Protein binding to the low density lipoprotein receptor promoter in vivo is differentially affected by gene activation in primary human cells.

Abstract

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 beyond the transcription start sites was observed in primary cultures of human skin fibroblasts and hepatocytes. Hypermethylation of guanine -59 within the sterol regulatory element-1 (SRE-1, repeat 2) occurred within a 4.0 h incubation of fibroblasts with media containing lipoprotein-deficient serum (LPDS) and cholesterol synthesis inhibitors. Methylation 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 fibroblasts, neither cholesterol synthesis inhibitors nor oxysterols produced consistent effects on the DMS-reactivity of guanine -59 in hepatocytes 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 guainine -59 within the SRE-1 is positively associated with activation of LDL receptor gene transcription in skin fibroblasts. Furthermore, the absence of demonstrable changes in DMS-reactivity of other purines within this region suggests that the LDL receptor promoter is poised to activate transcription with only minimal changes of protein binding to the proximal promoter in vivo.