Loss of Lysine Histone Methyltransferase Setd2 Disrupts Normal Hematopoiesis, Lineage Commitment and Reveals a Novel Role for H3K36me3 in Immunoglobulin VDJ Recombination

The post-translational modification consisting of trimethylated Histone H3 lysine-36 (H3K36me3) is a chromatin mark associated with actively transcribed genes and implicated in many biological processes, including transcription initiation and elongation, splicing, and DNA damage repair response via homologous recombination. H3K36me3 is catalyzed by the non-redundant histone methyltransferase, SETD2. Loss of function mutations in SETD2 or dominant negative "onco-histone" mutations in the H3K36 residue have been described in a broad array of solid tumors. More recently, SETD2 has been found to be commonly mutated in acute and chronic leukemias (B-ALL, ETP-ALL, CLL, AML), indicating its potential role as a tumor suppressor in hematopoietic malignancies. In order to study the role of Setd2 in normal and malignant hematopoiesis, we generated Vav1-cre and Mx1-cre Setd2 conditional knockout mice. Expression of Cre-recombinase resulted in loss of Setd2 and ablation of H3K36me3. While heterozygous mice had no overt phenotype, homozygous loss of Setd2(Setd2KO) in early hematopoietic development resulted in pancytopenia, splenomegaly, and overall bone marrow hypocellularity. Significant reduction in the total number of hematopoietic stem cells (HSCs) was also observed. Hematopoietic reconstitution assays in competitive and non-competitive transplant settings additionally revealed a qualitative defect in Setd2KO HSCs. Within the LSK-defined HSC fraction (Lin^-Kit⁺Sca1⁺), a significant depletion of the lymphoid primed progenitor population (MPP4) was observed while the erythroid primed progenitor (MPP2) population was significantly expanded. Interestingly, while myelopoiesis was largely unaffected (in number of progenitors and hematopoietic output), B-cells (B220⁺) and T-cells (CD4⁺CD8⁺) were significantly depleted in Setd2KO mice (8 and 10-fold respectively). In both the bone marrow and spleen, a significant expansion of early erythroid progenitors was observed. Gene set enrichment analysis of RNA-Sequencing of the LSK HSC fraction of Setd2KO mice revealed strong correlations to signatures of HSC differentiation, particularly significant enrichments in erythroid priming signatures, (eg. Gata1) and signatures related to proliferation and loss of stem cell quiescence. Altogether, these data indicate that Setd2is important in regulating normal HSC self-renewal and lineage commitment, with differential effects in each lineage.

We wanted to further explore the defect in lymphopoiesis and observed a profound block of B-cell development at the pro-B cell stage (B220⁺CD43⁺IgM^-), with a 25-fold depletion of pre-B cells (B220⁺CD43^-IgM^-) and an almost complete ablation of immature IgM⁺ B-cells in the bone marrow of our Setd2KO mice. Interestingly, Mb1-cre and Cd19-cre Setd2KO mice had no arrest in early B cell development, indicating that loss of Setd2 prior to pro-B cell stage was necessary for the block in differentiation. In Setd2KO mice, V to DJ rearrangements of immunoglobulin heavy-chain locus (IgH) were not detectable by a non-quantitative PCR method, suggesting that the block at the pro-B stage of development in our Setd2KO mice was due to the impairment of complete VDJ recombination at this locus. To prove this, we crossed our knockout mice with an IgHelMD4⁺ mouse strain and found that the block at the pro-B stage could be bypassed by the expression of a transgenic, fully rearranged IgH locus in Setd2KO mice, indicating the requirement of Setd2 and H3K36me3 in VDJ recombination. Additionally, we observed that loss of Setd2 later in B cell development impairs class switch recombination by LPS and IL-4 co-stimulation of Setd2KO Cd43^- splenocytes of Mb1cre Setd2KO mice. This defect in VDJ recombination as a result of loss of H3K36me3 is associated with impaired recruitment of NHEJ machinery to the IgH locus. Similarly, Setd2KO mice exhibited significantly reduced thymic size and a comparable block in T-cell development at the DN3 stage, where TCRb rearrangements initiate.

Altogether our studies demonstrate not only a critical role for Setd2 in the HSC compartment in determining hematopoietic differentiation, but also the requirement of Setd2 and H3K36me3 in lymphopoiesis, specifically for fully competent NHEJ-driven VDJ recombination and class switch recombination.