Post genome study for mesangium-predominant functional genes

阐明megsin的转录调控有助于我们理解其组织特异性，并为细胞类型依赖性基因表达机制提供重要见解。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5 '-侧翼区4021 bp的序列和报告基因分析表明，该区域存在一个共有启动子片段，并在人肾小球系膜细胞中具有较强的启动子活性，此外，定点突变和缺失突变分析结合电泳迁移率变动分析鉴定了一个正调控基序，在-120至-112区域内的不完全激活蛋白-1（AP-1）结合基序（CTGATTCAC）。PDTC是AP-1的主要激活剂，激活了megsin启动子。相比之下，我们的研究与megsin启动子载体的缺失突变体表明，YB-1结合位点，这是以前被证明是一个主要的，细胞类型特异性的反式激活基质金属蛋白酶-2（MMP-2）的基因表达在肾小球系膜细胞，是不重要的megsin基因转录。这些结果表明，在megsin的5 '-侧翼区的顺式作用元件参与megsin的激活，AP- 1是megsin的一个很好的候选转录因子。然而，具有5'-侧翼区的报告基因构建体也诱导非系膜细胞中报告基因的温和表达，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。我们目前正在对megsin启动子上游区域以及内含子进行广泛的研究，以了解megsin的系膜优势表达机制。

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.