Hewetson Aveline, Chilton Beverly S
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
J Biol Chem. 2003 Oct 10;278(41):40177-85. doi: 10.1074/jbc.M303921200. Epub 2003 Jul 30.
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 ATTGG 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.
类固醇调节兔RUSH/SMARCA3（一种与SWI/SNF相关的转录因子）的可变剪接。在2057 bp的基因组序列中评估了反式激活。截短分析在瞬时转染试验中鉴定出一个具有强基础启动子活性的最小252 bp区域。通过引物延伸分析确定了5'-非翻译区的大小（233 bp）和转录起始位点。转录起始位点定位于一个无TATA区域中的共有起始子（Inr）元件，其下游有一个启动子元件（+29）。这些元件通过突变/缺失分析得到验证。Inr/下游启动子元件组合在人直系同源物的假定核心启动子（-35/+35）中是保守的，这表明转录起始同样是保守的。在人SMARCA3的假定启动子中也保守的两个Sp1位点以及兔启动子特有的一个RUSH结合位点（-616/-611）抑制基础转录。这些位点通过凝胶迁移和染色质免疫沉淀试验得到了不同程度的验证。近端启动子分析表明，在瞬时转染试验中，-162/+90区域是孕酮反应性所必需的。随后的突变/缺失分析揭示了孕酮受体半位点介导的孕酮诱导作用。一个重叠的Y盒（反向ATTGG方向）在存在Sp1位点的情况下抑制基础转录和孕酮诱导的转录激活。通过凝胶迁移试验验证了孕酮受体和转录因子NF-Y结合的特异性。染色质免疫沉淀试验证实Y盒效应是以DNA结合依赖的方式介导的。这代表了一种独特的调控模式，其中孕酮受体的配体依赖性反式激活受到Sp1/NF-Y的抑制。
