The Lysine Histone Methyltransferase SETD2 Is Required for Appropriate Immunoglobulin VDJ Recombination

Rearrangement of the immunoglobulin heavy chain locus (IgH) through non-homologous end-joining (NHEJ)-mediated VDJ recombination is a requisite step in normal lymphocyte development. This process is initiated through cleavage of DNA by RAG1/2 recombinases and repair by classical NHEJ pathway factors. Loss or defects of these factors can impair end-processing (DNA-PKcs, ARTEMIS) or end-ligation (KU70/80, XRCC4, XLF, LIGIV) of rearrangement and are drivers of disease, notably, severe combined immunodeficiency (SCID). In addition to these core NHEJ factors, other proteins, such as ATM kinase, have been shown to be critical for the stabilization of the post-cleavage VDJ intermediates to ensure proper signal joint formation. While epigenetic regulation of this process has been described through the maintenance of accessibility for transcription factors and RAG1/2, very little is known about the role of chromatin modifications on NHEJ-mediated VDJ recombination.

Histone H3 lysine-36 tri-methylation (H3K36me3) is a histone modification associated with actively transcribed genes and has been shown to be critical for many biological processes, including transcription, mismatch repair and homologous recombination. Tri-methylation 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 tumors, including several hematopoietic malignancies. To study the role of Setd2 in hematopoiesis, we generated conditional knockout mice, where loss of Setd2 results in the ablation of the H3K36me3 mark. While heterozygous mice had no overt phenotype, mice with homozygous loss of Setd2(Setd2KO) displayed aberrant hematopoiesis, with a significant depletion of lymphoid primed progenitor population and B/T-cells in hematopoietic tissues. Setd2 loss early in hematopoiesis lead to a profound block of B-cell development at the proB cell stage and an early block in T-cell development, in a compartment where TCRβ rearrangements initiate. In Setd2KO mice, V to DJ rearrangements of the IgH locus were impaired, suggesting that the block at the proB stage of development was due to defective VDJ recombination. To assess this, we crossed our knockout mice with an IgHelMD4 mouse strain, and found that the block at the proB stage could be bypassed by the expression of a transgenic, fully rearranged IgH locus.

We confirmed global loss of H3K36me3 in proB cells from our Setd2KO mice by ChIP-Seq and found that the ablation of this mark did not affect germline IgH locus accessibility (by ATAC-seq) or local chromatin architecture (H3K4me3 or H3K9ac) at a well-studied critical regulatory region near the Eμ enhancer. Loss of Setd2 and H3K36me3 only mildly effected sterile transcription of IgH genes and did not effect the overall cellular proliferative rate or the recruitment of NHEJ factors. Furthermore, loss of Setd2 did not cooperate to generate B-cell lymphomas in a p53-/- background. However, sequencing of VDJ recombination products from knockout mice indicated not only decreased clonality of products and variable gene usage but also resulted in alterations of complementarity defining region-3 (CDR3) hypervariable sequences. These results phenocopy defects or loss of NHEJ end-ligation factors and suggest that Setd2 and H3K36me3 could play a pivotal role in the end-ligation steps of NHEJ.

Defects in end-ligation and resolution of VDJ intermediates have previously been shown to result in the formation of aberrant coding-signal hybrid joints. To see if this was occurring with loss of Setd2, we generated knockout murine proB v-Abl transformed lines where we could induce κ light chain rearrangement upon addition of an Abl kinase inhibitor. Remarkably, upon induction in Setd2KO v-Abl lines, we detected the formation of hybrid joints by both PCR and by the loss of a GFP-based coding-joint formation signal from a reporter expressed in these cells. We additionally could detect hybrid joint formation in the endogenous κ light chain locus of Setd2KO splenocytes, further indicating a role for Setd2/H3K36me3 in the end-ligation process. These studies demonstrate a novel and critical role for SETD2 and H3K36me3 in preventing aberrant, non-functional VDJ end joining of NHEJ-induced DNA breaks during lymphocyte development.