Transcriptional regulation of the TATA-less NADPH cytochrome P-450 oxidoreductase gene
Transcriptional regulation of the TATA-less NADPH cytochrome P-450 oxidoreductase gene
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DOI：
10.1006/abbi.1996.0253
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发表时间：
1996-06-15
影响因子：
3.9
通讯作者：
Kasper, CB
中科院分区：
文献类型：
--
作者：
OLeary, KA;McQuiddy, P;Kasper, CB
使用与氯霉素乙酰转移酶基因相关的启动子缺失构建子，在瞬时转染试验中鉴定了调节大鼠肝脏nadph -细胞色素P-450氧化还原酶基因表达的多个顺式作用DNA序列。无tata启动子具有9个cc -box，其中包含转录因子Spl的一致序列。虽然7个远端cc -box的缺失对转录活性的影响很小，但从-206到-172的下一个35 bp的缺失导致启动子活性损失高达90%。该区域包含一个Spl结合位点，表明：(1)这个特定的一致序列对于转录是必需的，(2)两个近端GC盒协同作用，或者(3)在这个35-bp的片段中存在一个尚未确定的调控元件。此外，转染实验表明，两个独立的远端区域（-622至-1167和-1500至-2300）含有负调控元件，它们以位置无关的方式下调基因转录。迁移转移分析和dna酶足迹鉴定了启动子近端区域的序列，这些序列结合了核提取物中存在的蛋白质。在转录起始位点上游的前100 bp内观察到4个受保护的片段；这些包括(1)包含转录起始位点（-7至+4）的区域，(2)通常由TATA-box占据的区域（-38至-18），(3)包含调控元件CACC的-78至-60碱基，以及(4)包含Spl结合位点的-105至-92碱基。因此，nadph -细胞色素P-450氧化还原酶基因的调控受到正调控元件和负调控元件的控制，并且，在9个Spl共识位点中，两个近端位点足以支持基础转录。(C) 1996学术出版社，Inc.
Multiple cis-acting DNA sequences regulating expression of the rat liver NADPH-cytochrome P-450 oxidoreductase gene have been identified in transient transfection assays using promoter deletion constructs linked to the chloramphenicol acetyl transferase gene. The TATA-less promoter possesses nine CC-boxes which contain the consensus sequence for the transcription factor Spl. While loss of the seven distal CC-boxes had minimal effect on transcriptional activity, deletion of the next 35 bp, from -206 to -172, resulted in similar to 90% loss of promoter activity. Contained within this region is an Spl binding site indicating that either (1) this particular consensus sequence was essential for transcription, (2) the two proximal GC boxes act in concert, or (3) a yet unidentified regulatory element resides within this 35-bp stretch. In addition, transfection experiments demonstrated that two separate distal regions (-622 to -1167 and -1500 to -2300) contain negative regulatory elements which down-regulate gene transcription in a position-independent manner. Mobility-shift analyses and DNase footprinting identified sequences in the proximal region of the promoter that bound proteins present in nuclear extracts. Four protected segments were observed within the first 100 bp upstream of the transcription start site; these include (1) the region encompassing the transcription start site (-7 to +4), (2) the region normally occupied by a TATA-box (-38 to -18), (3) the bases from -78 to -60 which contain the regulatory element CACC, and (4) bases -105 to -92 which include an Spl binding site. Hence, regulation of the NADPH-cytochrome P-450 oxidoreductase gene is controlled by both positive and negative regulatory elements, and, of the nine Spl consensus sites, the two proximal sites are sufficient to support basal transcription. (C) 1996 Academic Press, Inc.