Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXR alpha gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXR alpha promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXR alpha promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 alpha (HNF-4 alpha) in this area. Overexpression of HNF-4 alpha in HER 293T cells increased the expression of all LXRa promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4 alpha in HepG2 cells was associated with reduced LXR alpha protein levels and reduced activity of the -111/+384 LXR alpha promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4 alpha specific binding motif (H4-SBM) in the -50 to -40 region of the human LXR alpha promoter. A triple mutation in this H4-SBM abolished HNF-4 alpha binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4 alpha. In conclusion, our data indicate that HNF-4 alpha may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXR alpha in hepatic cells. (C) 2015 Elsevier Inc. All rights reserved.