核受体辅活化子PNRC与孤儿核受体SF1相互作用位点的鉴定

为了阐明核受体辅活化子 (proline richnuclearreceptorcoactivatorprotein ,PNRC)在孤儿核受体类固醇生成因子 1(steroidogenicfactor1,SF1)基因表达调控中的作用 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法鉴定了PNRC与SF1的相互作用位点 .结果显示 ,PNRC中氨基酸 2 78～ 30 0区域是与SF1相互作用的位点 .该区域富含脯氨酸 ,其中有 1个SH3结合模体 (motif) ,单独的SH3模体不足以与SF1产生有效的相互作用 .瞬时转染分析表明 ,PNRC 2 70 32 7对野生型PNRC的辅激活功能具有负显性抑制效应 .研究结果表明 ,含SH3结合模体的PNRC 2 78 30 0区域是与SF1相互作用的位点     

Functional dissection of the ETS transcription factor MEF

We previously indicated that myeloid elf-1-like factor (MEF) but not elf-1, specifically activated lysozyme gene expression in epithelial cells. MEF is highly homologous at the nucleotide and amino acid level, with elf-1 especially in the ETS domain. Here, we report the functional analysis of the nuclear localization and transactivation properties of MEF. To investigate the intracellular localization of MEF, we transiently transfected MEF-green fluorescence protein (GFP) fusion protein expression vector into HeLa cells. A region spanning residues 177-291 is required for nuclear localization. We produced deletion mutants of MEF to determine the transactivation domain. The data showed that the N-terminal region, encompassing amino acids 1-52 is a potent transactivation domain. The C-terminal region spanning residues 477-663 can also mediate transactivation but not as strongly as the N-terminal region. The activity of the amino acid residues 1-52 was confirmed by experiments with fused constructs of MEF to the DNA binding-domain of the yeast GAL4 protein. These results, which determined the localization of the functional domains of MEF, will provide us with new clues to its transactivation mechanisms to regulate lysozyme gene expression in epithelial cells.