Figure 3. KAP1 functions downstream of switch region transcription and upstream of DSB formation. (A) qRT-PCR for germline (IH-CH) tran- scripts in CD19Cre/+Kap1F/F relative to CD19Cre/+Kap1F/+ stimulated B cells (72 h). Expression is normalized to Cd79b and relative to CD19Cre/+Kap1F/+ B cells, set as 1. P-values were determined by the Student’s t test. Data are from three independent experiments. Error bars indicate SD. (B) Western blot for AID and -actin in Kap1F/F, CD19Cre/+Kap1F/+, CD19Cre/+Kap1F/F, AID+/+, AIDCre/+, and AID-deficient (AIDCre/Cre) B cells. Numbers below the panel reflect the inten- sity of bands representing AID protein levels relative to controls after normalization to -actin. Molecular mass markers in kilodaltons are indicated. Data are representative of three independent experiments. (C) KAP1 phosphorylation (-KAP1) by Western blot in wild-type B cells cultured with LPS + IL-4. As a positive control, CH12 cells treated with neocarzinostatin (NCS). (D) -KAP1 was immunoprecipitated from activated AID+/+ and AID-deficient (AIDCre/Cre) B cells and blotted for KAP1. Data are representative of two independent experiments. IP, immunoprecipitation; WB, Western blot. (E) Percentage of S/S3 switch junction sequences with indicated nucleotide overlap from CD19Cre/+Kap1F/+ and CD19Cre/+Kap1F/F LPS-stimulated B cells (72 h) from five indepen- dent experiments. Mean length of overlap in base pairs and the number of sequences analyzed (n) is indicated. (F) Quantification of IgH locus breaks as determined by IgH-FISH on metaphases prepared from control (n = 780) and CD19Cre/+Kap1F/F (n = 593) B cells cultured for 3 d with anti-CD40 and IL-4. Mean (+SEM) is shown. Data are from two independent experiments (Table S1). (E and F) P-values were determined by the Mann-Whitney test. (G) Frequency of IgH/c-myc translocations in CD19Cre/+Kap1F/F and CD19Cre/+Kap1F/+ B cells determined by long-range PCR and Southern blot in three inde- pendent experiments. The p-value was determined by the one-tailed exact Fisher’s test. of AID expression, -KAP1 was not detected in activated B cells (Fig. 3 C), indicating that KAP1 phosphorylation, if any, is below detection threshold. To determine whether a small fraction of KAP1 is phosphorylated during CSR, we immuno- precipitated -KAP1 and blotted for KAP1 (Fig. 3 D).Although -KAP1 was detected under these conditions, KAP1 phos- phorylation was not dependent on AID expression (Fig. 3 D). We conclude that the role of KAP1 in CSR is independent of its phosphorylation status at serine 824. CSR requires the generation of DSBs in switch regions and their resolution by nonhomologous end joining (NHEJ; Yan et al., 2007). To determine whether CSR junctions are affected by Kap1 deficiency, we compared IgG3 CSR junctions from CD19Cre/+Kap1F/F (n = 52) and control (n = 46) B cells. 1652 Sequence analysis revealed no significant differences (P = 0.9175) in the amount of microhomology at the junctions between groups (Fig. 3 E). We conclude that CSR junctions are unaffected by Kap1 deficiency and that DNA ends are re- paired normally by NHEJ. Inactivation of core NHEJ or DDR components results in global and IgH-specific genomic instability (Franco et al., 2006;Yan et al., 2007).To determine whether Kap1 deficiency results in the accumulation of unresolved DNA breaks trig- gered by AID in switch regions, we performed two-color IgH-specific fluorescence in situ hybridization (IgH-FISH; Franco et al., 2006; Yan et al., 2007) on metaphase spreads prepared from CD19Cre/+Kap1F/F (n = 593) and control (n = 780) activated B cells.Contrary to Atm/ or Xrcc4/ B cells Epigenetic tethering of AID to the  switch region | Jeevan-Raj et al.