A r t i c l e Figure 4. AID binding to S is impaired by Kap1 deficiency and results in reduced levels of DNA damage. (A) ChIP analysis for AID occupancy at the S switch region in CD19Cre/+Kap1F/+, CD19Cre/+Kap1F/F, and AIDCre/Cre B cells cultured in vitro with LPS + IL-4 for 60 h. Normalized AID-ChIP data from three experiments assayed with two different primer sets are shown. For each sample, AID-ChIP values were normalized to the input control and subtracted from the bead-only negative control. AID-ChIP signal in CD19Cre/+Kap1F/+ B cells was assigned an arbitrary value of 1. P-values were determined by the one- tailed Student’s t test. Error bars indicate SD. (B and C) Mutation analysis in the 5 end of S in CD19Cre/+Kap1F/F and CD19Cre/+Kap1F/+ LPS + IL-4–stimulated B cells (72 h) as determined by Sanger sequencing (B) or HTS (C). Data are from five and four independent experiments, respectively. Background mutation frequency was determined on tail genomic DNA. Pie charts in B are as in Fig. 2. P-values were determined by the two-tailed Student’s t test. (Yan et al., 2007), we found no significant increase (P = 0.9) in the percentage of metaphases with abnormalities in Kap1- deficient B cells (Fig. 3 F and Table S1). Consistent with this, we found that Kap1 disruption did not result in increased fre- quency of AID-dependent IgH/c-myc translocations (Fig. 3 G). We conclude that in the absence of KAP1, DSBs generated at switch regions are efficiently repaired and do not lead to global or IgH-specific genomic instability. Therefore, KAP1 functions upstream of the AID-induced DNA breaks that initiate CSR. AID binding to S is impaired by Kap1 deficiency To determine whether AID recruitment to the S switch re- gion is dependent on KAP1, we performed chromatin immuno- precipitation (ChIP) analysis using an anti-AID antibody (Pavri et al., 2010) on chromatin prepared from CD19Cre/+Kap1F/+, CD19Cre/+Kap1F/F, and AIDCre/Cre activated B cells (Fig. 4 A). We found that AID occupancy at the S switch region was significantly reduced (P < 0.0001) in CD19Cre/+Kap1F/F when compared with CD19Cre/+Kap1F/+ B cells (Fig. 4 A).AID-ChIP signal in CD19Cre/+Kap1F/F B cells relative to controls ranged between 20 and 50% (depending on the experiment; not de- picted) and was specific, as only background levels of immuno- precipitated AID protein were observed at S in AIDCre/Cre B cells. We conclude that AID binding to the donor switch region is impaired in Kap1-deficient B cells. Impaired AID binding results in reduced levels of DNA damage at the S To determine whether impaired AID recruitment in Kap1- deficient B cells translates into reduced levels of DNA dam- age, we measured the mutation frequency in the 5 end of S by cloning and sequencing.This assay has been used to assess JEM Vol. 208, No. 8 the level of AID-induced DNA damage in switch regions (Barreto et al., 2003; Guikema et al., 2007).We found that the proportion of mutated sequences and the mutation frequency were reduced (P = 0.040) in CD19Cre/+Kap1F/F (F = 0.78 × 103 mutations/bp; n = 170) when compared with controls (F = 1.07 × 103 mutations/bp; n = 169; Fig. 4 B). A signifi- cant reduction (P = 1.5 × 1019) in mutation frequency in the 5 end of S was also found in Kap1-deficient B cells when sequences were analyzed in a larger dataset by high- throughput amplicon sequencing (HTS; Fig. 4 C). We con- clude that impaired AID recruitment to S results in reduced levels of DNA damage and consequently in defective CSR. AID forms a complex with KAP1 and HP1 that binds H3K9me3 Epigenetic modifications at the IgH locus have been suggested to target the CSR machinery to switch regions by relaxing chromatin or by providing binding motifs for factors involved in CSR (Odegard et al., 2005; Wang et al., 2006; Fraenkel et al., 2007; Chowdhury et al., 2008; Kuang et al., 2009; Wang et al., 2009). Among these, H3K9me3, which is usually associated with silent genes and heterochromatin, was found at transcribed donor and acceptor switch regions (Chowdhury et al., 2008; Kuang et al., 2009).We hypothesized that AID could be tethered to transcribed switch regions bearing this modification through the association between AID, KAP1, and HP1 and the ability of the latter to bind H3K9me3 (Bannister et al., 2001). To deter- mine whether AID also associates with HP1, we performed reciprocal coimmunoprecipitation experiments using nuclear extracts prepared from cells expressing AIDFlag-HA and antibodies specific for the different HP1 isoforms. Although AIDFlag-HA coprecipitated with all HP1 isoforms, only HP1- reciprocally 1653