Abstract
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.
Highlights
CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins
To identify the proximal promoter region required for CD81 transcription, the upstream DNA sequence (1.63 kbp) of the CD81 gene (NG_023386.1 Reference Sequence Gene) and its deleted sequences were used for the construction of luciferase reporter plasmids (Fig. 1a and Supplementary Fig. S1)
We found two potential Pax5-binding sites, (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48), between − 130 bp and − 39 bp upstream of CD81-transcriptional start site (TSS) (Fig. 1c)
Summary
CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. 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. Electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax[5] bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax[5] is required for upregulation of B-cell lineage-specific transcripts such as Iga (CD79a, mb-1)[17], BLNK (SLP-65)[18], and CD1919. These transcripts are involved in pre-BCR signaling and the developmental transition from pro-B cells to pre-B cells
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