title: DNA elements and protein factors involved in the transcription of the beta 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBP alpha.
abstract: Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
authors.forename: L
authors.initials: L
authors.lastname: Jiang
authors.forename: B
authors.initials: B
authors.lastname: Gao
authors.forename: G
authors.initials: G
authors.lastname: Kunos
mesh_terms: D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006498:Hepatectomy; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*; D012260:Ribonucleases; D017421:Sequence Analysis; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D018343:Receptors, Adrenergic, beta-2; D014157:Transcription Factors; D011061:Poly A; D015500:Chloramphenicol O-Acetyltransferase; D012260:Ribonucleases