Figure 2. KAP1 is required for efficient CSR but is dispensable for SHM. (A) IgG1 surface expression and CFSE dilution by flow cytometry in CD19Cre/+, Kap1F/F, CD19Cre/+Kap1F/+, and CD19Cre/+Kap1F/F B cells stimulated with LPS + IL-4 for 72 h. The percentage of switched cells is indicated in each plot. (B) Percentage (+SD) of CSR after 72 h in CD19Cre/+Kap1F/F rela- tive to CD19Cre/+Kap1F/+ for the different isotypes tested (CSR to IgA and IgE could not be determined). CSR in CD19Cre/+Kap1F/+ B cells was set to 100%. Data are from five independent experiments. (C) qRT-PCR for post- switch (I-CH) transcripts in CD19Cre/+Kap1F/F relative to CD19Cre/+Kap1F/+ stimulated B cells (72 h). Expression is normalized to Cd79b expression and JEM Vol. 208, No. 8 A r t i c l e The function of KAP1 in CSR is upstream of DSB resolution CSR is dependent on transcription (Chaudhuri et al., 2007), and KAP1 is a transcriptional corepressor (Cammas et al., 2000). To determine whether switch region transcription is affected by Kap1 deficiency, we measured by quantitative RT-PCR (qRT-PCR) the level of switch region germline tran- scripts in activated B cells.We found no significant reduction in the level of these transcripts in Kap1-deficient B cells (Fig. 3 A and Fig. S3). However, occasional increases in the levels of 3, 1, 2b, and 2a transcripts were found (Fig. 3 A and Fig. S3). This is similar to what is observed in Atm/, H2ax/, or 53bp1/ B cells in which CSR is reduced despite normal or increased levels of switch region transcripts (Reina-San-Martin et al., 2003, 2004; Ward et al., 2004). We conclude that switch regions continue to be efficiently transcribed in Kap1- deficient B cells and that switch regions are accessible for DNA deamination by AID. Therefore, the function of KAP1 is downstream of switch region transcription. To determine whether Kap1 deficiency affects AID ex- pression, we measured the level of AID protein in activated B cells by Western blot. For comparison, we used AID-deficient (AIDCre/Cre) B cells, which were obtained from mice bearing a targeted insertion of the CRE recombinase cDNA into AID’s exon 1 (Robbiani et al., 2008) and which displayed the same phenotype as AID/ mice (Muramatsu et al., 2000). Reduced levels in AID protein were found in AIDCre/+ B cells (Fig. 3 B), as has been reported for AID+/ B cells (Sernández et al., 2008). In contrast, Kap1 deficiency had no deleterious effect on AID protein levels (Fig. 3 B). Consistent with this, retroviral overexpression of full-length AID in Kap1-deficient B cells did not rescue CSR, whereas it did in AID-deficient B cells (Fig. S5). As expected, overexpression of C-terminal truncation AID mutants had no effect on CSR in either AID- or in Kap1-deficient B cells (Fig. S5). We conclude that defective CSR in Kap1-deficient B cells is not caused by reduced levels of AID and that KAP1 functions downstream of AID expression. KAP1 is phosphorylated at serine 824 (-KAP1) by the ATM kinase and accumulates in -H2AX–containing foci in response to DNA damage (Ziv et al., 2006). Furthermore, inactivation of DDR components (i.e., ATM and H2AX) results in defective CSR (Ramiro et al., 2007). To determine whether KAP1 is phosphorylated during CSR, we assayed for -KAP1 by Western blot. KAP1 phosphorylation was readily detected in control cells exposed to the DSB-inducing chemical neocarzinostatin (Fig. 3 C). Despite robust levels is presented relative to the expression in CD19Cre/+Kap1F/+ B cells, set as 1. Data are representative of three experiments. Error bars indicate SD. (D) JH4 intron sequences from germinal center B cells (B220+Fas+GL-7+) sorted from the lymph nodes of immunized CD19Cre/+Kap1F/F and CD19Cre/+Kap1F/+ mice were analyzed for mutations. Segment sizes are proportional to the number of sequences bearing the indicated mutations. Mutation frequency (mutations/base pair [m/bp]) and number of sequences analyzed (in center) are indicated. Sequences were obtained from four independent experi- ments. (B–D) P-values were determined by the Student’s t test. 1651