Transcription factor specificity protein 1 (SP1) and activating protein 2l (AP-2l) regulate expression of human KCTD10 gene by binding to proximal region of promoter

摘要

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase t, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2l could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCRand western blot analysis, both overexpression and RNA interference of SP1 and AP-2l indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2l to this region showed opposite effects.