DOT1L-mediated H3K79me2 directs B-cell repertoire establishment, marginal zone development and germinal center function

DOT1L is an epigenetic regulator that promotes gene expression by methylating lysine 79 on histone H3 and recruits transcription factors to gene targets. DOT1L is also an oncogenic driver in cancers that affect developing lymphocytes, yet how DOT1L activity regulates B-cell maturation remains poorly defined. Here, we use deep-sequencing and conditional knockout models to elucidate gene targets of H3K79me2, and the mechanistic contribution of DOT1L, during key stages of murine B-cell development. In the bone marrow, Dot1l was upregulated in pro B-cells. Deep-sequencing revealed that H3K79me2 accumulated during maturation. The genomic distribution of H3K79me2 peaks indicating that DOT1L regulates transcription and the cell cycle across different stages of B-cell development. As such, DOT1L was found to be essential for pre B-cell expansion, leading to a significant decrease in pre B-cells in the absence of DOT1L and a skewing of the B-cell receptor repertoire to favor proximal VH usage. In addition to the effective generation of a diverse B-cell pool, DOT1L was also required to establish the marginal zone (MZ) B-cell gene expression program. Attenuation of MZ B-cells and a bottlenecking at the pre-MZ B-cell stage in Dot1L-deficient mice correlated to H3K79me2 peaks at key MZ-regulatory genes such as Lfng and Dock10 in developing B cells. In contrast, Dot1l was re-expressed in germinal center (GC) B-cells post-immunization to deposit H3K79me2 at key GC B-cell gene loci during GC differentiation. Together, these data demonstrate a vital role for DOT1L during B cell lymphopoiesis, MZ B-cell generation and GC B-cell biology. Overall design: B-cell subsets were sorted from naïve or immunized mice and H3K79me2 ChIP was performed and sequenced. Input and H3K79me2 ChIP files were generated for each sample. Follicular B cells were sorted and purified from mice bearing a B cell-specific conditional Dot1l deletion (Dot1lf/fMb1Cre/+) and from controls (Mb1Cre/+). BCR sequencing was performed to assess the impact of deleting Dot1l during early B-lymphopoiesis on the VH gene usage in mature B-lymphocytes.