Figure 4. Cyclin D1b promotes chromosomal confirmations associated with active transcription in response to androgen.

(A) LNCaP cells were starved of hormone for 72 hours and treated with 10 nM DHT for 3 hours. Cells were fixed, digested with the HindIII endonuclease, and ligated; total DNA was purified. Relative distance between the constant region (proximal to AROR1 of the SNAI2 gene) and 4 test regions spanning the SNAI2 gene and downstream sequences was determined using TaqMan qPCR. Top: 100 ng of purified ligated DNA was used to test individual primer sets for each test site to ensure formation of single bands specific to ligation products. Bottom: representative images of PCR products of each ligated site in the presence or absence of DHT. A control region lacking HindIII restriction sites serves as a genomic loading control. (B) LNCaP-Vec and LNCaP-D1b cells were treated as in A, and frequency of ligation is plotted as relative to ligation frequency of parental EtOH controls, after normalizing for total DNA content (control region). (C) ChIP sequencing data of AR occupancy across the cell cycle (C. McNair and K.E. Knudsen, unpublished observations) in LNCaP cells in response to 3 hours of (10 nM) DHT. AR occupancy within the SNAI2 gene is indicated by peaks, and proximal 3C test sites are designated by triangles. Error bars represent mean ± SEM. ***P < 0.001.