Histone H2A and H2B are monoubiquitinated at AID-targeted loci

Abstract

Background: Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.

Methodology/principal findings: Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and Sgamma3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.

Conclusions/significance: Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.

Figure 1. Diagram of the IgH loci in Ramos Burkitt's lymphoma.

A reciprocal translocation exchanging the ends of chromosomes 8 and 14 in Ramos resulted in the formation of MYC₁₄ and V₈. The chromosome 14 break point occurred within the heavy chain (IgH) switch region (indicated by jagged edges) just 5′ of the µ constant exon (IgHM) while the chromosome 8 breakpoint occurred in a c-MYC allele promoter. (R), silent, non-coding RNA; ×1, ×2, ×3, c-MYC exons. Arrows indicate transcriptional direction. Double bar denotes amplicon location. Primers were designed to amplify the IgH constant region mu (V_H1), the functional heavy chain V_H4-34/D/J6 rearrangement (V_H2), and an upstream non-rearranged V-region sequence (V_H3). Chromosome 8 primer sets amplify three c-MYC exons (MYC1–3), and a sequence 50 kb 5′ of MYC3 (MYC4). One primer set was designed for the non-expressed unrearranged V_H 4–34 on chromosome 8 (V₈).

Figure 2. Ramos Burkitt's lymphoma cells constitutively hypermutate at distinct genomic loci.

A. Flow cytometry measure of loss of surface IgM (sIgM) display over time for cultured Ramos cells. sIgM was detected using FITC conjugated antibody specific for human IgM (FL1-H). Ramos cells were continuously cultured for 90 days after which 10,000 cells were analyzed. B. Sequence analysis of the translocated and expressed MYC₁₄. Each pie wedge represents a unique sequencing read with the number of mutations identified in individual reads indicated. The total number of mutations identified in 10 unique sequence reads corresponding to individual exons is shown in the central circles.

Figure 3. ChIPs identify mUb-H2A and mUb-H2B at Ramos hypermutating loci.

A. Normalized template enrichment upon anti-AID ChIP. Template obtained from AID-IP was amplified and analyzed by qPCR using primer sets diagramed in Figure 1, above. Results were normalized to non-specific IgG IP and relative to input. Each bar indicates the mean of the values obtained from six amplifications (two separate experiments analyzed in triplicate) with standard deviation. B. Normalized template enrichment upon mUb-H2A ChIP. C. Normalized template enrichment upon mUb-H2B ChIP. D. Normalized template enrichment upon pol II ChIP. Enrichments were analyzed by qPCR as in Figure 3A. E. Ethidium bromide stained gel of traditional PCR amplifications from ChIP template. F. Antibodies used to ChIP mub-H2A and mub-H2B and AID are specific for monoubiquitinated H2A, H2B and AID protein. Western blot analysis of endogenous AID (24 kDa), mUb-H2A (27 kDa) and mUb-H2B (27 kDa) in Ramos cell lysates. Visible on the AID blot, the white negative stained bands of the protein marker correspond to 55, 40, 36, 25, 15 and 10 kilodaltons (top to bottom).

Figure 4. mUb-H2A and mUb-H2B associate with hypermutation in activated mouse primary B cells.

A. Cartoon depicting the engineered rearranged heavy chain allele sequences in quasi-monoclonal mouse . The rearranged allele (17.2.25) is compared to a normal heavy chain arrangement (wt). The 17.2.25, allele formed by the targeted replacement of the joining cluster with a defined V(D)J rearrangement. J_HΔ, allele formed by the targeted deletion of the joining cluster. S, Sγ3 switch region. IGHM, µ constant exon. V_H additional 5′ variable sequence segments. DQ52, diversity sequence segment immediately 5′ of the joining sequence cluster. Arrows indicate transcriptional direction. Double bar denotes amplicon location. B. ChIPs identify monoubiquitinated histones with hypermutation. Murine splenocytes were cultured for 72 hours with LPS, crosslinked, and then subjected to ChIP analysis with anti-mUb-H2A and anti-mUb-H2B, and anti-AID antibody. Normalized enrichment of the 17.2.25, Sγ3, triose phosphate isomerase (TPI) and neural-specific striatum-enriched protein-tyrosine phosphatase (STEP) loci from qPCR are shown relative to input DNA. C. Ethidium bromide visualization of traditional PCRs with primer sets corresponding to the qPCRs.

Figure 5. AID and monoubiquitinated H2B colocalize in discrete nuclear foci.

Representative immunofluorescence confocal microscopy images of Ramos cells stained for AID and mUb-H2B. Ramos cells were incubated with rabbit antibody specific to AID and murine mUb-H2B antibody followed by Alexa-488 conjugated rabbit secondary and Alexa-555 conjugated murine secondary antibody. Upper left, Ramos cells imaged with 488 nm filter. Upper right, Ramos cells imaged with 555 nm filter. Lower left, merged image with white scale bar (2 microns). Lower right nuclear envelope imaged using wheat germ agglutinin (Blue). Arrows point to colocalizations of AID and mUb-H2B.