CtIP promotes microhomology-mediated alternative end joining during class-switch recombination

Abstract

Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment.

Figure 1

CtIP knock-down alters CSR in CH12 cells. (a) Extracts derived from unstimulated (−) or CIT stimulated (+) CH12 cells expressing two different shRNAs (CtIP-1 or CtIP-2) against CtIP or control “scrambled” shRNA were analyzed by western blotting using antibodies against CtIP or GAPDH (loading control). The arrow indicates the polypeptide corresponding to CtIP. (b) CSR to IgA was measured by flow cytometry. CSR frequency in scrambled shRNA infected cells was assigned an arbitrary unit (AU) of 100. The data is a mean of 14 independent experiments with error bars depicting standard deviation from the mean. (c) Levels of AID transcripts of scrambled or CtIP knock-down cells were measured 48h after CIT stimulation by quantitative real-time PCR. The values are the mean of three independent experiments and the error bars represent standard deviation from the mean. (d) Empty lentiviral vector or one harboring human CtIP (hsCtIP) cDNA was introduced into control or CtIP knock-down cells and western blot was performed to determine expression of CtIP. (e) ChIP was performed from CIT-stimulated CH12 cells with anti-AID and the amount of AID bound to Sµ was determined by real-time quantitative PCR. The values represent the average of 3 independent experiments with error bars representing standard deviation from the mean. (f) CSR was measured by flow cytometry in hsCtIP or empty expression vector transduced CtIP knock-down cells.

Figure 2

CtIP knock-down alters end-joining during CSR. (a) Sµ-Sα junctions from CIT-stimulated CH12 cells infected with control or CtIP-1 shRNA were analyzed. Sequence data was compiled from six independent experiments. (b) The spectrum of junctions in control versus CtIP knock-down cells was tabulated. The difference in the percentage of junctions with microhomology of 4 nucleotides or more was statistically significant (p=0.02). (c) Sµ-Sα junctions in CtIP knock-down cells transduced with empty vector or vector encoding hsCtIP were analyzed.

Figure 3

CtIP knock-down alters end-joining in Ku70-deficient cells. (a) CIT-stimulated CH12 cells transduced with scrambled or shRNAs (Ku70-1, Ku70-2) against Ku70 were analyzed by western blotting using Ku70 or GAPDH (loading control) antibodies. (b) CSR to IgA in Ku70-1 or Ku70-2 shRNA transduced cells was measured by flow cytometry. CSR frequency in cells transduced with scrambled shRNA was assigned an arbitrary unit (AU) of 100. The data is a mean of at least three independent experiments with error bars indicating standard deviation from the mean. (c) Ku70 knock-down or control cells were transduced with scrambled or CtIP-1 shRNA and expression of Ku70, CtIP and GAPDH were determined by western blot analysis. (d) CH12 cells with the indicated shRNAs were stimulated with CIT for 72 hours and CSR to IgA was measured by flow cytometry. CSR frequency in cells transduced with scrambled shRNA was assigned an AU of 100. The data is a mean of nine independent experiments with error bars representing standard deviation from the mean. (e) Sµ-Sα junctions from Ku70-1/scrambled and Ku70-1/CtIP-1 cells were cloned, sequenced and the percentage of cells with the indicated lengths of microhomology was plotted. (f) The distribution of microhomology at the Sµ-Sα junctions is tabulated. The difference in the number of junctions with microhomology of 4 nucleotides or more between the Ku70-1/scrambled and Ku70-1/CtIP-1 knock-down cells was statistically significant (p=0.04).

Figure 4

CtIP binds to switch region DNA. (a) ChIP was carried out in CIT-stimulated CH12 cells with AID, CtIP or control non-specific IgG antibodies. DNA from the ChIP samples was diluted 3-fold, amplified by PCR and the presence of Sµ, µpromoter (Iµ promoter) or p53 was determined by analyzing the PCR products on agarose gels. (b) The amount of CtIP bound to Sµ in the indicated cells was measured by real-time quantitative PCR. Data are the mean of three independent experiments with error bars representing standard deviation from the mean. (c) CtIP binding to Sµ in unstimulated or CIT-stimulated CH12 cells was assayed by ChIP and quantified by real-time PCR. The values represent the mean of three independent experiments with error bars representing standard deviation from the mean. (d) Splenic B cells from wild type or AID^−/− mice were stimulated with α-CD40 and IL-4 for 48hrs and ChIP was performed with CtIP, AID, H3 and control non-specific IgG antibodies. Immunoprecipitated DNA was diluted 4-fold and amplified by PCR for the presence of Sµ, Sγ1, Iµ promoter or p53.

Figure 5. A model for the role of CtIP in CSR

AID activity induces formation of staggered DSBs in two distinct S regions. The DSBs are initially processed by the Mre11/Rad50/Nbs1 (MRN) complex, possibly to generate blunt ends and then channeled into either the C-NHEJ or the A-NHEJ pathway. For C-NHEJ, blunt DSBs are bound by Ku (and other C-NHEJ proteins) and subsequently ligated by DNA ligase IV/XRCC4 that do not require microhomology at the DNA ends. For A-NHEJ, CtIP alone, or in conjunction with MRN, further processes the DNA ends to reveal stretches of microhomology prior to ligation. Since CtIP binding to S regions is enhanced when Ku protein is depleted (fig. 4), Ku could possibly compete with or suppress the A-NHEJ pathway. Additional components of A-NHEJ that participate in CSR, including the ligase that seals the DNA ends are yet to be elucidated.