The human glucocorticoid receptor promoter upstream sequences contain binding sites for the ubiquitous transcription factor, Yin Yang 1

Studies on the human glucocorticoid receptor (GR) promoter were carried out so as to understand the regulation of GR expression. A -2738 to +19 fragme nt of the human GR promoter was used to identify important regulatory eleme nts involved in the control in GR expression in NIH 3T3 cells (mouse fibrob lasts) and HeLa cells (human cervical carcinoma cells). Important regulator y domains in the distal region of the human GR promoter were identified by sequential 5' end deletion analysis. A region between -2490 and -2025 contr ibuted 50% of the measured transcriptional activity to the promoter. Using DNase I footprint analysis, four sites in this region were identified: -236 2 to -2339 (mouse footprint, mFP); -2301 to -2293 (distal YY1, dYY1); -2130 to -2122 (middle YY1, mYY1); and, -2086 to -2078 (proximal YY1, pYY1). Thr ee sites contained an identical core sequence, CCAAGATGG and were identifie d as Yin Yang 1 (YY1) binding sites. The site located at -2362 to -2339 was footprinted in NIH 3T3 cells only. The sequence of this site is a direct r epeat with a 2-nucleotide spacer region, and it does not share homology wit h any known transcription factor binding sites. Computer analysis of the en tire promoter sequence revealed an additional YY1 site located at -260 to - 249 (initiator YY1, iYY1) with the sequence CTCCTCCATTTTG. Electrophoretic mobility supershift assays, with an anti-YY1 antibody, were used to confirm YY1 binding to these four putative YY1 binding sites. Site-directed deleti on of all three upstream YY1 sites but not the iYY1 site, or the iYY1 site alone, showed a similar to 60% decrease in transcriptional activity of the hGR promoter in HeLa cells but had no effect in NIH 3T3 cells. A similar (5 0%) decrease in the expression of a full-length hGR/luciferase reporter gen e was obtained when HeLa cells were cotransfected with a full-length antise nse YY1 expression plasmid. Additionally, a region between -1841 and -1689 contributed to hGR promoter activity in both cell types tested. An Sp1 bind ing site was identified in this region (-1748 to -1733) by DNase I footprin t acid mobility supershift analyses. The presence of four YY1 binding sites in the human GR promoter suggests that these sites play a critical role in GR gene regulation, (C) 1999 Elsevier Science Ltd. All rights reserved.