Dynamic Changes of Sp6 Transgene Expression in Dental Epithelial Cells During Long-term Culture

To investigate the function of specificity protein 6 (SP6) transcription factor by gain-of-function procedure, we established cytomegalovirus (CMV) promoter-driven Sp6 stable transformants, C9 cells, using dental epithelialderived cells. Initially, C9 cells produced a significant amount of SP6 protein. However, SP6 expression was reduced in these cells upon long-term culture. We could detect Sp6 transcripts in C9 cells by RT-PCR throughout the passages, although the CMV promoter is known to be epigenetically silenced. We recently found that SP6 was a short-lived protein that was degraded by a ubiquitin-independent proteasome pathway, although it is yet unclear how Sp6 expression was regulated during culture. Thus, we studied the possibility of epigenetic regulation of Sp6 expression. Comparative analysis of endogenous and exogenous Sp6 mRNA expressions demonstrated the specific down-regulation of exogenous Sp6 mRNA levels during culture passages. A DNA methyltransferase inhibitor, 5-Aza-2\u27-deoxycytidine (5AC), and a histone deacetylase inhibitor, valproic acid (VPA), enhanced or induced SP6 protein expression up to passage 28 without enhancing the mRNA level. The dramatic up-regulation of exogenous Sp6 mRNA was uniquely observed only at passage 50 by 5AC or VPA treatment. These findings indicate that multiple epigenetic regulatory mechanisms operate to fine-tune Sp6 expression during long-term culture

Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5\u27 ends of the Sp6 cDNA by 5\u27 RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions