Setd2-Mediated Epigenetic Regulation of Erythropoiesis and HSC Maintenance in Mouse Models

SET domain containing 2 (SETD2) is a methyltransferase for histone 3 lysine 36 tri-methylation (H3K36me3). Using a conditional knockout allele (Setd2^Δ/Δ), we recently demonstrated that their SLAM-hematopoietic stem cells (SLAM-HSCs) have intrinsic defects in maintaining quiescence and multi-lineage differentiation potential. Setd2^Δ/Δ mice show decreased lymphoid and myeloid progenitors except increased pre-colony-forming-unit-erythroid (Pre CFU-E) and erythroblasts in bone marrow (BM); they suffer from lethal macrocytic anemia. A decreasing H3K36me3, but increasing H3K36me1 and H3K36me2, were observed in Setd2^Δ/Δ mice. As a consequence, RNA polymerase II elongation is enhanced at a subset of genes, including Myc which encodes a transcriptional factor essential for erythropoiesis and HSC activation (Zhou Y., Haematologica, 2018). In the current study, we aim to investigate the detailed mechanisms of Setd2 deficiency in the intrinsic and extrinsic mechanisms of erythropoiesis and in the maintenance of HSC from definitive HSCs to adult.

Erythropoiesis from the BFU-E/CFU-E stage to mature erythrocytes is sequentially regulated by an erythropoietin (EPO)-dependent early stage and macrophages-dependent late stage. To understand the function of Setd2 in erythroid differentiation, we generated two Setd2 conditional knockout mice: Setd2^flox/flox/Vav1-Cre⁺ (Setd2^Δ/Δ) and Setd2^flox/flox/EpoR-Cre⁺ (Setd2^Δ/Δ/EpoR). Setd2^Δ/Δmice harbor a pan-hematopoietic deletion, while Setd2^Δ/Δ/EpoR mice harbor an erythroid stage specific deletion from CFU-Es, proerythroblasts and basophilic erythroblasts. In Setd2^Δ/ΔBM, the absolute number of basophilic erythroblasts and polychromatic erythroblasts were significantly increased but mature red blood cells were decreased. Surprisingly, Setd2^Δ/Δ/EpoR mice showed normal proportions of erythroid blasts and did not suffer from anemia, suggesting that Setd2 is not required for the proliferation and differentiation of EPO-dependent erythroblasts and thereafter mature erythrocytes. Central macrophages are known to play a crucial role in terminal erythropoiesis by supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. We then analyzed macrophages in Setd2^Δ/Δ mice. The total F4/80⁺ macrophages were not changed, however, the proportion of type II macrophages (CD206⁺CD80^-F4/80⁺) were significantly increased in Setd2^Δ/Δ BM. Myc is a well-known factor for polarizing type II immune phenotypes. Our data suggest that Setd2 deficiency causes a shift of central macrophage to type II, which possibly impedes the terminal maturation of erythrocytes and causes macrocytic anemia, even with significantly increased BFU-E, CFU-E, and erythroblasts.

Next, we addressed whether Setd2 was required for HSC maintenance from definitive HSCs to adult HSCs in an autonomous manner. It has been shown that genes regulating the maintenance of HSC at different developmental stages are diverse. To clarify in which developmental stages Setd2 is critical for HSC maintenance, we examined the HSC populations in Setd2^Δ/Δ embryos at E14.5, and in Setd2^Δ/Δ mice BM at 3 weeks (wks), 8 wks and 34-38 wks of age. HSC populations from fetal liver (FL) at E14.5 and BM at 3 wks were not different from Setd2^flox/flox control littermates. Significant reductions in the absolute number of SLAM-HSCs, CD201⁺SLAM-HSCs and multipotent progenitors were observed at 8 wks, eventually HSCs were completely exhausted for mice at 34-38 wks. We further evaluated the HSC function in a competitive bone marrow transplantation assay. Similar levels of multi-lineage reconstitution were observed in recipient mice with Setd2^Δ/Δ donor cells from FL or BM transplanted at 3 wks. However, Setd2^Δ/Δ HSCs from 8 wk and 34 wk BM failed to support a long-term reconstitution and there is a complete absence of donor derived Setd2^Δ/Δ LSKs. Altogether, our data suggest that Setd2 is required for HSC maintenance at adult stage, but not at fetal or neonatal stages.

In summary, our study revealed a previously unknown extrinsic function of Setd2 in terminal erythroid maturation; and also identified Setd2 as a critical intrinsic regulator for adult HSC maintenance. Our findings may provide unique insights into the defects in erythroid lineages, such as anemia or erythrocytosis, and also epigenetic mechanism of adult HSC maintenance.