Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription
Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription
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DOI：
10.1042/0264-6021:3560571
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发表时间：
2001-06-01
影响因子：
4.1
通讯作者：
Fernandez, MP
中科院分区：
文献类型：
--
作者：
Carcedo, MT;Iglesias, JM;Fernandez, MP
人膜联蛋白A5是一种普遍存在的蛋白质，参与与细胞生长和分化相关的多种信号转导过程，并且其基因调控是该功能的重要组成部分。针对人膜联蛋白A5基因的宽5 '部分（相对于几个离散转录起始点的最5 '的bp-1275至+79）测定启动子转录活性。在HeLa细胞中进行的转染实验将bp-202至+79的片段鉴定为赋予最佳转录活性的最小启动子。在CpG岛的紧邻5 '侧翼区域中的两个典型Spl位点是显著转录所需的。在bp -717至-1153之间的远端启动子区域中的强抑制活性归因于与长末端重复47 B同源的内源性逆转录病毒长末端重复的存在。非翻译外显子1中从bp位置+31至+79的下游序列对于转录也是必需的，因为其从任何质粒构建体中的缺失都消除了转染测定中的活性。电泳迁移率变动分析，Southwestern印迹分析和亲和色谱法被用来确定一个蛋白质的相对分子质量为35 kDa，结合在外显子1的八核苷酸回文序列的双联体。DNA顺式元件类似于E盒，但不结合更高分子量的转录因子，如上游刺激因子或激活蛋白4。膜联蛋白A5基因转录的关键下游元件的发现，及其与潜在的新的转录因子pr复合物的相互作用，可能提供一个线索，了解转录的起始TATA少，多起始位点启动子。
Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function, Promoter transcriptional activity was determined for a wide 5 ' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5 ' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Spl sites in the immediate 5 ' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor pr complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.