HIRA-dependent H3.3 deposition and its modification facilitate somatic hypermutation of immunoglobulin gene by maintaining the proper chromatin context and transcription

Abstract The histone variant H3.3 and its chaperone HIRA are involved in active transcription but their detailed roles in regulating somatic hypermutation (SHM) in human B cells have not been fully elucidated. Here we found that the knockout (KO) of H3.3 HIRA chaperone significantly decreased SHM in the Ramos human germinal center like B cell line without changing the levels of AID and other major proteins known to be involved in SHM. The HIRA KO was associated with a decrease at the immunoglobulin (Ig) heavy chain locus of many factors related to transcription elongation including H3.3 and this was accompanied by decreased nascent transcription of the Ig heavy chain locus. However, H3K79me2/3 elongation modification was not affected. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the Ig locus was also reduced by the HIRA KO, suggesting that the HIRA loss decreased the level of H3.3K36me3. However, the chromatin accessibility of Ig locus was increased by the HIRA depletion. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit the trimethylation of H3.3K36 reduced SHM in Ramos cells, while the H3.3K79M mutant did not. Consistent with HIRA KO, H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on Ig heavy chain variable and the downstream switching regions. These results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and further confirm the central role of transcription-coupled chromatin accessibility in SHM in human B cells.