IDENTIFICATION OF A NOVEL CIS-ACTING ELEMENT PARTICIPATING IN MAXIMALINDUCTION OF THE HUMAN LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE-TRANSCRIPTION IN RESPONSE TO LOW CELLULAR CHOLESTEROL LEVELS

In this paper, we present both in vivo and in vitro evidence for the p resence 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 pr otection from -145 to -126, 5'-GAGCTTCACGGGTTAAAAAG-3' (referred to as FP1 site). Second, transient transfections of HepG2 cells with promot er luciferase reporter constructs containing the FP1 site resulted in significant enhancement (approximately 375%) of reporter gene expressi on in response to low levels of sterols compared with parallel plasmid without the FP1 site. In addition, this response was markedly attenua ted on nucleotide substitutions within the FP1 site. Third, by electro phoretic mobility shift assays, the FP1 sequence was found to bind pro tein(s) from HepG2 nuclear extracts in a sequence-specific manner. In vitro binding of the FP1 mutants paralleled the results obtained for t heir in vivo transcription. On the basis of competition profiles, the FP1-binding factor is different from the known transcription factors b inding to the AT-rich CArG and GArC motifs. Further more, the FP1-bind ing protein is not specific to HepG2 cells because nuclear factor(s) w ith the same specificity was observed in nuclear extracts of non-hepat ic HeLa cells. We conclude that transcriptional induction of the LDL r eceptor gene in response to sterol depletion is mediated, in part, by an highly conserved novel cis-acting element through the binding of sp ecific nuclear protein(s).