Coactivator-associated arginine methyltransferase 1 regulates fetal hematopoiesis and thymocyte development

Abstract

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

Figure 1. CARM1 deficiency results in an early thymopoiesis block in E18.5 embryos

(A) Representative flow cytometric analysis of CD45⁺Lin⁻ thymocytes from E18.5 Carm1^−/− and Carm1^+/− or Carm1^+/+ littermate controls. Arrows indicate gating strategy. DN thymocyte subsets are specified. Dot plots were depicted at low resolution when subset cellularity was low. (B) Cellularity of thymocyte subsets from E18.5 Carm1^−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 4 independent experiments with 2 or more mice per experiment. P values: * P<0.05; ** P<0.01; *** P<0.001.

Figure 2. CARM1 deficiency in the thymic stromal compartment does not impair thymocyte development

E15.5 Carm1^−/− or littermate control thymic lobes were incubated in 2-deoxyguanosine for 5 days and transplanted under the kidney capsule of athymic recipients. After 8 weeks, thymocytes recovered from the grafts were analyzed by flow cytometry. Data shown are from 5 individual experiments with 2 or more animals per experiment. Mean values are indicated by the horizontal bars. P values: * P<0.05; ** P<0.01; *** P<0.001.

Figure 3. Carm1^−/− fetal thymi contain fewer thymocyte progenitors than Carm1^+/+ littermates

Transverse cryosections of Carm1^−/− and Carm1^+/+ fetal thymi were costained for thymocyte and TEC markers as indicated. (A) Distribution of CD45+ progenitors in E12.5 thymus rudiment. Scale bar = 100mm; (B) Distribution of c-kit+ (DN1 or DN2) thymocytes in E13.5 thymus rudiment (denoted by dashed line). Scale bar = 200mm; (C) Distribution of CD25+ (DN2 or DN3) thymocytes and K5+ TECs in E13.5 (Scale bar = 100mm) E15.5 (Scale bar = 500mm) and E17.5 thymi (Scale bar = 1000mm). Three experiments were performed with thymi from two Carm1^−/− and 1 Carm1^+/+ per experiment. Sections were mounted in VECTASHIELD mounting medium (Vector Laboratories) and staining was analyzed at room temperature using an Olympus ProVis AX70 microscope with UPlanFl 40/0.75, UPlanFl 20/0.50 and UPlanFl 10/0.30 objectives, DP Controller and DP Manager software.

Figure 4. CARM1 deficiency results in reduced cellularity of hematopoietic progenitors in E18.5 bone marrow

(A) Representative flow cytometric analysis of Lin⁻ bone marrow cells from E18.5 Carm1^−/− null mice and littermate controls. The top row is a representative stain from C57Bl6/J adult bone marrow, which was used to define gating strategy for progenitor populations. Arrows indicate gating strategy. Hematopoietic subsets are specified. Dot plots were depicted at low resolution when subset cellularity was low. (B) Cellularity of bone marrow progenitors from E18.5 Carm1^−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 5 independent experiments with 2 or more animals per experiment. P values: * P<0.05; ** P<0.01; *** P<0.001.

Figure 5. CARM1 is not necessary for maintenance of hematopoietic progenitors in fetal liver

(A) Representative flow cytometric analysis of Lin⁻ fetal liver cells from E18.5 Carm1^−/− mice and littermate controls. The top row is duplicated from Figure 4 to show the gating used to identify hematopoietic progenitor subsets. Arrows indicate gating strategy. Hematopoietic subsets are specified. (B) Cellularity of fetal liver progenitors from E18.5 Carm1^−/− embryos and littermate controls. Each point represents an individual embryo. Mean values are indicated by the horizontal bars. Results are combined from 5 independent experiments using the same animals as were used in Figure 4. P values: * P<0.05; ** P<0.01; *** P<0.001.

Figure 6. Competitive reconstitution reveals a cell autonomous requirement for CARM1 in hematopoiesis

(A) Schematic of competitive reconstitution assay. Equal numbers of EGFP negative C57Bl6/J fetal liver cells were mixed with EGFP positive fetal liver cells from Carm1^−/− or control E14.5 embryos. The cell mixture was injected i.v. into sublethally irradiated (4.5 Gy) RAG-2^−/− γc^−/− mice. Eight to twelve weeks post-injection, recipient thymocytes and bone marrow cells were analyzed for the relative EGFP+ versus EGFP− chimerism. (B) The ratio of EGFP+ to EGFP− chimerism within each thymocyte subset in competitive reconstitution recipients was determined by flow cytometric analysis. Results are from 4 independent experiments with 2 or more animals per experiment. (C) The ratio of EGFP+ to EGFP− chimerism within each bone marrow progenitor subset in competitive reconstitution recipients was determined by flow cytometric analysis. Results are from 3 independent experiments with 2 or more animals per experiment. Error bars show ± SEM; *P < .05; **P < .01; ***P < .001.

Figure 7. CARM1 deficiency impairs survival of E14.5 fetal liver progenitors during in vitro T cell differentiation

500 sorted KLS from E14.5 Carm1^−/− or control fetal livers were cultured on OP9-DL1 stroma in the presence of 5ng/ml IL-7 and Flt3L for 6 days. (A) Cellularity of DN1, DN2, DN3, and DN4 subsets was determined by flow cytometry. Each point represents an average of 3 replicate wells plated from a single embryo. Mean values are indicated by horizontal bars. Data are combined from 6 independent experiments. (B) Percentage of cycling cells was determined using flow cytometric analysis of DNA content in recovered cells. Each point represents an average of 3 replicate wells plated from a single embryo. (C) Percentage of annexin V+ cells was determined by flow cytometric analysis of recovered cells. Each point represents an average of 3 replicate wells plated from a single embryo. P values: ** P<0.01; *** P<0.001. (D) Representative flow cytometric histograms of IL-7R expression on E18.5 fetal liver and thymocyte progenitors. IL-7R expression is comparable on fetal liver Flk2+ MPP, CLP, and on thymocyte DN3 subsets in Carm1^−/− versus control embryos. IL-7R expression is reduced on the Carm1^−/− CD44⁺CD25⁻ thymocyte progenitor population, including the c-Kit⁺ DN1 subset. Fetal liver data are representative of 3 separate experiments, including a total of 6 control and 5 Carm1^−/− embryos. Thymocyte data are representative of 4 separate experiments, including a total of 5 control and 6 Carm1^−/− embryos.