Hosokawa Kohei, Ishimaru Hanako, Watanabe Tadashi, Fujimuro Masahiro
Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto-shi, Kyoto, 607-8412, Japan.
Department of Virology, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan.
Sci Rep. 2021 Nov 25;11(1):22919. doi: 10.1038/s41598-021-02082-9.
CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between - 130 and - 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between - 130 and - 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.
CD81是四跨膜蛋白家族的一种整合膜蛋白，可与多种其他细胞表面膜蛋白形成复合物。CD81参与细胞迁移和B细胞活化。然而，CD81基因的转录调控机制仍不清楚。在此，我们发现CD81转录激活是转录因子Pax5结合在CD81基因转录起始位点（TSS）上游的Pax5结合序列（-54）GCGGGAC（-48）所必需的。报告基因检测表明，CD81基因TSS上游-130至-39 bp之间的DNA序列具有CD81转录的启动子活性。CD81基因TSS上游-130至-39 bp之间的DNA序列含有两个潜在的Pax5结合序列（-87）GCGTGAG（-81）和（-54）GCGGGAC（-48）。报告基因、电泳迁移率变动和染色质免疫沉淀（ChIP）检测表明，Pax5与CD81基因启动子区域的（-54）GCGGGAC（-48）结合以激活CD81转录。Pax5过表达增加了CD81蛋白的表达水平，而shRNA介导的Pax5敲低则降低了CD81的表达。此外，我们发现CD81的表达水平与人类肿瘤细胞系中Pax5的表达呈正相关。由于据报道CD81参与细胞迁移，我们通过伤口愈合和Transwell检测评估了Pax5过表达的影响。数据显示，Pax5或CD81的过表达均促进上皮细胞迁移。因此，我们的研究结果为通过转录因子Pax5了解CD81基因的转录机制提供了见解。
