PROTEIN-BINDING TO THE LOW-DENSITY-LIPOPROTEIN RECEPTOR PROMOTER IN-VIVO IS DIFFERENTIALLY AFFECTED BY GENE ACTIVATION IN PRIMARY HUMAN-CELLS

Protein-DNA interactions within a region of the LDL receptor promoter involved in sterol-mediated feedback repression of transcription were examined using in vivo genomic footprinting with dimethylsulfate (DMS) . A broad region of protein-DNA contacts spanning from repeat 1 to bey ond the transcription start sites was observed in primary cultures of human skin fibroblasts and hepatocytes. Hypermethylation of guanine -5 9 within the sterol regulatory element-1 (SRE-1, repeat 2) occurred wi thin a 4.0 h incubation of fibroblasts with media containing lipoprote in-deficient serum (LPDS) and cholesterol synthesis inhibitors. Methyl ation of this residue was reduced to control levels within 2.0 h after the addition of a mixture of 25-hydroxycholesterol and mevalonic acid . The time-dependent changes in DMS-reactivity of guanine -59 induced by the cholesterol synthesis inhibitors or oxysterols were paralleled by alterations in LDL receptor mRNA. In contrast to the results with f ibroblasts, neither cholesterol synthesis inhibitors nor oxysterols pr oduced consistent effects on the DMS-reactivity of guanine -59 in hepa tocytes despite induction or repression of LDL receptor mRNA in these cells. Interestingly, no other changes in the protection pattern over repeats 1, 2, and 3 were apparent in either fibroblasts or hepatocytes . These results demonstrate that hypermethylation of guanine -59 withi n the SRE-1 is positively associated with activation of LDL receptor g ene transcription in skin fibroblasts. Furthermore, the absence of dem onstrable changes in DMS-reactivity of other purines within this regio n suggests that the LDL receptor promoter is poised to activate transc ription with only minimal changes of protein binding to the proximal p romoter in vivo.,