Mechanistic Exploration Of The Chromatin Environment In Targeting Somatic Hypermutation In Human B Cells

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Somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes is initiated by activation induced deaminase (AID) and allows B cells to make antibodies that protect us against a wide variety of pathogens. AID mutates transcription coupled single stranded DNA (ssDNA) in heavy chains and is largely restricted to the variable (V) and switch (S) regions of IgH genes. The mechanism of this targeting is not fully understood. We used the Ramos human Burkitt’s lymphoma cell line (Rep161) to show that the H3K79me2/3 histone modification and its methyltransferase Dot1L are more abundant on the chromatin of the V region than the constant (C) region, which does not mutate. The knockout (KO) and inhibition of Dot1L in Rep161 cells significantly reduce V region mutation and is associated with a reduction of factors involved with elongation but did not affect the chromatin accessibility as measured by ATAC-seq. To further explore the roles of chromatin accessibility and other histone modifications, we extended our studies to the histone H3.3 chaperone HIRA which is also involved in active transcription. The KO of HIRA significantly decreases SHM in the Rep161 cells by decreasing the abundance of factors involved in active transcription in IgH locus, but increases the chromatin accessibility of the IgH V region. Furthermore, stable expression of ectopic H3.3G34 mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduces SHM. Our results reveal unrecognized roles of Dot1L, HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription associated chromatin changes contribute to SHM in human B cells but do not fully explain how AID is activated and restricted to mutate the V region exon.

To identify and quantify additional functional protein complexes specifically located on V region by MASS Spectrometry, we implemented the new dCas9-APEX system that will separately biotinylate proteins associated with the V or the C region chromatin in the Rep161 intact cells. Using this platform, we identified many additional proteins that are enriched on either V or C regions and verified some of them by ChIP-qPCR. Moreover, the inhibitor for Dot1L was used in this dCas9-APEX system and revealed changes in the relative abundance of some factors that are regulated by Dot1L. By using these datasets, we hope to find additional proteins or combinations of proteins specifically on the V or C region chromatin that regulate the mutational and repair processes and to identify additional factors involved in V region SHM.

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Please contact the Grand Round coordinator listed below:

Nancy Strein
Department: Pathology
Phone: (631) 444-3000
Email: nancy.strein@stonybrookmedicine.edu