mediating RNA editing (Doi et al., 2009; Nonaka et al., 2009) or by recruiting factors that promote efficient DNA repair (Barreto et al., 2003; Ta et al., 2003; Ito et al., 2004; Shinkura et al., 2004; Kracker et al., 2010). In addition, epi- genetic modifications at the IgH locus, including histone H3 trimethylation at lysine 9 (H3K9me3), have been suggested to target the CSR machinery to switch regions (Wang et al., 2006, 2009; Chowdhury et al., 2008; Kuang et al., 2009). However, a direct causal link between these modifications and AID function in CSR has not been established. RESULTS AID associates with KAP1 (KRAB domain–associated protein 1) To identify nuclear proteins associating with AID, we per- formed sequential immunoprecipitations followed by mass spectrometry identification using nuclear extracts prepared from CH12 B cells expressing tagged AID (AIDFlag-HA; Fig. S1). As negative controls, we used cell lines expressing the tags alone (Flag-HA) or an irrelevant tagged protein (EGFPFlag-HA). Among the identified proteins specifically coprecipitating with AID but not with the negative controls, we found known AID partners, DNA-PKCS (Wu et al., 2005) and CRM1 (Brar et al., 2004; Ito et al., 2004; McBride et al., 2004; Geisberger et al., 2009), proteins previously implicated in CSR, Ikaros (Sellars et al., 2009), MSH2 (Chaudhuri et al., 2007), and MSH6 (Chaudhuri et al., 2007), and proteins with no known function in SHM or CSR, like KAP1 (Fig. S1). KAP1 (also known as TRIM28, KRIP1, or Tif1) was selected for analy- sis because it has been previously implicated as an effector of the DDR (Ziv et al., 2006). KAP1 is also a transcriptional co- repressor (Cammas et al., 2000) that associates with members of the HP1 (heterochromatin protein 1) family to participate in chromatin packaging and heterochromatin formation (Nielsen et al., 1999).The association between AID and KAP1 was confirmed by reciprocal immunoprecipitations and West- ern blotting (Fig. 1 A) and was specific, as KAP1 did not co- precipitate with EGFPFlag-HA (Fig. 1 B), despite relatively higher expression of EGFPFlag-HA (Fig. S1). To determine whether AID associates with KAP1 through its C terminus, we performed immunoprecipitation and Western blot experi- ments using extracts prepared from CH12 cells expressing an AID C-terminal truncation mutant (AIDFlag-HA 182–198). Although the expression level of AIDFlag-HA 182–198 is significantly lower than AIDFlag-HA or EGFPFlag-HA (Fig. S1), we found that C-terminal deletion in AID did not disrupt its association with KAP1 (Fig. 1 C).To determine whether the association between AID and KAP1 can take place within chromatin, we performed a coimmunoprecipitation experiment on chroma- tin fractions prepared from CH12 B cells expressing tagged AIDFlag-HA.We found that chromatin-bound AID reciprocally coimmunoprecipitated with KAP1 (Fig. 1 D). We conclude that endogenous KAP1 associates with nuclear and chromatin- bound tagged AID through interactions that do not require its C-terminal domain. KAP1 is required for efficient CSR but is dispensable for SHM To determine whether KAP1 plays a role in CSR and SHM, we bred Kap1 floxed mice (Kap1F/F; Cammas et al., 2000) with CD19Cre/+ transgenic mice (Rickert et al., 1997) to in- activate Kap1 in developing B cells. Despite efficient Cre- mediated deletion, no differences were observed in cellularity in the bone marrow or the spleen (not depicted), and B cell development appeared to be unaffected (Fig. S2). To deter- mine whether CSR is affected by Kap1 deficiency, we cul- tured in vitro CFSE-labeled splenic B cells isolated from CD19Cre/+Kap1F/F and control mice under conditions that induce CSR to various isotypes. We found that Kap1 defi- ciency had no effect on proliferation (Fig. 2 A) or survival (not depicted). However, Kap1 deficiency resulted in a 40– 50% reduction in CSR to all isotypes tested (Fig. 2, A–C). Consistent with this, the level of postrecombination I-CH transcripts, which appear only after recombination, was sig- nificantly reduced in CD19Cre/+Kap1F/F B cells (Fig. 2 C and Fig. S3). We conclude that Kap1 deficiency results in a B cell–intrinsic defect in CSR that is independent of abnormali- ties in survival or proliferation. To determine whether SHM is also affected by Kap1 de- ficiency, we immunized CD19Cre/+Kap1F/F and control mice with NP-CGG, sorted germinal center B cells from the lymph nodes, and analyzed J 4 intron sequences for muta- H tions (Jolly et al., 1997). We did not find significant differ- ences in mutation frequency (Fig. 2 D; P = 0.12), distribution, or pattern (Fig. S4) between sequences obtained from CD19Cre/+Kap1F/F (n = 83) and control (n = 91) animals. We conclude that KAP1 is involved in mediating CSR but ap- pears to be dispensable for SHM. Figure 1. KAP1 associates with AID. (A–C) Nuclear extracts prepared from CH12 cells stably expressing AIDFlag-HA (A), EGFPFlag-HA (B), or AIDFlag-HA 182–198 (C) were immunoprecipitated and blotted with anti-KAP1 and anti-Flag antibodies. Because of the lower expression levels of AIDFlag-HA 182–198 when compared with AIDFlag-HA or EGFPFlag-HA (Fig. S1), loading for Western blot (WB) analysis was adjusted accordingly in C. (D) Chromatin fractions prepared from CH12 cells expressing AIDFlag-HA were immunoprecipitated and blotted with anti-KAP1 and anti-Flag antibodies. Input control represents 1% of ma- terial used for immunoprecipitation (IP). Molecular mass markers in kilodaltons are indicated. Data are representative of five independent experiments. 1650 Epigenetic tethering of AID to the  switch region | Jeevan-Raj et al.