anemia, we focused our analysis on known cell-extrinsic growth factors which are crucial to support FL erythropoiesis and identified a dramatic and almost complete loss of mKitL, a crucial cytokine for supporting erythropoiesis.^{29, 33} Crucially, this loss of mKitL was not restricted to the endothelial cells, but was also present on hepatoblasts which were not targeted by Tie2-Cre. This global loss of mKitL in Tie2-Ezh2-KO FL was caused by overexpression of Mmp9 by Ezh2-deleted FL endothelium which exerts a hematopoietic impact by cleaving mKitL.^{23, 37} We also demonstrate that Ezh2 directly regulates the expression of Mmp9 in embryonic endothelial cells and adult BM stromal cells. Notably, mKitL has been suggested to be more efficient at supporting erythropoiesis than sKitL,³³ potentially due to a more persistent activation of c-Kit signaling,⁴³ helping to explain the failure of fetal erythropoiesis upon the loss of mKitL in the Tie2-Ezh2-KO model. We also utilized a mKitL specific KO mouse¹⁷ to demonstrate that mKitL loss recapitulated the loss of erythroid supporting capability of endothelial cells in a novel co-culture assay. Furthermore, global loss of mKitL in utero has been reported to be associated with severe developmental and hematopoietic abnormalities normally associated with Steel alleles (with complete loss of Kit signaling);^{17, 36} Steel null mice die in utero at E14 to E16 with severe anemia.²⁹ Moreover, Mmp9-specific inhibitor treatment was able to fully rescue the erythropoietic supporting capability of Tie2-Ezh2-KO endothelial cells in vitro by restoring the mKitL cell-surface expression. Thus providing conclusive evidence that cell-extrinsically mediated loss of mKitL is a key mechanism of anemia in Tie2-Ezh2-KO embryos, although other mechanisms may also be involved.

To our knowledge, this is the first description of a cell-extrinsic role of an epigenetic factor in the regulation of hematopoiesis. This is of relevance for a number of reasons. First, this highlights the importance of considering possible cell-extrinsic mechanism when studying models of epigenetic regulators to avoid making false assumptions. Second, due to the key role of Ezh2 in the development of hematologic cancers, three Ezh2-specific inhibitors (GSK2816126⁴⁴, EPZ-6483⁴⁵ and CPI-1205⁴⁶) are in clinical trials. However, despite the encouraging outcome from both GSK2816126 and EPZ-6483 treatment on patients with advanced Non-Hodgkin lymphoma or solid tumors, they have some undesired hematopoietic side effects which including anemia.^{47, 48} Our findings provide a mechanistic explanation for some of these unfavorable adverse effects where global suppression of Ezh2 expression will increase MMP9 production which subsequently depletes mKitL in the HSC niche. This “off-target” toxicity could be particularly important when combining such agents with cytotoxic chemotherapy.

Collectively, our study has definitively established that Ezh2 is dispensable for HSC function in fetal hematopoiesis and has also identified a previously undescribed potential for disruption of epigenetic regulators to exert a major cell-extrinsic impact on hematopoiesis mediated through non-hematopoietic cell types. This finding has potentially important implications for analysis of mouse models and understanding of hematologic toxicity of epigenetic therapies.

Key Points.

• Ezh2 is dispensable for fetal hematopoietic stem cells

• Inactivation of Ezh2 in fetal liver endothelium leads to cell-extrinsically mediated anemia