coprecipitated AIDFlag-HA (Fig. 5 A). To determine whether KAP1, HP1, and AID exist in a complex, we fractionated Flag immunoprecipitates by gel filtration chromatography using a Superose 6 column and assayed the different fractions for AID, KAP1, and HP1 isoforms by Western blot (Fig. 5 B).We found that AIDFlag-HA, KAP1, and HP1- coelute in fractions corre- sponding to a complex of 670 kD (Fig. 5 B). However, the ma- jority of HP1- was eluted in low molecular weight fractions (Fig. 5 B), indicating that only a small fraction of the complex contains HP1- or that the in vitro association of HP1- within the complex is weaker. HP1- and HP1- were not detected (Fig. 5 B).To determine whether the AID–KAP1–HP1 complex is able to bind H3K9me3 residues, we performed peptide pull- down experiments using nuclear extracts from cells expressing AIDFlag-HA, EGFPFlag-HA, or AIDFlag-HA 182–198 and biotinylated histone H3 peptides either unmodified or trimethylated at lysine 9. We found that the H3K9me3 peptide precipitated KAP1, all the HP1 isoforms, and AIDFlag-HA (Fig. 5 C). Precipitation of AIDFlag-HA was specific, as this peptide did not precipitate EGFPFlag-HA (Fig. 5 C).The H3K9me3 peptide also precipitated AIDFlag-HA 182–198 (Fig. 5 C), indicating that the C-terminal do- main of AID is not required for the recognition of H3K9me3 resi- dues by the AID–KAP1–HP1 complex.We conclude that KAP1, HP1, and AID exist in a complex that binds H3K9me3 in vitro. Therefore, this modification could serve as a tethering motif for a complex formed by AID, KAP1, and HP1 during CSR in vivo. H3K9me3 and KAP1 mark the donor switch region before and during CSR To determine whether KAP1 and HP1 are recruited to switch regions during CSR in vivo and whether this correlates 1654 Figure 5. AID forms a complex with KAP1 and HP1 that binds H3K9me3. (A) Nuclear extracts prepared from CH12 cells expressing AIDFlag-HA were immunoprecipitated and blotted with anti–HP1-, anti–HP1-, anti–HP1-, and anti-Flag antibodies. Data are representative of three independent experi- ments. (B) Flag immunoprecipitates eluted with the Flag peptide were fractionated with a Superose 6 gel filtration column. The indicated fractions were analyzed by Western blot using antibodies specific for KAP1, HP1-, HP1-, HP1-, and AID. Arrows indicate the elution position of calibration proteins of known mo- lecular mass. Data are representative of two independent experiments. (C) Peptide pull- downs (PPD) using biotinylated unmodified (H3) or modified H3 peptides (H3K9me3) and avidin-agarose beads (Avidin) on nuclear extracts prepared from CH12 cells stably expressing AIDFlag-HA, EGFPFlag-HA, or AIDFlag-HA 182–198. Precipitated proteins were separated by SDS-PAGE and blotted with anti- bodies specific for KAP1, HP1-, HP1-, HP1-, and Flag. Data are representative of three independent experiments. (A–C) Molecular mass markers in kilodaltons are indicated. IP, immunoprecipitation; WB, Western blot. with histone modi- fications associated with the IgH locus, we performed ChIP experiments on resting and activated con- trol and CD19Cre/+Kap1F/F B cells (Fig. 6 and Figs. S6 and S7). Consistent with a previous study (Wang et al., 2009), we found that histone H3 associated with the pair of recombin- ing switch regions is acetylated at lysine 9 and 14 (H3K9/ K14Ac) in B cells undergoing CSR (Fig. 6 A) and that this correlated with switch region transcription (Fig. 3 A). Con- sistent with normal levels of germline transcripts in Kap1- deficient B cells (Fig. 3 A), we found that H3K9/K14Ac was not affected by Kap1 deficiency (Fig. S6). Although H3K9me3 has been reported to be induced at the donor and acceptor switch regions during CSR (Chowdhury et al., 2008; Kuang et al., 2009), we found that H3K9me3 was already present at the IgH locus before recombination in resting B cells (Fig. 6 B). Upon stimulation, the level of H3K9me3 was substantially reduced throughout the locus but was specifically retained over the donor switch region (S; Fig. 6 B and Table S2) when compared with sequences located immediately downstream of the variable region (JH4), the I exons, the CH exons, or regions surrounding the Mest promoter, which were included as positive and negative con- trols (Riclet et al., 2009). Surprisingly, H3K9me3 retention was not observed at S3 when cells were stimulated to switch to IgG3 with LPS, nor at S1 when cells were stimulated to switch to IgG1 with LPS + IL-4 (Fig. 6 B). The H3K9me3 pattern was similar in Kap1-deficient B cells (Fig. S6 and Table S3), indicating that this modification is not dependent on KAP1. Interestingly, we found that KAP1, together with Epigenetic tethering of AID to the  switch region | Jeevan-Raj et al.