An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene
An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene
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DOI：
10.1074/jbc.m303921200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Chilton, BS
中科院分区：
文献类型：
--
作者：
Hewetson, A;Chilton, BS
类固醇调节兔RUSH/SMARCA 3的选择性剪接，一种SWI/SNF相关的转录因子。在2057 bp的基因组序列中评价反式激活。截断分析确定了一个最小的252 bp的区域，在瞬时转染试验中具有强的基础启动子活性。通过引物延伸分析确定5 '非翻译区（233 bp）的大小和转录起始位点。转录起始位点定位于具有下游启动子元件（+29）的无TATA区域中的共有起始子（Inr）元件。通过突变/缺失分析鉴定这些元件。Inr/下游启动子元件组合在人直系同源物的推定核心启动子（-35/+35）中是保守的，表明转录起始是类似保守的。两个Sp1位点也在人SMARCA 3的推定启动子中保守，一个RUSH结合位点（-616/-611）是兔启动子所特有的，抑制基础转录。这些网站进行了不同的凝胶位移和染色质免疫沉淀试验验证。近端启动子的分析表明，-162/+90区域是瞬时转染试验中孕酮反应所必需的。随后的突变/缺失分析显示孕激素受体半位点介导的孕酮诱导。一个重叠的Y-盒（在反向AT-TGG方向）抑制基础转录和甾醇诱导的转录激活的Sp1位点的存在下。孕酮受体和转录因子NF-Y结合的特异性通过凝胶迁移试验进行验证。染色质免疫沉淀试验证实，Y盒效应介导的DNA结合依赖的方式。这代表了一种独特的调节方案，其中孕酮受体的配体依赖性反式激活受到Sp1/NF-Y抑制。
Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5'-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse AT-TGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression.