Functional Impairment of Hematopoietic Stem/Progenitor Cells By Loss of Uhrf2 in Mice

A growing body of evidence has underlined the involvement of epigenetic regulations that include histone modification in hematopoiesis. We previously identified Ubiquitin Like with PHD and Ring Finger Domains 2 (UHRF2) as a crucial multi-domain E3 ubiquitin ligase that facilitates coordination of the cell-cycle machinery and the epigenetic regulations via interaction with cyclins, TP53, DNA methyltransferases, and G9A H3K9 methyltransferase in non-hematopoietic cells (Mori et al, FEBS Letters, 2012). In fact, H3K9 trimethylation (H3K9me3) is reduced in Uhrf2^-/- embryonic stem (ES) cells (Pichler et al, J Cell Biochem, 2011). Recently, Lu et al. have also reported that UHRF2 is highly expressed in common lymphoid progenitors and mature lymphocytes rather than myeloid cells, and expression of UHRF2 is reduced in some acute myeloid leukemia (AML) cells and a variety of cancers (Oncotarget, 2016). However, the association of UHRF2 with hematopoiesis has not yet been studied. Here, to clarify the role of UHRF2 in hematopoiesis, we investigated hematopoietic cells and organs in Uhrf2^-/- mice.

We did not find a significant difference in peripheral blood leukocyte and platelet counts and hemoglobin values, and spleen sizes between wild-type (WT) and Uhrf2^-/- mice at young (12 weeks) and old (50 weeks) ages. The total cell counts and proportions of hematopoietic stem/progenitor cell (HSPC)-enriched lineage^-Sca1⁺Kit⁺ (LSK) cells, common myeloid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in bone marrow (BM) were also similar between WT and Uhrf2^-/- mice. We next performed competitive repopulation assay with BM transplants (BMT) by infusing the same numbers of Ly5.1⁺ WT BM cells and Ly5.2⁺Uhrf2^-/- BM cells into lethally irradiated Ly5.1⁺ WT recipients (n = 4). Of note, at 12 weeks after the first BMT, the proportions of Ly5.2⁺Uhrf2^-/- cells were significantly less in all lineages compared with control BMT that used Ly5.1⁺ WT BM cells and Ly5.2⁺Uhrf2^+/+ BM cells. After the second BMT, Uhrf2^-/- cells were almost rejected in neutrophils while 20-30% Uhrf2^-/- T cells and B cells were still detected. After the third BMT, however, no Uhrf2^-/- myeloid or lymphoid cells were found in peripheral blood of recipients. These findings indicate functional impairment of Uhrf2^-/- HSPCs with defective self-renewal capacity.

We then investigated the mechanism of impairment in the function of HSPCs in Uhrf2^-/- mice. As reported in Uhrf2^-/- ES cells, we found reduced H3K9me3 in Uhrf2^-/- BM cells. We also performed RNA sequencing for LSK cells, which showed downregulations of the genes that are crucial for maintenance and proliferation of HSPCs, such as Flt3 (2.2-fold) and Stat5a (2.3-fold). GSEA (gene set enrichment analysis) revealed significant enrichment of targets of H3K9me3 (FDRq value = 0.045). Furthermore, the pathway analysis identified global reductions in the canonical immunological pathways associated with differentiation of B cells, including Role of NFAT in Regulation of the Immune Response (Z score = -3.258, P = 0.009), B Cell Receptor Signaling (Z score = -2.197, P = 0.021), and PI3K Signaling in B Lymphocytes (Z score = -2.041, P = 0.024) in Uhrf2^-/- LSK cells. In agreement, it has been recently reported that reduction of H3K9me3 in HSPCs results in impairments of their abilities to differentiate and generate B cells (Ugarte et al, Stem Cell Reports, 2015; Djeghloul et al, Stem Cell Reports, 2016).

Collectively, Uhrf2 possibly play some roles in self-renewal and differentiation of HSPCs in part through the trimethylation of H3K9. Defect of Uhrf2 might contribute to the impairment of differentiation rather than growth advantage in some cancer or AML cells.