Directed Ig class switch recombination in activated murine B cells.

Winter, Edward; Krawinkel, Ulrich; Radbruch, Andreas

doi:10.1002/j.1460-2075.1987.tb02415.x

Cited by 96 publications

(45 citation statements)

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“…5a; P = 0.0006 for Sμ mutation in B cells stimulated with LPS and IL-4 for 72 h compared with resting B cells (52 mutations/18,660 bp versus 10 mutations/16,542 bp, respectively); P = 0.0003 for Sμ mutation in stimulated B cells at 72 h compared with Eμ plus Cμ mutation in B cells stimulated under the same conditions (33 mutations/59,196 bp for Eμ plus Cμ); P-values were determined by a twotailed t-test assuming unequal variance; the frequency of mutation in the 5′ region of Sμ was 5 × 10 -4 when measured with a proofreading polymerase). Consistent with previous reports, we did not find deletions in this part of Sμ 22 . The induced mutations were AID dependent because no significant increase in mutation over background was observed in Sμ in AID -/-B cells cultured with LPS and IL-4 (Fig.…”

supporting

confidence: 93%

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

Petersen

Casellas

Reina‐San‐Martin

et al. 2001

Nature

462

438

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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...

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supporting

confidence: 93%

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

Petersen

Casellas

Reina‐San‐Martin

et al. 2001

Nature

462

438

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“…The interruption of Sm may lead to Ighm/c-myc recombination and chromosomal translocation t(12;15) on the non-productive Igh allele, followed by deletional remodeling' of the t(12;15) by aberrant class switch recombination and normal class switching on the productive Igh allele (see Figure 7 for more details). The exact sequence of these events is currently unclear, but the ®nal result of the processes illustrated in Figure 7 appears to be in agreement with one of the general rules of class switching, namely that both nonproductive and productive alleles frequently rearrange to the same C H gene (Winter et al, 1987). The proposed model also agrees with the empirical ®nding in BALB/c plasmacytomas that most tumors have switched both the heavy-chain encoding and the translocated Igh allele to Ca.…”

Section: Discussionsupporting

confidence: 73%

Deletional remodeling of c-myc-deregulating chromosomal translocations

1997

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Evidence is presented for the existence of a novel remodeling-by-deletion mechanism that alters the ®ne structure of c-myc-deregulating chromosomal translocations in t(12;15)-positive BALB/c plasmacytomas. DNA sequence analysis of the t(12;15) in ®ve primary tumors revealed the co-existence of precursor cells harboring genetic recombinations between the immunoglobulin heavy-chain m locus (Ighm) and c-myc with clonally related progenitors containing rearrangements between the immunoglobulin heavy-chain a locus (Igha) and cmyc. Clonal relatedness was based upon unique junction fragments between the switch region of Ighm and c-myc. Sm/c-myc junctions are thus useful clonotypic markers for monitoring the conversion of Ighm/c-myc-positive tumor precursor clones into Igha/c-myc-positive plasmacytomas. Aberrant isotype switch recombination appears to be the most likely mechanism e ecting this conversion event (other possibilities are discussed) which may help to explain the preferred usage of the Igha locus in recombinations with c-myc in t(12;15)-positive plasma cell tumors in BALB/c mice.

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“…Different knockout experiments also highlighted the importance of germline promoters as well as of the splice donor site for efficient CSR [16][17][18][19][20][21][22][23][24]. CSR is often directed to the same S sequences on both homologous chromosomes [25][26][27]. The question then arises whether germ-line transcription initiates from both I promoters or from the one on the expressed allele only.…”

Section: Introductionmentioning

confidence: 99%

Germ‐line transcription occurs on both the functional and the non‐functional alleles of immunoglobulin constant heavy chain genes

et al. 2003

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In B cells undergoing class switching, recombination is usually directed to the same switch sequences on both homologous chromosomes and is generally preceded by germ-line transcription initiated from I promoters upstream of immunoglobulin constant heavy chain genes. Whether germ-line transcription occurs on both alleles or is restricted to the productive one has not been directly addressed before. To clearly distinguish between these two possibilities, a system is needed in which one might detect allele-specific germ-line transcripts. We used homologous recombination to set up two in vivo systems in which one + 3 allele was marked by a loxP sequence whereas the other allele was silenced by inhibiting ? heavy chain production. Here, we provide evidence that germ-line transcription occurs on both the functional and the non-functional alleles.

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Directed Ig class switch recombination in activated murine B cells.

Cited by 96 publications

References 44 publications

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

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

Deletional remodeling of c-myc-deregulating chromosomal translocations

Germ‐line transcription occurs on both the functional and the non‐functional alleles of immunoglobulin constant heavy chain genes

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