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.
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.
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参与大鼠肝脏 β2-肾上腺素能受体基因转录的 DNA 元件和蛋白质因子。
DOI：
10.1021/bi960844o
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
George Kunos
中科院分区：
文献类型：
--
作者：
Liu Jiang;Bin Gao;George Kunos
大鼠β2肾上腺素能受体(β2AR)基因的引物延伸和核糖核酸酶保护分析确定了两个转录起始点，分别位于-和-220nt。将可能的启动子/PCAT片段瞬时导入DDT1MF-2细胞，发现-36~-100(PI)和-186~-312(P2)片段足以促进转录，而-911~-1122含有负调控元件(S)。3‘非翻译区(3’-UTR)的核糖核酸酶保护分析表明，存在两个转录本，其3‘-UTR111和604个核苷酸不包括聚(A+)尾部。使用全长和部分cDNA探针进行的Northern杂交表明，主要的2.2kb和次要的1.6kb的物种是由不同的启动子和不同的多聚腺苷信号产生的。使用大鼠肝核提取液的DNase I足迹和DNA迁移率改变分析(DMSA)确定了许多转录因子与P1和P2内或其上游的序列元件结合，包括Sp1、Cre、Cp1、AP-2、NF-1、NF-kappa B和C/EBP。用C/EBPα和C/EBPβ抗体进行的超移位分析和突变分析表明，与C/EBP共识识别位点-925到-933结合的蛋白质是C/EBPα。将C/EBPα、C/EBPα和C/EBPβ共转染入DDT1 MF-2细胞或大鼠原代肝细胞，可显著降低含有C/EBP识别位点的启动子/CAT的活性。肝部分切除后，残留肝组织中C/EBPα一过性降低，β2受体基因表达水平和转录速率升高。因此，通过C/EBPα的去抑制可能参与了再生大鼠肝脏中β2AR的上调。
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.