Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

Abstract

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Locus-specific proteomics identifies new aspects of the chromatin context involved in V region somatic hypermutation

“…S2F-S2H). The increase in H3K79me1 in the V region but not the C region with inhibitor treatment was reported in our previous paper (Duan et al, 2021). We found that the EPZ004777 treatment significantly but very slightly decreased the abundance of dCas9-APEX on chromatin in C-gRNA cells after the inhibitor treatment (Fig.…”

“…Encouragingly, the abundance of MLLT1, a component of super elongation complex, was increased on V region after Dot1L inhibitor treatment. We had shown previously that the Dot1L inhibitor EPZ004777 only inhibited its enzymatic activity but did not change its physical abundance on the V region, and the EPZ004777 treatment only decreased the SHM by 30% compared with the 70% decrease of SHM in Dot1L KO cells (Duan et al, 2021). The Dot1L molecule has been shown to both form its own complex and to be a component of SEC where its function appears to be independent of its histone methyltransferase activity (Cao et al, 2020).…”

“…It is widely accepted that the transcription-related processes play a central role in generating ssDNA that is the substrate for AID and in recruiting the factors that are responsible for the mutational process (Bransteitter et al, 2003; Sun et al, 2013). Cis-acting DNA regions, transcription factors, several transcription elongation factors, histone modifications and a histone chaperone which contributes to active transcription have been shown to play important roles in this process (Duan et al, 2021; Feng et al, 2020; Tang and MacCarthy, 2021; Yu et al, 2021a; Yu et al, 2021b). For example, Spt5 was reported to facilitate the transition from pausing to elongation and to promote V region SHM both through its physical interaction with AID and by inducing RNA polymerase II (RNA Pol II) stalling (Methot et al, 2018; Pavri et al, 2010; Pham et al, 2019).…”

“…The Ramos germinal center like Burkitt’s lymphoma cell line has proven to be a good system to study the role of individual candidate genes and processes such as transcription as well as the role of larger genomic regions such as topologically associating domains (TADs) in the SHM of the human heavy chain V region (Dinesh et al, 2020; Duan et al, 2021; Senigl et al, 2019; Tarsalainen et al, 2022; Yu et al, 2021b). In order to obtain a broader more quantitative and less biased view of chromatin environment in which AID induced mutation is occurring in situ, we used Mass Spectrometry that would allow us to compare and quantify both known and unknown proteins that were associated with the chromatin in the mutating V and non-mutating C regions.…”

Somatic hypermutation patterns in immunoglobulin variable regions are established independently of the local transcriptional landscape

“…SHM has also been correlated with the epigenetic landscape especially with activating histone marks. In particular, depletion of histone modifying enzymes resulted in decreased SHM and/or CSR accompanied with a decrease in activating histone modifications (24,(28)(29)(30)(31)(32)(33)(34)(35)(36). However, the precise role of these marks in SHM is unknown.…”