Prox1 co-localizes with LSD1/NuRD complex components on CYP7A1 promoter.

(A) Occupancy of HNF4α, Prox1, and LSD1/NuRD components on CYP7A1 promoter in HepG2 cells. Chromatin immunoprecipitation (ChIP) was performed on chromatin fragments prepared from HepG2 cells using specific antibodies as indicated and corresponding normal IgG as non-specific control. (B) Prox1 co-localizes with LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Sequential ChIP was performed on chromatin fragments prepared from HepG2 cells using anti-Prox1 antibodies for first round ChIP (A) and antibodies to HNF4α LSD1 and HDAC2 for second round ChIP, respectively. (C) Occupancy of Prox1, LSD1 and HDAC2 on CYP7A1 promoter in mouse liver cells. Chromatin fragments prepared from mouse liver cells were subjected to ChIP using antibodies to Prox1, HNF4α LSD1 and HDAC2 as indicated. In panels A-C, corresponding normal mouse or rabbit IgG was used as non-specific background control for each antibody used. Precipitated CYP7A1 promoter segments were detected using quantitative real-time PCR and relative chromatin occupancy was calculated as %input as described in Materials and Methods. In panel A, a control region in downstream CYP7A1 mRNA coding sequences (CDS) was also quantitated using real-time PCR in parallel as further demonstration of assay specificity. Means and SD from three independent experiments are presented. Statistically significant enrichment by specific antibodies (P<0.05 in student’s t test) were indicated (*).