Post genome study for mesangium-predominant functional genes

阐明巨噬蛋白的转录调控有助于我们了解其组织特异性，并为了解细胞型依赖基因表达的机制提供重要见解。我们通过引物延伸分析确定了megsin的转录起始位点，该位点位于起始密码子上游391 bp处。对megsin基因4021 bp长度的5’侧区域的序列分析和报告基因分析表明，该区域存在一个一致的启动子片段，并且在人系膜细胞中具有较强的启动子活性。此外，位点定向和缺失诱变分析结合电泳迁移率转移分析确定了一个正调控基序，即-120至-112区域内的不完全激活蛋白-1 （AP-1）结合基序（CTGATTCAC）。AP-1的显性激活因子PDTC激活了megsin启动子。相比之下，我们对巨噬蛋白启动子载体缺失突变体的研究表明，先前被认为是肾小球系膜细胞中基质金属蛋白酶-2 （MMP-2）基因表达的主要细胞类型特异性反激活因子的YB-1结合位点对巨噬蛋白基因转录并不重要。这些结果表明，这个位于巨噬蛋白5'侧区的顺式作用元件参与了巨噬蛋白的激活，并且AP- 1是巨噬蛋白转录因子的一个很好的候选。然而，具有5'侧翼区域的报告基因构建也诱导了非系膜细胞中报告基因的轻度表达，这表明细胞类型特异性转录调控并不仅仅位于该启动子区域。目前，我们正在对巨噬蛋白启动子更上游的区域以及内含子进行广泛的研究，以找出巨噬蛋白在系膜显性表达的机制。

Elucidation of the transcriptional regulation of megsin should help us to understand its tissue specificity and provide important insights into the mechanisms of cell-type dependent gene expression. We determined the transcriptional start site of megsin by primer extension analysis, and showed that the site lied 391 bp upstream from the start codon. The sequence and reporter analyses on 4021 bp-length 5'-flanking region of megsin gene demonstrated a consensus promoter segment within this region and a relatively strong promoter activity in human mesangial cells.Furthermore, site-directed and deletion mutagenesis analyses in combination with electrophoretic mobility shift assay identified one positive regulatory motif, an incomplete activator protein-1 (AP-1) binding motif (CTGATTCAC) within -120 to -112 region. PDTC, a dominant activator of AP-1, activated the megsin promoter. In contrast, our studies with deletion mutants of megsin promoter vectors showed that the YB-1 binding site, which was previously shown to be a major, cell type-specific transactivator of matrix metalloprotease-2 (MMP-2) gene expression in glomerular mesangial cells, is not important for megsin gene transcription. These results suggest that this cis-acting element in the 5'-flanking region of megsin is involved in activation of megsin, and that AP- 1 is a good candidate as a transcriptional factor of megsin.However, the reporter construct with the 5'-flanking region also induced a mild expression of a reporter gene in non-mesangial cells, suggesting that the cell type specific transcriptional regulation in not located solely in this promoter region. We are currently performing extensive investigation of more upstream of megsin promoter region as well as introns to find out the mechanism of mesangium-predominant expression of megsin.