Epigenetic regulation of human alpha1d-adrenergic receptor gene expression: a role for DNA methylation in Sp1-dependent regulation.
A growing body of evidence implicates alpha1-adrenergic receptors (alpha1ARs) as potent regulators of growth pathways. The three alpha1AR subtypes (alpha1aAR, alpha1bAR, alpha1dAR) display highly restricted tissue expression that undergoes subtype switching with many pathological stimuli, the mechanistic basis of which remains unknown. To gain insight into transcriptional pathways governing cell-specific regulation of the human alpha1dAR subtype, we cloned and characterized the alpha1dAR promoter region in two human cellular models that display disparate levels of endogenous alpha1dAR expression (SK-N-MC and DU145). Results reveal that alpha1dAR basal expression is regulated by Sp1-dependent binding of two promoter-proximal GC boxes, the mutation of which attenuates alpha1dAR promoter activity 10-fold. Mechanistically, chromatin immunoprecipitation data demonstrate that Sp1 binding correlates with expression of the endogenous gene in vivo, correlating highly with alpha1dAR promoter methylation-dependent silencing of both episomally expressed reporter constructs and the endogenous gene. Further, analysis of methylation status of proximal GC boxes using sodium bisulfite sequencing reveals differential methylation of proximal GC boxes in the two cell lines examined. Together, the data support a mechanism of methylation-dependent disruption of Sp1 binding in a cell-specific manner resulting in repression of basal alpha1dAR expression.
Michelotti
Gregory A
GA
Department of Pharmacology/Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA. miche007@mc.duke.edu
Brinkley
D Marshall
DM
Morris
Daniel P
DP
Smith
Michael P
MP
Louie
Raphael J
RJ
Schwinn
Debra A
DA
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors; D019175:DNA Methylation; D000077209:Decitabine; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D018340:Receptors, Adrenergic, alpha-1 / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013447:Sulfites / Q000494:pharmacology; D014158:Transcription, Genetic
C547719:ADRA1D protein, human; D002843:Chromatin; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D013447:Sulfites; D000077209:Decitabine; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine; C009279:sodium bisulfite