in close proximity to its substrate by KAP1, HP1, and H3K9me3, thus increasing the probability of cytidine deamination, the efficiency of DSB formation, and consequently of CSR. Our results thus provide a mechanism linking AID to epigenetic modifications associated with the IgH locus during CSR. MATERIALS AND METHODS Mice. Kap1F/F (Cammas et al., 2000), CD19Cre/+ (provided by K. Rajewsky, Harvard Medical School, Boston, MA; Rickert et al., 1997), Kap1V488A-L490A/+ (Herzog et al., 2011), and AIDCre/Cre (Robbiani et al., 2008) mice were on a C57BL/6 background and were bred and maintained under specific patho- gen-free conditions. Age-matched littermates (8–12 wk old) were used in all experiments. All animal work was performed under protocols approved by the Direction des Services Vétérinaires du Bas-Rhin, France (Authorization no. 67–343). Splenic B cell purification, CSR assays, and two-color IgH-FISH. Resting splenic B cells were isolated, cultured in vitro with LPS (CSR to IgG3 and IgG2b), LPS + IL-4 (CSR to IgG1 and IgE), or LPS + IFN- (CSR to IgG2a), and assayed for CSR as described previously (Robert et al., 2009). Switch transcripts, switch junctions, and IgH/c-myc translocations were analyzed as described previously (Robert et al., 2009). Metaphase spreads were prepared and subjected to two-color IgH-FISH as described previously (Franco et al., 2006). Nuclear extracts and tandem afinity purification. Nuclear extracts and chromatin fractions were prepared from 12 × 109 cells using standard tech- niques. 20 mg of clarified extracts was taken into immunoprecipitation buffer (IP-300: 20 mM Tris, pH 7.9, 300 mM KCl, 20% glycerol, 0.25 mM EDTA, 0.125 mM EGTA, 0.5 mM PMSF, 1 mM DTT, 0.025% NP-40, 1× protease inhibitor cocktail [Roche], and 100 U/ml Benzonase [EMD]) and precleared with mouse IgG and protein G–agarose (GE Healthcare) for 1 h at 4°C. Flag M2 agarose beads (100 µl, 50% slurry; Sigma-Aldrich) were added and incu- bated overnight at 4°C. Immune complexes were eluted three times with 100 µl of 0.2 mg/ml Flag peptide (30 min at 4°C). Elutions were pooled and subjected to immunoprecipitation with anti-HA agarose beads (40 µl, 50% slurry; 4 h at 4°C; Roche). Immune complexes were eluted twice with 40 µl of 0.1 M glycine, pH 2.4 (10 min), or by overnight incubation with 40 µl of 0.6 mg/ml HA peptide. Proteins were fractionated by SDS-PAGE in 4–12% gradient gels (Invitrogen) and stained with Coomassie blue (G250; Bio-Rad Laboratories) before processing for mass spectrometry. Mass spectrometry analysis. Whole lanes from one-dimensional electro- phoresis were systematically excised in 5-mm bands. Proteins in bands were subjected to in-gel reduction, alkylation, and trypsin digestion. Pep- tides were dried, resuspended in 0.1% formic acid, and injected into the nano liquid chromatography (Ultimate 3000; Dionex) coupled with a lin- ear ion trap mass spectrometer equipped with a nanoelectrospray source (LTQ XL ETD; Thermo Fisher Scientific). Tandem mass spectrometry spectra were recorded in the data-dependent mode on the five most intense ions observed in mass spectrometry scan with collision-induced dissocia- tion and electron transfer dissociation fragmentation. Peptides selected for tandem mass spectrometry acquisition were then dynamically excluded for 30 s. Protein ID was performed using Proteome Discoverer 1.1 (Thermo Fisher Scientific) and SEQUEST (Thermo Fisher Scientific) searching against the mouse Swiss-Prot database (version 57.9) with the following fixed parameters: precursor mass tolerance of ±0.5 D, product ion mass tol- erance of ±0.8 D, and two missed cleavages, and carbamidomethylation of cysteine as fixed modification and methionine oxidation as variable modi- fication. SEQUEST results were filtered with Xcorr versus charge state 1.5-1, 1.6-2, 3-3, 3.2-4. SHM analysis. Germinal center B cells (B220+Fas+GL-7+) were sorted from the lymph nodes of NP-CGG–immunized mice and analyzed for mutation in JH4 intron sequences (Jolly et al., 1997) with SHMTool (Maccarthy et al., 2009). 1658 Real-time quantitative PCR. RNA and cDNA were prepared using standard techniques. Quantitative PCR was performed using SYBR green JumpStart Taq ReadyMix (Sigma-Aldrich) or QuantiTect SYBR green PCR kit (QIAGEN). Approximately 3 ng cDNA or 3–5 × 106 cell equiva- lents of de–cross-linked chromatin were run (in triplicate) and analyzed on a LightCycler 480 (Roche). Transcript or DNA quantities were calculated relative to standard curves and normalized to CD79b transcripts or input DNA. Gene of interest/CD79b or region of interest/input DNA ratios were averaged across experiments and normalized to the appropriate controls. S mutation analysis. The 5 end of S was amplified by PCR using Pfu Turbo as described previously (Reina-San-Martin et al., 2004) or using Platinum Taq polymerase (Invitrogen) and bar-coded primers (Table S5) for HTS. HTS amplification conditions were as follows: 30 cycles of 94°C (15 s), 60°C (15 s), and 68°C (1 min). PCR products were purified with AmPure beads (Agencourt), quantified using Quan-iT Pico Green (Invitrogen), and mixed in equimolar ratios before sequencing with a 454 GS FLX sequencer (Roche; GATC). Sequences were aligned with Lasergene (DNASTAR) and analyzed with SHMTool (Maccarthy et al., 2009). Peptide pull-downs. Avidin-conjugated agarose beads (Thermo Fisher Sci- entific) were coupled to biotinylated unmodified or modified H3 peptides (A1RTKQTARK9[me3]STGGKAPRKQLATK23-biotin; letters in bold indi- cate the modified amino acid, and the type of modification is in brackets) for 3 h at 25°C with rotation, washed in PBS 1× NP-40 0.1% and resuspended as a 50% slurry. 5 mg of nuclear extracts was precleared with avidin-agarose beads and incubated overnight with peptide-bound resin (40 µl, 50% slurry). Resin was washed and resuspended in 20 µl of Laemmli buffer. Proteins were fractionated by SDS-PAGE and analyzed by Western blot. ChIP. The protocol was adapted from Millipore (http://www.millipore. com/userguides/tech1/mcproto407). In brief, 3 × 107 resting or stimulated B cells were cross-linked at 37°C for 10 min in 5 ml PBS/0.5% BSA with 1% formaldehyde. The reaction was quenched with 0.125 M glycine. After lysis, chromatin was sonicated to 0.5–1 kb using a Vibracell 75043 (Thermo Fisher Scientific). After 2× dilution in ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, and 167 mM NaCl), chromatin was precleared by rotating for 2 h at 4°C with 80 µl of 50% pro- tein A/G slurry (0.2 mg/ml sheared salmon sperm DNA, 0.5 mg/ml BSA, and 50% protein A/G; GE Healthcare). 0.3–0.5 × 106 cell equivalents were saved as input, and 3–5 × 106 cell equivalents were incubated overnight with specific or control antibodies (Table S6). Immune complexes were precipi- tated by the addition of protein A/G or Dynabeads protein A (for AID) and processed according to the Millipore protocol. Gel filtration chromatography. 20 mg of nuclear extract was immuno- precipitated with Flag M2–agarose beads in IP-300 buffer, washed, and eluted with 0.2 mg/ml Flag peptide in 100 µl. Eluted proteins were fraction- ated using an micro Superose 6 PC 3.2/30 column (ÄKTA). 100 µl of pro- teins in each fraction was analyzed by Western blot. Online supplemental material. Fig. S1 shows Western blot analysis of nuclear and cytoplasmic extracts prepared from CH12 cells expressing AIDFlag-HA, Western blot analysis of nuclear extracts from CH12 cells expressing AIDFlag-HA, EGFPFlag-HA, and AIDFlag-HA 182–198, SDS-PAGE (silver stain) analysis of proteins eluted after tandem affinity purification, and the KAP1 peptides identified by mass spectrometry. Fig. S2 shows that Kap1 deficiency has no major impact on B cell development. Fig. S3 displays two additional experiments showing that Kap1 deficiency results in reduced levels of post- recombination transcripts without decreasing germline transcription. Fig. S4 shows that Kap1 deficiency has no effect on the frequency of germinal center B cells and the distribution or pattern of somatic mutations. Fig. S5 shows that AID overexpression in Kap1-deficient B cells does not rescue CSR. Fig. S6 displays two independent experiments showing that Kap1 deficiency does not alter the pattern of H3K9me3 or H3K9/K14Ac marks at the IgH locus. Epigenetic tethering of AID to the  switch region | Jeevan-Raj et al.