HNF-4 plays a pivotal role in the liver-specific transcription of thechipmunk HP-25 gene
HNF-4 plays a pivotal role in the liver-specific transcription of thechipmunk HP-25 gene
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DOI：
10.1046/j.1432-1327.2000.01499.x
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发表时间：
2000-08-01
期刊：
影响因子：
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通讯作者：
Shiba, T
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文献类型：
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作者：
Kojima, M;Takamatsu, N;Shiba, T
金花鼠冬眠特异性蛋白HP-25的基因在肝脏中特异性表达。为了了解HP-25基因表达的转录调节，我们分离了其基因组克隆，并对其结构组织和5'侧翼区进行了表征。该基因跨度约7 kb，由三个外显子组成。通过引物延伸分析确定的转录起始位点位于翻译起始密码子上游113 bp处。在HepG 2细胞中的瞬时转染研究表明，80 bp的5'侧翼序列足以使肝特异性启动子活性。在使用HepG 2核提取物的凝胶阻滞测定中，从-74至-46的5'侧翼序列显示出移动的条带。通过酵母单杂交系统分离的所有cDNA克隆都能与编码HNF-4的5'侧翼序列结合。体外合成的HNF-4在凝胶阻滞测定中结合到该序列。此外，用抗（HNF-4）IG进行的超移测定证实了HepG 2或花栗鼠肝细胞核提取物中与该序列结合的蛋白质是HNF-4。当转染到HeLa细胞中时，HNF-4反式激活HP-25基因启动子的转录，并且HNF-4结合位点的突变消除了HNF-4的反式激活，表明HNF-4在HP-25基因表达中起重要作用。
The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5' flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.