T-cell specific chromatin looping between Bcl11b and the downstream Major Peak. (A) Chromatin conformational capture was performed on samples from T-lineage cells (B6 thymocytes, Rag1^−/− or Rag2^−/− thymocytes, P2C2 cells) and non-T cells (Rag1^−/− or Rag2^−/− splenocytes, Raw264.7 cells and NIH3T3 cells) to measure the physical interactions between Bcl11b and Major Peak regions. Rag-knockout thymocytes were included as naturally arrested DN3 thymocyte populations, the in vivo equivalent of P2C2 pro-T cells. Panels show the PCR signals resulting from linkage between the R3, R4, and R5 primers within the Major Peak region and the indicated forward primers spanning the Bcl11b promoter and first two introns. For schematics and controls, see supplemental Figures 6 and 7; sequences presented in supplemental Table 1. P values for cell-type specificity were calculated by Mann-Whitney U test, comparing the five independent T-lineage samples with the four indicated non-T samples; *P < .02. The lineage nonspecific R5-F5 signal is caused by a background anomaly with this specific primer combination. Additional negative controls, samples of normal and Rag-knockout thymocytes processed for 3C without addition of ligase (“unligated”), are shown for reference. (B) Map of positions of primers used.