a complex formed by AID, KAP1, and HP1 is tethered to the transcribed donor switch region bearing the H3K9me3 mark in vivo. DISCUSSION We have found that AID forms a complex with KAP1 and HP1 and that Kap1 inactivation results in a CSR-specific de- fect that phenocopies mice deficient for components of the DDR (i.e., defective CSR and normal SHM; Ramiro et al., 2007). Because KAP1 has been reported to be an effector of the DDR downstream of ATM (Ziv et al., 2006) and because Kap1 inactivation results in a CSR-specific defect similar to Atm/ mice (Lumsden et al., 2004; Reina-San-Martin et al., 2004), we hypothesized that the function of KAP1 in CSR is to respond to AID-induced DNA damage. However, we found that the role of KAP1 in CSR is independent of its phosphorylation status at serine 824 and that its inactivation does not result in defective DNA repair or increased genomic instability. Therefore, KAP1 does not appear to participate in the repair of AID-induced DNA damage during CSR. Impaired CSR in the absence of KAP1 could be ex- plained by a global reactivation of genes normally silenced by a KAP1-dependent mechanism (Nielsen et al., 1999) or by a substantial deregulation of chromatin structure (Ziv et al., 2006) at the IgH locus. However, this is unlikely as the tran- scriptional repressor activity of KAP1 requires its association with HP1 (Sripathy et al., 2006), and we show that the ex- pression of a KAP1 mutant protein that cannot associate with HP1 (Cammas et al., 2004) was unable to restore CSR to wild-type levels. Furthermore, no significant alterations in switch region transcription were observed because of Kap1 deficiency. We believe that the long-range interactions be- tween switch region promoters and IgH enhancers, which are controlled by transcription (Wuerffel et al., 2007), are most likely not affected and that the global three-dimensional structure of the IgH locus is not altered by Kap1 deficiency. The local access of AID to the switch region is most likely not affected in the absence of KAP1 because AID targeting to switch regions appears to be dependent on its association with Spt5 and RNA polymerase II (Pavri et al., 2010) and because Kap1 inactivation reduces but does not abolish the recruitment of AID to S. We pinpoint the role of KAP1 in CSR to a step that facili- tates AID recruitment and the subsequent generation of DNA damage at S. We find a substantial reduction in AID recruit- ment that is in contrast with the 50% reduction in CSR that we observe in Kap1-deficient B cells. This might relate to dif- ferences in the immunoprecipitation efficiency between ex- periments. As the AID-ChIP signal in Kap1-deficient B cells (relative to controls) ranged between 20 and 50% depending on the experiment, we think that it might be underestimated. We show that AID forms a complex with KAP1 and HP1 that selectively recognizes H3K9me3 in vitro. We also show that H3K9me3, KAP1, and HP1 are recruited to the IgH locus in resting B cells and that upon activation H3K9me3 and KAP1 are released but retained over S. With respect to the JEM Vol. 208, No. 8 A r t i c l e pattern of H3K9me3, our results differ from a previous study (Kuang et al., 2009) as we find that this modification is not inducible. Although it is difficult to pinpoint the reason for this discrepancy, it most likely relates to technical issues such as differences in culture conditions, time points assayed, de- tection methods, etc. Nevertheless, the specific enrichment of KAP1 binding at S that we observe suggests that H3K9me3 can serve as a recognition motif for an AID–KAP1–HP1 complex in vivo at the donor switch region during CSR. Al- though we cannot provide an explanation as to why KAP1 recruitment expands up to C or why it is not recruited to the acceptor switch regions, this suggests that additional fac- tors and/or histone modifications may be required to limit the range of AID recruitment and/or to modulate the effi- ciency of its retention. As we did not observe recruitment of KAP1 at S3 or S1, it is possible that KAP1 deficiency has no impact in the tethering efficiency of AID to acceptor switch regions, the efficiency of cytidine deamination, and the subsequent generation of DSBs. As DSB formation at ac- ceptor switch regions has been suggested to be a rate-limiting step in CSR (Reina-San-Martin et al., 2003; Schrader et al., 2003), it might explain why the impairment in CSR in the absence of KAP1 is not more profound. H3K9me3 is usually associated with heterochromatin for- mation and gene silencing. However, increasing evidence suggests additional roles for heterochromatin (Grewal and Jia, 2007), for example in recruiting factors that facilitate access of RNA polymerase II to heterochromatic regions (Zofall and Grewal, 2006) or by acting as a recruitment platform for fac- tors regulating long-range chromosomal interactions (Jia et al., 2004). The presence of HP1- throughout the IgH locus before and during CSR is reminiscent of the mating-type switching in the fission yeast Schizosaccharomyces pombe, in which heterochromatin promotes the spreading of the re- combination-promoting complex across the mating locus and imposes structural constraints that are important for the choice of the recombination donor site (Jia et al., 2004). In- deed, H3K9me3 has been shown to be present, along with HP1-, at actively transcribed genes in mammalian cells (Vakoc et al., 2005) and at recombining switch regions in mouse and human B cells (Chowdhury et al., 2008; Kuang et al., 2009). Strikingly, we demonstrate that the in vivo KAP1–HP1 asso- ciation is required for efficient AID binding at S and to sus- tain CSR by expressing a KAP1 mutant that cannot associate with HP1 (in a Kap1-deficient background). Our observations are consistent with a model in which AID forms a complex with KAP1 and HP1 that is tethered to transcribed switch regions bearing the H3K9me3 mark.There is precedent for a model based on the recognition of a modi- fied histone, as recognition of hypermethylated histone H3 at lysine 4 by the PHD domain of RAG2 promotes efficient V(D)J recombination (Liu et al., 2007; Matthews et al., 2007). In our model, H3K9me3 and associated KAP1 and HP1- mark the S region before recombination. Upon stimulation, these marks are retained over the S and provide a docking motif for AID tethering in vivo. AID would then be retained 1657