Prox1 recruits LSD1/NuRD complex components to CYP7A1 promoter and engenders repressive epigenetic changes in histone modification patterns.
(A) Expression levels of LSD1/NuRD complex components LSD1 and HDAC2 in HepG2 are not affected by Prox1. HepG2 cells were infected with recombinant lentiviruses expressing Prox1-targeting siRNA precursors si258 or si1646, or scrambled control siSCR as indicated and protein levels of Prox1, HNF4α, LSD1 and HDAC2 were detected in Western blot 36 hours post infection. Beta-actin was used as loading control. (B) Knockdown of Prox1 decreases LSD1 and HDAC2 occupancy on CYP7A1 promoter. HepG2 cells infected with indicated recombinant lentiviruses were subjected to ChIP analysis using antibodies to LSD1 and HDAC2, respectively. (C) Knockdown of Prox1 increases the level of H3K4 methylation on CYP7A1 promoter. HepG2 cells infected with indicated recombinant lentiviruses were subjected to ChIP analysis using antibodies to di-methylated H3K4 (H3K4me2), acetylated H3 and acetylated H4, respectively. Precipitated CYP7A1 promoter segments in B and C were detected using quantitative real-time PCR and relative chromatin occupancy was calculated as %input as described in Materials and Methods. Normal mouse/rabbit IgG was used as non-specific control. Means and SD from three independent experiments are presented. Statistically significant changes (P<0.05 in student’s t test) were indicated (*). Results similar to B and C were obtained using lenti-si258 infection (Supplementary Figure S1).