H3K9me3, is recruited to the IgH locus in resting B cells. However, KAP1 binding was predominant over S when compared with downstream regions (Fig. 6 C). Upon stim- ulation, KAP1 was released from the locus but was Figure 7. The in vivo association between KAP1 and HP1 is required for efficient CSR. (A) IgG1 cell surface expression in CD19Cre/+ Kap1F/+, CD19Cre/+ Kap1V488A-L490A/+, CD19Cre/+Kap1F/F, and CD19Cre/+ Kap1V488A-L490A/F CFSE-labeled B cells stimulated with LPS + IL-4 for 3 d. The percentage of switched cells is indicated in each plot. (B) Percentage (+SD) of CSR in CD19Cre/+Kap1F/+, CD19Cre/+Kap1F/F, and CD19Cre/+ Kap1V488A-L490A/F relative to CD19Cre/+Kap1F/+. CSR in CD19Cre/+Kap1F/+ B cells was set to 100%. Data are from five independent experiments. P-values were 1656 preferentially retained over S (Fig. 6 C). KAP1 recruitment was specific as no significant ChIP signal above background was found in Kap1-deficient B cells (not depicted). HP1- binding differed from H3K9me3 and KAP1 in that it was distributed throughout the locus in resting and activated B cells (Fig. 6 D, Fig. S7, and Table S4). Increased HP1- binding at C in activated B cells was observed (Fig. 6 D) but was not reproducible (Fig. S7).We conclude that H3K9me3, HP1-, and KAP1 are present at the IgH locus as part of a heterochromatic signature in resting B cells and that H3K9me3 and KAP1 specifically mark the donor switch region (S) before and during CSR. The in vivo KAP1–HP1 association is required for efficient AID binding at S and CSR To determine whether the in vivo association between KAP1 and HP1 is required to sustain CSR, we bred CD19Cre/+ Kap1F/F mice with knockin mice (Kap1V488A-L490A; Herzog et al., 2011) expressing a mutated KAP1 protein that is un- able to associate with the HP1 proteins (Cammas et al., 2004) to generate CD19Cre/+Kap1V488A-L490A/F mice. Although B cells isolated from CD19Cre/+Kap1V488A-L490A/+ mice (express- ing a wild-type KAP1 and a mutant protein) underwent CSR at wild-type frequencies (similar to CD19Cre/+Kap1F/+), B cells isolated from CD19Cre/+Kap1V488A-L490A/F mice (express- ing only the KAP1 mutant protein, which cannot associate with HP1; Fig. S2) displayed the same CSR defect observed in CD19Cre/+Kap1F/F B cells (Fig. 7, A and B). We conclude that the in vivo association between KAP1 and HP1 is re- quired to sustain efficient CSR. To determine whether AID binding to S is dependent on the interaction between KAP1 and HP1, we performed AID-ChIP experiments on chromatin prepared from CD19Cre/+ Kap1F/+, CD19Cre/+Kap1V488A-L490A/F, and AIDCre/Cre activated B cells (Fig. 7 C). We found that AID occupancy at the S switch region was significantly reduced (P < 0.0001) in CD19Cre/+Kap1V488A-L490A/F when compared with CD19Cre/+ Kap1F/+ B cells (Fig. 7 C).The reduction in AID retention at S was similar to that observed in Kap1-deficient B cells (Fig. 4 A). We conclude that the in vivo association between KAP1 and HP1 is required to mediate the efficient binding of AID to the donor switch region and to sustain CSR and that determined by the one-tailed Student’s t test (*, CD19Cre/+Kap1F/+ vs. CD19Cre/+Kap1F/F: IgG3, P = 0.0154; IgG1, P = 0.0035; IgG2b, P = 0.0264; IgG2a, P = 0.001; **, CD19Cre/+Kap1F/+ vs. CD19Cre/+Kap1V488A-L490A/F: IgG3, P = 0.0017; IgG1, P = 0.0036; IgG2b, P = 0.0276; IgG2a, P = 0.0004). (C) ChIP analysis for AID occupancy at the S switch region in CD19Cre/+ Kap1F/+, CD19Cre/+Kap1V488A-L490A/F, and AIDCre/Cre B cells cultured in vitro with LPS + IL-4 for 60 h. Normalized AID-ChIP data from three experi- ments 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. Epigenetic tethering of AID to the  switch region | Jeevan-Raj et al.