Figure 6.

Activation of V_δ gene segment chromatin and rearrangement using a HDAC inhibitor. H3ac (A) and H4ac (B) were measured by ChIP using chromatin prepared from adult Rag2^−/− thymocytes that were cultured for 8 hrs with or without 3 ng/ml TSA. Values of bound/input were expressed relative to B2m (normalized to one) in each sample. The data represent the mean ± SEM of two to three independent chromatin preparations for each treatment. *, P<.05 by two-tailed Student’s t-test. C. Germline transcription was measured by quantitative real-time PCR using cDNA prepared from adult Rag2^−/− thymocytes that were cultured for 8 hrs with or without 3 ng/ml TSA. The data represent the mean ± SEM of four independent cDNA preparations for each genotype, all normalized to values for Actb. *, P<.05 by two-tailed paired Student’s t-test. D. Adult 129 DN1 thymocytes were placed in culture for 14 days with or without 3 ng/ml TSA on OP9-DL1 stromal cells; DN3 thymocytes were sorted from cultured cells for preparation of genomic DNA. Three-fold serial dilutions (wedges) of genomic DNA were analyzed by PCR followed by Southern blot. PCR was performed using TRDV2-2 or TRDV4 primers in combination with a J1 primer; a ³²P-labeled internal J1 oligonucleotide was used as a probe. Cd14 PCR insured use of similar amounts of DNA. (−), no DNA. The data is representative of three independent experiments.