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Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.

Boboila C, Yan C, Wesemann DR, Jankovic M, Wang JH, Manis J, Nussenzweig A, Nussenzweig M, Alt FW - J. Exp. Med. (2010)

Bottom Line: Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR.Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins.Our findings suggest that more than one form of A-EJ can function in CSR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

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Ku70−/−HL and Ku70−/−Lig4−/−HL mice posses mature splenic B cells which proliferate in response to stimulation by αCD40/IL-4. (a) Disruption of the Ku70 and Lig4 genomic loci and absence of Ku70 and Lig4 proteins in Ku70−/−Lig4−/− mice. (left) Southern blotting: BamHI-digested genomic DNA was probed with Ku70- or Lig4-specific probes. (right) Thymus protein extracts were assayed for the presence of Ku70 or Lig4 proteins. K, kidney protein extracts; BM, bone marrow protein extracts. Both the Southern and the Western blotting were repeated two times on independent samples. (b) WT-HL, Ku70−/−HL and Ku70−/−Lig4−/−HL mice have mature splenic B cells. Whole cell suspensions from spleens of WT-HL, Ku70−/−HL, and Ku70−/−Lig4−/−HL were used to quantify the percentage of B220+IgM+ mature B cells. Data shown are representative of more than 10 experiments, with at least one mouse of each genotype per experiment. (c) Ku70−/−HL mature B cells proliferate at levels comparable to WT-HL after αCD40/IL-4 stimulation. Splenic B cells were enriched with B220 magnetic beads and counted by Trypan blue staining starting at day 1.5 from the beginning of the culture, to allow for the disappearance of non–B cells. Data shown are based on two independent stimulations and a total of 4 WT-HL and 5 Ku70−/−HL mice were used. Error bars are based on the relative cell number for each mouse.
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fig1: Ku70−/−HL and Ku70−/−Lig4−/−HL mice posses mature splenic B cells which proliferate in response to stimulation by αCD40/IL-4. (a) Disruption of the Ku70 and Lig4 genomic loci and absence of Ku70 and Lig4 proteins in Ku70−/−Lig4−/− mice. (left) Southern blotting: BamHI-digested genomic DNA was probed with Ku70- or Lig4-specific probes. (right) Thymus protein extracts were assayed for the presence of Ku70 or Lig4 proteins. K, kidney protein extracts; BM, bone marrow protein extracts. Both the Southern and the Western blotting were repeated two times on independent samples. (b) WT-HL, Ku70−/−HL and Ku70−/−Lig4−/−HL mice have mature splenic B cells. Whole cell suspensions from spleens of WT-HL, Ku70−/−HL, and Ku70−/−Lig4−/−HL were used to quantify the percentage of B220+IgM+ mature B cells. Data shown are representative of more than 10 experiments, with at least one mouse of each genotype per experiment. (c) Ku70−/−HL mature B cells proliferate at levels comparable to WT-HL after αCD40/IL-4 stimulation. Splenic B cells were enriched with B220 magnetic beads and counted by Trypan blue staining starting at day 1.5 from the beginning of the culture, to allow for the disappearance of non–B cells. Data shown are based on two independent stimulations and a total of 4 WT-HL and 5 Ku70−/−HL mice were used. Error bars are based on the relative cell number for each mouse.

Mentions: Several independent studies from more than a decade ago reported that Ku70- or Ku80-deficient B cells had very severe CSR defects (Manis et al., 1998; Casellas et al., 1998), leading to the assumption that, as in V(D)J recombination, CSR end-joining is catalyzed strictly by C-NHEJ (Honjo et al., 2004). However, normal CSR requires B cell proliferation, and previously studied Ku70- or Ku80-deficient B cells had severe proliferation defects, with many dying when activated for CSR (Casellas et al., 1998; Manis et al., 1998; Reina San-Martin et al., 2003). Therefore, we have suggested that although Ku70 and Ku80 are required for normal CSR, their overall contribution via C-NHEJ remained to be determined (Manis et al., 2002; Chaudhuri and Alt, 2004; Chaudhuri et al., 2007). Our current protocols for activating B cells stimulate much higher CSR levels in WT B cells than those used in the original Ku70−/− and Ku80−/− B cell studies; e.g., in the earlier studies of Ku-deficient cells, only ∼10% of WT B cells underwent CSR to IgG1 (Casellas et al., 1998; Manis et al., 1998), whereas under current stimulation conditions (different purification of cells, sources of cytokines, and activators, etc.) we observe CSR to IgG1 in 60% or more of WT B cells (Cheng et al., 2009; see Fig. 2). Therefore, we have reexamined the requirement for Ku70 and Ku80 in CSR. As Ku70 and Ku80 are required for V(D)J recombination and generation of mature B cells, we generated Ku70−/− or Ku80−/− mice that harbored preassembled (“knock-in”) IgH (B1-8-HC) and IgL (3–83k-LC) variable region exons (Pelanda et al., 1997; Sonoda et al., 1997). Various studies have shown that the B1-8-HC V(D)J knock-in allele undergoes normal CSR (Casellas et al., 1998; Manis et al., 1998; Yan et al., 2007). Because of the complex breeding strategies necessary to generate these mice, we generated Ku70−/− and Ku80−/− strains that were either homozygous or heterozygous for the B1-8-HC knock-in allele (referred to, respectively, as H/HL and H/+L). The breakdown of results with these genotypes is indicated in each figure or in supplementary figures and tables. Because H/HL and H/+L lines gave similar results for most experiments, we generically refer to both genotypes as HL mice, except where otherwise noted. We verified homozygous disruption of the Ku70 and Ku80 locus and the absence of the Ku70 protein in the Ku70−/−HL mice and cells analyzed (Fig. 1 a and not depicted).


Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.

Boboila C, Yan C, Wesemann DR, Jankovic M, Wang JH, Manis J, Nussenzweig A, Nussenzweig M, Alt FW - J. Exp. Med. (2010)

Ku70−/−HL and Ku70−/−Lig4−/−HL mice posses mature splenic B cells which proliferate in response to stimulation by αCD40/IL-4. (a) Disruption of the Ku70 and Lig4 genomic loci and absence of Ku70 and Lig4 proteins in Ku70−/−Lig4−/− mice. (left) Southern blotting: BamHI-digested genomic DNA was probed with Ku70- or Lig4-specific probes. (right) Thymus protein extracts were assayed for the presence of Ku70 or Lig4 proteins. K, kidney protein extracts; BM, bone marrow protein extracts. Both the Southern and the Western blotting were repeated two times on independent samples. (b) WT-HL, Ku70−/−HL and Ku70−/−Lig4−/−HL mice have mature splenic B cells. Whole cell suspensions from spleens of WT-HL, Ku70−/−HL, and Ku70−/−Lig4−/−HL were used to quantify the percentage of B220+IgM+ mature B cells. Data shown are representative of more than 10 experiments, with at least one mouse of each genotype per experiment. (c) Ku70−/−HL mature B cells proliferate at levels comparable to WT-HL after αCD40/IL-4 stimulation. Splenic B cells were enriched with B220 magnetic beads and counted by Trypan blue staining starting at day 1.5 from the beginning of the culture, to allow for the disappearance of non–B cells. Data shown are based on two independent stimulations and a total of 4 WT-HL and 5 Ku70−/−HL mice were used. Error bars are based on the relative cell number for each mouse.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822597&req=5

fig1: Ku70−/−HL and Ku70−/−Lig4−/−HL mice posses mature splenic B cells which proliferate in response to stimulation by αCD40/IL-4. (a) Disruption of the Ku70 and Lig4 genomic loci and absence of Ku70 and Lig4 proteins in Ku70−/−Lig4−/− mice. (left) Southern blotting: BamHI-digested genomic DNA was probed with Ku70- or Lig4-specific probes. (right) Thymus protein extracts were assayed for the presence of Ku70 or Lig4 proteins. K, kidney protein extracts; BM, bone marrow protein extracts. Both the Southern and the Western blotting were repeated two times on independent samples. (b) WT-HL, Ku70−/−HL and Ku70−/−Lig4−/−HL mice have mature splenic B cells. Whole cell suspensions from spleens of WT-HL, Ku70−/−HL, and Ku70−/−Lig4−/−HL were used to quantify the percentage of B220+IgM+ mature B cells. Data shown are representative of more than 10 experiments, with at least one mouse of each genotype per experiment. (c) Ku70−/−HL mature B cells proliferate at levels comparable to WT-HL after αCD40/IL-4 stimulation. Splenic B cells were enriched with B220 magnetic beads and counted by Trypan blue staining starting at day 1.5 from the beginning of the culture, to allow for the disappearance of non–B cells. Data shown are based on two independent stimulations and a total of 4 WT-HL and 5 Ku70−/−HL mice were used. Error bars are based on the relative cell number for each mouse.
Mentions: Several independent studies from more than a decade ago reported that Ku70- or Ku80-deficient B cells had very severe CSR defects (Manis et al., 1998; Casellas et al., 1998), leading to the assumption that, as in V(D)J recombination, CSR end-joining is catalyzed strictly by C-NHEJ (Honjo et al., 2004). However, normal CSR requires B cell proliferation, and previously studied Ku70- or Ku80-deficient B cells had severe proliferation defects, with many dying when activated for CSR (Casellas et al., 1998; Manis et al., 1998; Reina San-Martin et al., 2003). Therefore, we have suggested that although Ku70 and Ku80 are required for normal CSR, their overall contribution via C-NHEJ remained to be determined (Manis et al., 2002; Chaudhuri and Alt, 2004; Chaudhuri et al., 2007). Our current protocols for activating B cells stimulate much higher CSR levels in WT B cells than those used in the original Ku70−/− and Ku80−/− B cell studies; e.g., in the earlier studies of Ku-deficient cells, only ∼10% of WT B cells underwent CSR to IgG1 (Casellas et al., 1998; Manis et al., 1998), whereas under current stimulation conditions (different purification of cells, sources of cytokines, and activators, etc.) we observe CSR to IgG1 in 60% or more of WT B cells (Cheng et al., 2009; see Fig. 2). Therefore, we have reexamined the requirement for Ku70 and Ku80 in CSR. As Ku70 and Ku80 are required for V(D)J recombination and generation of mature B cells, we generated Ku70−/− or Ku80−/− mice that harbored preassembled (“knock-in”) IgH (B1-8-HC) and IgL (3–83k-LC) variable region exons (Pelanda et al., 1997; Sonoda et al., 1997). Various studies have shown that the B1-8-HC V(D)J knock-in allele undergoes normal CSR (Casellas et al., 1998; Manis et al., 1998; Yan et al., 2007). Because of the complex breeding strategies necessary to generate these mice, we generated Ku70−/− and Ku80−/− strains that were either homozygous or heterozygous for the B1-8-HC knock-in allele (referred to, respectively, as H/HL and H/+L). The breakdown of results with these genotypes is indicated in each figure or in supplementary figures and tables. Because H/HL and H/+L lines gave similar results for most experiments, we generically refer to both genotypes as HL mice, except where otherwise noted. We verified homozygous disruption of the Ku70 and Ku80 locus and the absence of the Ku70 protein in the Ku70−/−HL mice and cells analyzed (Fig. 1 a and not depicted).

Bottom Line: Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR.Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins.Our findings suggest that more than one form of A-EJ can function in CSR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

Show MeSH
Related in: MedlinePlus