YY1 binding within the human HSD3132 gene intron 1 is required for maximal basal promoter activity:: Identification of YY1 as the 3β1-A factor
YY1 binding within the human HSD3132 gene intron 1 is required for maximal basal promoter activity:: Identification of YY1 as the 3β1-A factor
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DOI：
10.1677/jme.0.0330099
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发表时间：
2004-08-01
影响因子：
3.5
通讯作者：
Reichardt, JK
中科院分区：
文献类型：
--
作者：
Foti, DN;Reichardt, JK
3 -羟基类固醇脱氢酶的几种同工异构体催化了3 -羟基类固醇的氧化和异构化为酮-4-烯类固醇，这是所有激素类固醇合成的关键步骤。在人类中，存在两种高度同源的亚型，I型由外周组织中的HSD3B1基因表达，II型由类固醇生成器官中的HSD3B2基因表达。先前的研究表明，HSD3B1基因3beta1-A元件包含两个基因之间不完全保守的内含子1的24个核苷酸，并与保守的TG盒重叠，通过结合普遍存在且未识别的3beta1-A转录因子，有助于最大的基础启动子活性。在这项研究中，我们首次报道了类似的情况，在报告基因分析中，HSD3B2基因内含子1是最大基础启动子活性所必需的，因为缺少内含子1会导致启动子活性降低4- 10倍。凝胶转移分析的突变分析表明，3beta1-A因子仅需要3beta1-A元件保守片段的7个核苷酸就能结合HSD3B2和HSD3B1基因内含子1。通过竞争分析和使用抗YY1抗体在凝胶移位和Western blot实验中，我们确定3beta1-A蛋白是普遍存在的转录因子YY1。此外，我们还发现了另一个类似的YY1结合位点，它位于两个基因中3beta1-A元件的不同位置。瞬时转染实验的缺失和突变分析显示，与先前对HSD3B1基因的研究相反，YY1与II型3beta1-A元件缺乏结合只会导致基础启动子活性的轻微降低。相反，YY1与位于3beta1-A元件下游35个位置的第二个位点结合，强烈激活HSD382基因基础启动子活性，因为阻止YY1与该区域结合导致基础转录减少50%。II型内含子1中YY1结合的完全取消导致基因报告活性与缺乏整个内含子1的报告结构相同。这些结果表明YY1是负责内含子1介导的HSD3B2基因基础启动子活性增强的因子。我们讨论了YY1在HSD3B1和HSD382基因内含子1内结合的异同，包括保守的内含子1 TG盒，这表明YY1介导的刺激这两个基因基础启动子活性的机制不同。
The oxidation and isomerization of 3beta-hydroxy-5-ene steroids into keto-4-ene steroids, a pivotal step in the synthesis of all hormonal steroids, is catalyzed by several isoforms of 3beta-hydroxysteroid dehydrogenase. In humans, two highly homologous isoforms exist, type I expressed by the HSD3B1 gene in peripheral tissues, and type II expressed by the HSD3B2 gene in steroidogenic organs. Previously, it was shown that the HSD3B1 gene 3beta1-A element, encompassing 24 nucleotides of intron 1 not perfectly conserved between the two genes and overlapping with a conserved TG box, contributes to maximal basal promoter activity by binding the ubiquitous and unidentified 3beta1-A transcription factor. In this study for the first time we report that similarly, the HSD3B2 gene intron 1 is required for maximal basal promoter activity in reporter gene analyses, as lack of intron 1 results in a 4- to 10-fold reduction in promoter activity. Mutational analysis in gel shift assays revealed that the 3beta1-A factor binds both the HSD3B2 and HSD3B1 gene intron 1 by requiring only seven nucleotides of a conserved segment within the 3beta1-A element. By competition analysis and use of anti-YY1 antibody in both gel shift and Western blot experiments, we identified the 3beta1-A protein as the ubiquitous transcription factor YY1. In addition, we have characterized another similar YY1 binding site differently located with respect to the 3beta1-A element in both genes. Deletion and mutational analysis in transient transfections experiments revealed that contrarily to as previously shown for the HSD3B1 gene, lack of YY1 binding to the type II 3beta1-A element only results in a marginal reduction of basal promoter activity. Instead, YY1 binding to the second site, placed 35 by downstream from the 3beta1-A element, strongly activates the HSD382 gene basal promoter activity, as preventing YY1 binding to this region caused a 50% decrease of basal transcription. Complete abrogation of YY1 binding within type II intron 1 resulted in a gene reporter activity identical to a reporter construct lacking the whole intron 1. These results designate YY1 as the factor responsible for the intron 1-mediated boost of the HSD3B2 gene basal promoter activity. Similarities and dissimilarities between YY1 binding within the HSD3B1 and HSD382 gene intron 1 are discussed involving the conserved intron 1 TG box, that suggests different mechanisms are implicated in the YY1-mediated stimulation of these two genes basal promoter activity.