RAG down-regulation is associated with loss of transcription factor binding and chromatin conformation. (A) Rag1 and Rag2 transcript abundance evaluated by RT-PCR in control and PMA plus ionomycin (P+I)–treated VL3-3M2 cells. The data represent mean ± SEM of six independent experiments, with values for Rag1 and Rag2 normalized to those for Actb. (B) ASE transcription factor occupancy evaluated by ChIP in control and PMA plus ionomycin–treated VL3-3M2 cells. The data represent mean ± SEM of three independent experiments. (C) 3C analysis of interactions of BglII fragments with the ASE viewpoint in control and PMA plus ionomycin–treated VL3-3M2 cells. The data represent the mean ± SEM of three independent experiments, with interaction frequencies normalized to those of a nearest neighbor BglII fragment. (D) Rag1 and Rag2 transcript abundance as in A using control and PMA plus ionomycin–treated sorted DP thymocytes. The data represent mean ± SEM of six independent experiments. (E) ASE transcription factor occupancy evaluated by ChIP in control and PMA plus ionomycin–treated sorted DP thymocytes. The data represent mean ± SEM of three independent experiments. (F) 3C analysis as in C using control and PMA plus ionomycin–treated sorted DP thymocytes. The data represent mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired Student’s t test (A and D) or two-way ANOVA with Holm-Sidak’s multiple comparisons test (B, C, E, and F).