FIGURE 5.

Effect of cofactor silencing on chromatin looping VDRE containing regions to the TSS of the p21 gene. MDA-MB453 cells were transfected for 24 h with siRNA oligonucleotides against HDAC4, MED1, or LSD1 and subsequently stimulated with 10 nm 1α,25(OH)₂D₃ for the indicated times. Chromatin was extracted, cross-linked, and digested with either Hpy8I (upper and center graphs representing regions p21-1 and p21-2, respectively) or MvaI (lower graph representing region p21-3). After ligation, the DNA was extracted and analyzed by PCR with primers and FAM-labeled probes as indicated in Fig. 3. Values indicate looping as percentage to that in untreated cells transfected with non-targeting siRNA from indicated region to the p21 TSS. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the effects of the specific siRNAs in reference to control siRNA (^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001).