Setd2 deficiency impairs hematopoietic stem cell self-renewal and causes malignant transformation

Abstract

The histone H3 lysine 36 methyltransferase SETD2 is frequently mutated in various cancers, including leukemia. However, there has not been any functional model to show the contribution of SETD2 in hematopoiesis or the causal role of SETD2 mutation in tumorigenesis. In this study, using a conditional Setd2 knockout mouse model, we show that Setd2 deficiency skews hematopoietic differentiation and reduces the number of multipotent progenitors; although the number of phenotypic hematopoietic stem cells (HSCs) in Setd2-deleted mice is unchanged, functional assays, including serial BM transplantation, reveal that the self-renewal and competitiveness of HSCs are impaired. Intriguingly, Setd2-deleted HSCs, through a latency period, can acquire abilities to overcome the growth disadvantage and eventually give rise to hematopoietic malignancy characteristic of myelodysplastic syndrome. Gene expression profile of Setd2-deleted hematopoietic stem/progenitor cells (HSPCs) partially resembles that of Dnmt3a/Tet2 double knockout HSPCs, showing activation of the erythroid transcription factor Klf1-related pathway, which plays an important role in hematopoietic malignant transformation. Setd2 deficiency also induces DNA replication stress in HSCs, as reflected by an activated E2F gene regulatory network and repressed expression of the ribonucleotide reductase subunit Rrm2b, which results in proliferation and cell cycle abnormalities and genomic instability, allowing accumulation of secondary mutation(s) that synergistically contributes to tumorigenesis. Thus, our results demonstrate that Setd2 is required for HSC self-renewal, and provide evidence supporting the causal role of Setd2 deficiency in tumorigenesis. The underlying mechanism shall advance our understanding of epigenetic regulation of cancer and provide potential new therapeutic targets.

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo

“…In stark contrast, expression of H3K36M reduced the number of LKS cells, LT-HSCs and LMPPs ( Fig. 4a-f), thus suggesting a potent role of H3K36 methylation during very early lymphoid differentiation as well as stem cell maintenance, which is consistent with a recent report that implicated Setd2 in haematopoietic stem and progenitor cell (HSPC) self-renewal 28 . Together, these data show that H3K36 and H3K9 methylation have profound, partially opposing effects on early HSPC populations.…”

Loss of H3K36 Methyltransferase SETD2 Impairs V(D)J Recombination during Lymphoid Development

“…Here, we discovered a strong dependency of normal hematopoiesis and, in particular, lymphogenesis on Setd2/ H3K36me3 phenotypes consistent with previously published reports of three distinctly engineered knockout mouse models Zhang et al, 2018;Ji et al, 2019). Early loss of Setd2/H3K36me3 leads to severely impaired B and T cell development that could be partially rescued, in the case of B lymphogenesis, with the expression of a fully rearranged Igh locus, pointing to a role for Setd2/H3K36me3 in V(D)J recombination.…”

“…In addition, loss of Setd2 induced qualitative and quantitative defects in hematopoietic stem cells, as well as abnormal erythroid progenitor expansion in the bone marrow ( Figures 1D, 1G, 1H, S1C-S1F, and S2A-S2C). These hematopoietic phenotypes are consistent with other reports on Setd2 knockout mice Zhang et al, 2018;Ji et al, 2019). Altogether, these data indicate that loss of Setd2 disrupts normal hematopoiesis and severely impacts lymphoid development.…”

“…We and others Zhang et al, 2018;Ji et al, 2019) observed that loss of Setd2 early in hematopoiesis resulted in significant depletions of the lymphoid populations in the bone marrow, spleen, and thymus ( Figures 1C, 1D, 1G and S1). To rule out that these early developmental deficiencies were not solely the result of reduced numbers of early lymphoid progenitors ( Figure S1C), we crossed our knockout mice with multiple B lymphoid lineage-restricted Cre-recombinase-expressing lines and found that whereas an early and profound proB block in development was only observed upon early deletion of Setd2 (Mx1, Vav1cre) (Figure 2A), the deletion of Setd2 in later stages of B cell development (with hCD2, Mb1, and Cd19cre) resulted in abnormal lymphocytic output that was more apparent in more mature B cell populations (Figures 2B and 2C).…”

“…Thus, it is possible that the severity of the block in normal lymphocyte development observed in vivo could be influenced by factors and mechanisms beyond the role of Setd2/H3K36me3 in end-joining during the repair phase of V(D)J recombination. Also, we were unable to assess if there was a non-enzymatic role for the Setd2 protein itself in any of the NHEJ processes in lymphocyte development, as expression of a full-length Setd2 ($2,500 amino acids, $8-kb nucleotide coding sequence) in primary murine cells was unable to be achieved (by us and also not reported by other groups with genetically engineered Setd2 knockout mouse models, including but limited to Zhou et al, 2018;Zhang et al, 2018;Ji et al, 2019). We also did not fully and exhaustively investigate the role of A-NHEJ factors or the potential contribution of other roles that Setd2 and H3K36me3 play in normal cellular processes in both V(D)J recombination or post-mitotic neurogenesis settings.…”