title: Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α.
abstract: 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 LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα 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 LXRα 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 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α 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-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.
authors.affiliation: University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete, Greece.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Theofilatos
authors.affiliation: University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete, Greece.
authors.forename: Aristomenis
authors.initials: A
authors.lastname: Anestis
authors.affiliation: Department of Preemptive Medicine and Metabolism, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-city, Tokyo, 113-8510, Japan.
authors.forename: Koshi
authors.initials: K
authors.lastname: Hashimoto
authors.affiliation: University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, 71003, Crete, Greece. Electronic address: kardasis@imbb.forth.gr.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Kardassis
mesh_terms: D001665:Binding Sites; D005786:Gene Expression Regulation / Q000235:genetics*; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D051557:Hepatocyte Nuclear Factor 4; D000071518:Liver X Receptors; C000605213:NR1H3 protein, human; D057093:Orphan Nuclear Receptors
keywords: Gene regulation; HDL; HNF-4α; LXR; Oxysterols