Identification of a novel cis-acting element participating in maximal induction of the human low density lipoprotein receptor gene transcription in response to low cellular cholesterol levels

Abstract

In this paper, we present both in vivo and in vitro evidence for the presence of a novel cis-acting regulatory element that is required for maximal induction of the human low density lipoprotein (LDL) receptor gene following depletion of cellular sterols in HepG2 cells. First, in vivo dimethyl sulfate footprinting of the human LDL receptor promoter before and after transcriptional induction in HepG2 cells revealed protection from -145 to -126, 5'-GAGCTTCACGGGTTAAAAAG-3' (referred to as FP1 site). Second, transient transfections of HepG2 cells with promoter luciferase reporter constructs containing the FP1 site resulted in significant enhancement (approximately 375%) of reporter gene expression in response to low levels of sterols compared with parallel plasmid without the FP1 site. In addition, this response was markedly attenuated on nucleotide substitutions within the FP1 site. Third, by electrophoretic mobility shift assays, the FP1 sequence was found to bind protein(s) from HepG2 nuclear extracts in a sequence- specific manner. In vitro binding of the FP1 mutants paralleled the results obtained for their in vivo transcription. On the basis of competition profiles, the FP1-binding factor is different from the known transcription factors binding to the AT-rich CArG and GArC motifs. Furthermore, the FP1- binding protein is not specific to HepG2 cells because nuclear factor(s) with the same specificity was observed in nuclear extracts of non-hepatic HeLa cells. We conclude that transcriptional induction of the LDL receptor gene in response to sterol depletion is mediated, in part, by an highly conserved novel cis-acting element through the binding of specific nuclear protein(s).