Sp6 is a transcription factor of the SP/KLF family and an indispensable regulator of the morphological dynamics of ameloblast differentiation during tooth development. However, the underlying molecular mechanisms remain unclear. We have previously identified one of the Sp6 downstream genes,
Rock1, which is involved in ameloblast polarization. In this study, we investigated the transcriptional regulatory mechanisms of
Rock1 by Sp6. First, we identified the transcription start sites (TSS) and cloned the 5'-flanking region of
Rock1. Serial deletion analyses identified a critical region for
Rock1 promoter activity within the 249-bp upstream region of TSS, and chromatin immunoprecipitation assays revealed Sp6-binding to this region. Subsequent transient transfection experiments showed that
Rock1 promoter activity is enhanced by Sp6, but reduced by Sp1. Treatment of dental epithelial cells with the GC-selective DNA binding inhibitor, mithramycin A, affected
Rock1 promoter activity in loss of enhancement by Sp6, but not repression by Sp1. Further site-directed mutagenesis indicated that the region from -206 to -150 contains responsive elements for Sp6. Taken together, we conclude that Sp6 positively regulates
Rock1 transcription by direct binding to the
Rock1 promoter region from -206 to -150, which functionally distinct from Sp1. J. Med. Invest. 61: 306-317, August, 2014
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