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. 2003 Jun 16;197(12):1767-78.
doi: 10.1084/jem.20030569.

H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation

Bernardo Reina-San-Martin  1 , Simone Difilippantonio, Leif Hanitsch, Revati F Masilamani, Andre Nussenzweig, Michel C Nussenzweig
Affiliations

H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation

Bernardo Reina-San-Martin et al. J Exp Med. .

Abstract

Changes in chromatin structure induced by posttranslational modifications of histones are important regulators of genomic function. Phosphorylation of histone H2AX promotes DNA repair and helps maintain genomic stability. Although B cells lacking H2AX show impaired class switch recombination (CSR), the precise role of H2AX in CSR and somatic hypermutation (SHM) has not been defined. We show that H2AX is not required for SHM, suggesting that the processing of DNA lesions leading to SHM is fundamentally different from CSR. Impaired CSR in H2AX-/- B cells is not due to alterations in switch region transcription, accessibility, or aberrant joining. In the absence of H2AX, short-range intra-switch region recombination proceeds normally while long-range inter-switch region recombination is impaired. Our results suggest a role for H2AX in regulating the higher order chromatin remodeling that facilitates switch region synapsis.

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Figures

Figure 1.
Figure 1.
Mutations in Sμ. (a) Time course of Sμ mutation in wild-type B cells stimulated with LPS and IL-4. The number of mutations was: resting B cells: 7 mutations/59,301 bp; 24 h: 9 mutations/30,060 bp; 48 h: 15 mutations/36,000 bp; 72 h: 32 mutations/62,720 bp; 96 h: 30 mutations/37,066 bp. (b) Proportion of μ intronic enhancer (Eμ), VHB1–8, and Sγ3 sequences carrying mutations after 72 h of stimulation with LPS and IL-4. The number of mutations was: μ intronic enhancer: 0 mutations/22124 bp; Sγ3: 4 mutations/31812 bp and VHB1–8: 2 mutations/20,427 bp. Analysis of VHB1–8 sequences was performed on B cells obtained from mice carrying a prerearranged VHB1–8 gene (reference 45). Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of the charts. The frequency of mutations per bp sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart respectively. Statistical significance was determined by a two-tailed t test assuming unequal variance and comparing to background (Resting B cells). P values are indicated below each pie chart. (c) Distribution of point mutations in Sμ. The region sequenced is indicated with the first base corresponding to position 4600 in the Sμ germline sequence (GenBank/EMBL/DDBJ accession no. J0040). Lower-case letters above the line indicate independent mutations (141 total). Mutations occurring at any base within the RGYW motif (references and 8) were considered to be hotspot mutations. RGYW motifs containing mutations are underlined. A double head arrow underneath the line indicates deletions.
Figure 2.
Figure 2.
Analysis of CSR and mutations in Sμ of wild-type, AID− / −, Ku80− / −, Ku80− / −Bcl2+, and H2AX− / − B cells. (a) Flow cytometric analysis of IgG1 expression on CFSE-labeled wild-type (WT), AID−/−, H2AX−/−, Ku80−/−, and Ku80−/−Bcl2+ B cells stimulated with LPS plus IL-4 for 4 d. Cell division as measured by CFSE dye dilution is shown in the upper panel. The percentage of cells expressing IgG1 after a specific number of cell divisions is indicated on the lower panel. (b) Sμ mutations in wild-type B cells sorted for 1, 3, or 5 cell divisions. The number of mutations was: 1 division, 7 mutations/33,306 bp; 3 divisions, 14 mutations /30,818 bp; 5 divisions, 33 mutations/32,460 bp. (c) Sμ mutations in wild-type B cells sorted for 5 cell divisions and IgM or IgG1 cell surface expression. The number of mutations was: IgM+: 17 mutations/45,356 bp; IgG1+: 51 mutations/47,040 bp. (d) Sμ mutations in AID−/−, Ku80−/−, and Ku80−/−Bcl2+ B cells sorted for 5 cell divisions and expressing IgM. The number of mutations was: AID−/−, 2 mutations/34,124 bp; Ku80−/−, 7 mutations/31,360 bp; Ku80−/−Bcl2+, 4 mutations/27,740 bp. (e) Sμ mutations in H2AX−/− B cells sorted for 5 cell divisions and IgM or IgG1 cell surface expression. The number of mutations was: IgM+: 12 mutations/35,810 bp; IgG1+: 20 mutations/33,946 bp. Pie charts and statistics are as in Fig. 1.
Figure 3.
Figure 3.
Somatic hypermutation is unaffected in the absence of H2AX. (a) Wild-type and H2AX−/− germinal center B cells (B220+Fas+GL-7+) obtained from the PPs of unimmunized mice. Pie charts show proportion of JH4-intron sequences carrying different number of mutations. (b) Wild-type and H2AX−/− GC B cells (B220+Fas+GL-7+) from lymph nodes of NP-immunized mice. Pie charts show proportion of VHB1–8 sequences carrying different number of mutations. (c) Percent nucleotide substitutions in VHB1–8 from GC B cells adjusted for base composition. Percentage of mutations within hotspot motifs is indicated underneath each panel. Total number of mutations analyzed was: wild-type = 653 mutations; H2AX−/−= 475 mutations. Pie charts and statistics are as in Fig. 1.
Figure 4.
Figure 4.
Switch region accessibility is normal in the absence of H2AX. Real time RT-PCR for (a) μ sterile transcript, (b) γ1 sterile transcript, and (c) γ1 circle transcript in wild-type (solid circles) and H2AX−/− (open circles) B cells stimulated with LPS and IL-4 over 3 d. Mean results from two independent cultures are expressed as fold induction relative to resting B cells. (d) Proportion of mutations in the Sγ1 5′ region in wild-type, AID−/−, and H2AX−/− B cells stimulated with LPS and IL-4 and sorted for IgM surface expression and 5 divisions. The number of mutations was: Resting B cells, 5 mutations/59,301 bp; wild-type, 26 mutations/53,624 bp; AID−/−, 5 mutations/52,164 bp; H2AX−/−, 21 mutations/53,838 bp. Pie charts and statistics are as in Fig. 1.
Figure 5.
Figure 5.
Switch recombination junctions are normal in the absence of H2AX. Sμ-Sγ1 switch recombination junctions from wild-type (a) and H2AX−/− (b) B cells. Overlap was determined by identifying the longest region at the switch junction of perfect uninterrupted donor/acceptor identity. The Sμ and Sγ1 germline sequences are shown above and below each junction sequence respectively. Lower-case letters indicate mutations, (|) indicates identity between nucleotides and (−) indicates a deletion. Homology at the junctions is boxed. (c) Length of microhomologies at Sμ/Sγ1 junctions in wild-type and H2AX−/− B cells. (d) Scatter analysis of the μ/γ1 breakpoints derived from in vitro–stimulated B cells. The axes indicate the position relative to GenBank/EMBL/DDBJ sequences J00440 (Sμ) and D78340 (Sγ1). Open circles denote breakpoints from H2AX−/−, filled circles from wild-type controls.
Figure 6.
Figure 6.
Resolution of Sμ internal deletions is independent of H2AX but requires AID and Ku80. Southern blot analysis of the Sμ region in IgM secreting hybridomas derived from (a) wild-type (WT), (b) AID−/−, (c) Ku80−/−, (d) Ku80−/−Bcl2+, and (e) H2AX−/− B cells. Restriction enzymes and probes used are indicated in the top left panel. Δ indicates deletions in Sμ. The SP2/0Ag-14 (SP2) cell line has a deletion in Cμ and shows no hybridization. The same deletions in Sμ were found using an Eμ probe (unpublished data). Number of deletions over hybridomas screened is indicated below each panel. Molecular weight markers in kilobase pairs are indicated on the left side of each panel.
Figure 7.
Figure 7.
The Sγ1 locus is intact in IgM secreting hybridomas stimulated for CSR. Southern blot analysis of the Sγ1 region in IgM secreting hybridomas from (a) wild-type (WT), (b) AID−/−, (c) Ku80−/−, (d) Ku80−/−Bcl2+, and (e) H2AX−/− mice. Restriction enzymes and probes used are indicated in the top left panel. Molecular weight markers in kilobase pairs are indicated on the left side of each panel. Number of deletions over hybridomas screened is indicated below each panel.
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