The role of HIRA-dependent H3.3 deposition and its modifications in the somatic hypermutation of immunoglobulin variable regions

Abstract

The H3.3 histone variant and its chaperone HIRA are involved in active transcription, but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells are not yet fully understood. In this study, we show that the knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of the immunoglobulin heavy chain (IgH) in the human Ramos B cell line without changing the levels of activation-induced deaminase and other major proteins known to be involved in SHM. Except for H3K79me2/3 and Spt5, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells, and this was accompanied by decreased nascent transcription in the IgH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the IgH locus was also reduced in the HIRA KO. Somewhat surprisingly, HIRA loss increased the chromatin accessibility of the IgH V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with the HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IgH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription-associated chromatin structure and accessibility contribute to SHM in human B cells.

Keywords: HIRA and H3.3 modifications; chromatin accessibility; immunoglobulin variable region; somatic hypermutation; transcription elongation.

Fig. 1.

HIRA and H3.3 genes are up-regulated in germinal center B cells and the abundance of H3.3 is slightly higher in the variable region of heavy chain than the downstream constant region in the Rep161 cell line. (A–D) The fold difference of RNA expression of HIRA, UBN1, CABIN1, and H3F3A and H3F3B between germinal center B cells and naïve B cells in both human and mouse. The RNA-seq data were downloaded from the GEO (human: GSE45982 and mouse: GSE71702) website and analyzed as described in SI Appendix, SI Materials and Methods. Three independent datasets for human and four for mouse were analyzed. The error bar represents the SEM among these. The y axis is the fold difference of expression for each gene between GC and naïve B cells. *padj ≤ 0.05, **padj ≤ 0.01, ***padj ≤ 0.001, ns, no significance. (E) The occupancy pattern of H3.3 in the IgH locus in Rep161 cells by ChIP-qPCR. The amplicon of CD4 is in the first intron, which is ∼230 bp from the beginning of the first exon. The amplicon of PAX5 is also in the first intron, which is ∼860 bp from the beginning of the first exon. The exact primer information for ChIP-qPCR is in SI Appendix, Table S1. (F) ChIP-qPCR of H3 in the IgH locus in Rep161. The x axis shows the amplification regions as displayed in the IgH locus carton at the Bottom. CD4, not expressed in B cells, is a negative control and PAX5 is a potential AID off-target gene. The y axis is the percentage of input for each antibody that was normalized to input. IgG background was subtracted for each site. For E and F, error bars in each figure represent the SEM among three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ns, no significance.

Fig. 2.

Knockout of HIRA significantly decreased the SHM measured by FACS and deep sequencing. (A) A representative figure of FACS showing the decreased SHM measured by the frequency of the cell population with decreased fluorescence intensity of mCherry. The number in each FACS figure shows the percentage of cells with decreased fluorescence intensity of mCherry. FSC-W means the width of forward scatter for each cell. The y axis represents the fluorescence intensity (Int) of mCherry. (B) Bar graph showing that the HIRA KO significantly decreased SHM by 65% compared with WT. Error bars represent the SEM among three independent experiments. ***P ≤ 0.001. (C) The overall frequency of mutation and mutations in different types of DNA motifs were analyzed by deep sequencing with UMIs as described in Materials and Methods. For mutation analysis, both WT and HIRA KO cells were treated with 0.25 μM 4-OHT for 7 d. In each group, “mutation frequency” means the absolute mutation number was divided by the total numbers of nucleotides, “mutated V regions” means the absolute number of mutated V sequences was divided by the total number of V regions sequenced, and “mutations/V region” means the absolute mutation number was divided by the total number of V sequences. For each row of the Bottom part of the table, the respective absolute number of the mutated sites was divided by the total number of motifs or sites in each group. For the WGCW, WRC, and GYW motifs, the frequency of mutation for the respective underlined nucleotide was calculated.

Fig. 3.

HIRA KO significantly reduced the abundance of transcription elongation–related factors and nascent transcription of the IgH gene in Rep161 cells. (A–H) The abundance of transcription-related factors associated with the IgH gene in both WT and HIRA KO cells was measured by ChIP-qPCR assays in three independent experiments. The normal IgG background was subtracted and PCR amplification sites are indicated in the IgH gene cartoon at the Bottom. (I) HIRA KO significantly decreased the nascent transcription of IgH locus in Rep161 cells. For RT-qPCR of nascent transcription, the detailed information is described in SI Appendix, SI Materials and Methods. Error bars in each panel represent the SEM among three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ns, no significance.

Fig. 4.

The effect of HIRA KO on chromatin accessibility was measured in three different independent ways. (A) The HIRA KO did not significantly affect the chromatin accessibility genome-wide by ATAC-seq, but slightly though significantly decreased it in distal intergenic regions. The y axis shows the frequency of enrichment of the unique reads that are aligned to each genomic region. Details of the analysis are described in SI Appendix, SI Materials and Methods. Error bars represent the SEM among two independent experiments. *padj < 0.05. ns, no significance. (B) Genomic tracks displaying the ATAC-seq distribution pattern in the IgH locus from both WT and HIRA KO cells. The y axis indicates the relative signal of ATAC-seq profiles and the x axis represents the position of the IgH locus in the reporter Ramos cell line. The dark blue ribbons show the locations of subregions in the IgH locus. For the constant region (Cμ), each ribbon represents an exon. (C) The KO of HIRA increased the ability of MNase to digest parts of the V region. The x axis shows the amplification sites of the V region in Rep161 cells that are illustrated in the cartoon. The y axis represents the MNase digestion that is normalized to the undigested whole genome from each group. The bigger the value on the y axis, the greater the degree of MNase digestion. FW1, FW2, and FW3 represent framework 1, 2, and 3 of the V region, respectively. Error bars represent the SEM among three independent experiments. ***P ≤ 0.001. (D) The KO of HIRA increased the frequency of single-stranded DNA in the V region in Rep161 cells as measured by bisulfite ssDNA deep sequencing with UMIs. The details of the bisulfite assay are described in SI Appendix, SI Materials and Methods. “Patch frequency of top/bottom” means the comparison between frequency of bisulfite-accessible patches in the Top strand and in the Bottom strand. If the number is close to 1, it means the bisulfite treatment detected equal amounts of ssDNA in both Top and Bottom strands. “Top-average patch size” means the average patch size in Top strand defining the patch size as having at least two consecutive bisulfite-accessible Cs. Similarly, for the “bottom-average patch size.” “Ratio of V with one patch” means the frequency of V regions in Rep161 that contain one bisulfite patch. P represents P value and it means significance of the difference between WT and HIRA KO if the P ≤0.05. Those that are statistically significant are colored red.

Fig. 5.

Ectopic expression of H3.3G34 mutants significantly decreased SHM of the V region and reduced the abundance of transcription elongation–related factors on the IgH locus. (A) Ectopic expression of H3.3G34V and H3.3G34R mutants each significantly decreased SHM compared with H3.3 WT. (B–D) The abundance of transcription-related factors on the IgH gene was measured by ChIP-qPCR assays in three independent experiments for both the H3.3 WT and the H3.3G34V mutant. The Bottom Right cartoon shows the subregions of the IgH locus and the Bottom letters A, C, E, G, H, and I represent the amplification sites for ChIP-qPCR. Error bars in each panel represent the SEM among three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, and ns, no significance.