AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching

Abstract

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (CH) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream CH genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction 1 . Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair 2-4 , form nuclear foci at the CH region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX -/-mice. Localization of Nbs1 and γ-H2AX to the IgH locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sμ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.Correspondence and requests for materials should be addressed to A.N. (andre_nussenzweig@nih.gov) or M.C.N. (nussen@mail.rockefeller.edu).. Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. To determine whether DNA repair factors associate with DSBs at the switch regions, we first examined the intracellular localization of γ-H2AX, Nbs1, Rad51 and Brca1 in activated B cells by immunofluorescence. Brca1 and Rad51 are required for homologous recombination, the Mre11-Rad50-Nbs1 complex has been implicated in both homologous recombination and non-homologous end-joining (NHEJ), and γ-H2AX is critical for recruiting these repair factors to DSBs 6 and facilitates NHEJ in Saccharomyces cerevisiae 4 . All four proteins showed diffuse nuclear staining in most of the resting B cells from C57BL/6 wild-type mice. High local concentrations of these factors (nuclear foci) were detected in a very small percentage of cells (<5%), which increased significantly when the cells were stimulated to undergo CSR in vitro with lipopolysaccharide (LPS) and interleukin (IL)-4 (Fig. 1a). After 3 days of stimulation, 37% of the B cells contained discrete Brca1 foci (12 ± 6 per cell) and 43% contained Rad51 foci (7 ± 3 per cell), consistent with previous results 7 ; the remaining cells exhibited a weak, diffuse pattern of nuclear staining (Fig. 1a). Many of the stimulated B cells also formed Nbs1 foci (32% contained, on average, 3 ± 2 per cell) and γ-H2AX foci (40% contained, on average, 4.5 ± 3 per cell). To determine which of these repair factors are co-localized in activated B cells, we performed two colour immunofluoresence experiments (Fig. 1b). Only 20% of the cells that contained Rad51 and Nbs1 (n = 687) or Brca1 and Nbs1 foci (n = 431) exhibited co-localization. In contrast, γ-H2AX foci co-localiz...

The γ‐H2A.X: Is it just a surrogate marker of double‐strand breaks or much more?

“…The DNA damage signaling kinases, ataxia telangectasia mutated (ATM), ATM-and Rad3-related protein (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-Pkcs) were found to be responsible for the generation of g-H2A.X [Burma et al, 2001;Stiff et al, 2004]. H2A.X phosphorylation appears at sites of DSBs during DSB repair, meiotic recombination [Hunter et al, 2001;Mahadevaiah et al, 2001], apoptotic digestion , V (D) J splicing [Chen et al, 2000;Gellert, 2002], and class switch recombination [Petersen et al, 2001]. It therefore seems to be a universal cellular response to DSBs.…”

ERK activity facilitates activation of the S-phase DNA damage checkpoint by modulating ATR function

“…These foci are essential in facilitating the assembly of repair factors, including Brca1 and the MRE11-RAD50-NBS1 complex, on damaged DNA (Paull et al, 2000;Celeste et al, 2002), and also aid in the transduction of DNA damage signals by binding to 53BP1 and MDC1 (FernandezCapetillo et al, 2002;Stewart et al, 2003). gH2AX foci also form at sites of physiological DSBs in lymphocytes and germ cells (Chen et al, 2000;Mahadevaiah et al, 2001;Petersen et al, 2001;Fernandez-Capetillo et al, 2003). Knocking out H2AX produces mice with immune deficiency and male infertility , while loss or reduction of H2AX compromises genome stability and facilitates tumorigenesis Bassing et al, 2003;Celeste et al, 2003).…”

V(D)J and immunoglobulin class switch recombinations: a paradigm to study the regulation of DNA end-joining

“…As eluded to in the introduction, experimental evidence has demonstrated that CSR, like V(D)J recombination, involves the generation of DNA dsb: (1) an episomal circle is generated during CSR (Chaudhuri and Alt, 2004); (2) AID-dependent DNA dsb have been detected by ligated mediated PCR assays in Switch regions of mitogen-activated B cells (Wuerffel et al, 1997;Catalan et al, 2003;Rush et al, 2004); (3) phosphorylation of histone H2AX (gH2AX), which spreads around DNA dsb, is detected at the IgH locus in an AID-dependent manner, in B cells activated for CSR (Petersen et al, 2001); (4) CSR is impaired in mice deficient for H2AX, ATM, p53 binding protein 1 and Nbs1, which are DDR early components (Reina-San-Martin et al, 2003, 2004Lumsden et al, 2004;Manis et al, 2004;Ward et al, 2004;Kracker et al, 2005). The link between AIDdriven DNA cytidine deamination and the introduction of DNA dsb in Ig S regions is not fully established yet.…”