Prox1 directly interacts with LSD1 and recruits the LSD1/NuRD complex to epigenetically co-repress CYP7A1 transcription.

Abstract

Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the first and rate-limiting step in the classical pathway of bile acids synthesis in liver and is crucial for maintaining lipid homeostasis. Hepatocyte nuclear factor 4α (HNF4α) and α1-fetoprotein transcription factor (FTF) are two major transcription factors driving CYP7A1 promoter activity in hepatocytes. Previous researches have shown that Prospero-related homeobox (Prox1) directly interacts with both HNF4α and FTF and potently co-represses CYP7A1 transcription and bile acid synthesis through unidentified mechanisms. In this work, mechanisms involved in Prox1-mediated co-repression were explored by identifying Prox1-associated proteins using immunoprecipitation followed by mass spectrometry (IP-MS) methodology. Multiple components of the epigenetically repressive lysine-specific demethylase 1 (LSD1)/nucleosome remodeling and histone deacetylase (NuRD) complex, most notably LSD1 and histone deacetylase 2 (HDAC2), were found to be associated with Prox1 and GST pulldown assay demonstrated that Prox1 directly interacts with LSD1. Sequential chromatin immunoprecipitation (ChIP) assays showed that Prox1 co-localizes with HNF4α, LSD1 and HDAC2 on CYP7A1 promoter in HepG2 cells. Furthermore, by using ChIP assay on HepG2 cells with endogenous Prox1 knocked down by RNA interference, Prox1 was shown to recruit LSD1 and HDAC2 onto CYP7A1 promoter and cause increased H3K4 demethylation. Finally, bile acids treatment of HepG2 cells, which significantly repressed CYP7A1 transcription, resulted in increased Prox1 and LSD1/NuRD complex occupancy on CYP7A1 promoter with a concurrent increase in H3K4 demethylation and H3/H4 deacetylation. These results showed that Prox1 interacts with LSD1 to recruit the repressive LSD1/NuRD complex to CYP7A1 promoter and co-represses transcription through epigenetic mechanisms. In addition, such Prox1-mediated epigenetic repression is involved in the physiologically essential negative feedback inhibition of CYP7A1 transcription by bile acids.