These data comprise the first report to provide that tuberin regulates OGG1 expression through the transcription factors AP4 in cultured cells and in rat kidney cortex. Several approaches were utilized to study the potential role of tuberin in the regulation of OGG1. First, we showed that decreased tuberin expression is associated with decreased AP4 and OGG1 expression in renal proximal tubular cells isolated from Eker rat as compared with wild-type cells. Second, decreased tuberin expression is associated also with decreased AP4 and OGG1 expression as well as significantly increased accumulation of 8-oxodG in kidney cortex of Eker compared with wild-type rat. In the kidney tumours of Eker rat, loss of tuberin is associated with loss of AP4 and OGG1 expression and an 8-fold increase in 8-oxodG compared with kidney tissue of wild-type rat. Third, we show that reduction of tuberin expression in human epithelial cells (293) using siRNA resulted in a marked decrease in the expression of OGG1 and AP4. Fourth, introduction of Tsc2 complementary DNA into tuberin-deficient cells restored OGG1 and AP4 expression. In addition, reduction of AP4 using siRNA results in significant decrease in OGG1 protein expression. Our data also show that AP4 is associated with tuberin in cells. We investigated the mechanism by which tuberin regulates OGG1 by gel shift analyses and ChIP and identified the transcription factor AP4 is a regulator of OGG1 promoter. Furthermore, we found that the DNA-binding activity of AP4 was significantly reduced in Tsc2−/− compared with Tsc2+/+ cells. In addition, the transcriptional activity of the OGG1 promoter was also decreased in Tsc2−/− cells. We find also that mutation(s) in AP4-binding region significantly inhibited OGG1 promoter activity suggesting that AP4 in an important enhancer of OGG1 promoter activity.
Tuberin expression in kidney from Eker rats is significantly reduced compared with expression in normal kidney tissues from wild-type littermates (37
). In the current study, we show that loss of tuberin was associated with abolished AP4 and OGG1 expression as well as accumulation of significant levels of 8-oxodG in the kidney tumours of Eker rats. Downregulation of tuberin using siRNA directed specific for the Tsc2
gene in 293 human renal epithelial cells was associated with decreased in AP4 and OGG1 expression. In addition, overexpression of tuberin by adenovirus (Ad
) shows an increased in AP4 and OGG1 expression, indicating that tuberin is upstream of AP4 and OGG1. The immunoprecipitation analysis data of AP4 show that tuberin co-precipitates with AP4 indicating that AP4 is associated with tuberin in cells.
AP4 is an ubiquitously expressed transcription factor. It binds to the consensus sequence ‘CAGCTG’ (34
). AP4 recognizes specific binding sites in the promoter as well as enhancer sequences of viral genes (34
). The enhancer activity is abolished by mutations in these regions that also affect binding of AP4 in vitro
). AP4 has recently been identified as a transcriptional repressor of HIV-1 (35
). In the present study, we screened for enhancer-binding proteins to explore the molecular mechanisms that regulate OGG1
gene expression. We found that the OGG1
promoter contains a putative binding site for AP4 close to 3′ of the promoter region (−163/−168 nt).
Further investigation to test the role of tuberin in the regulation OGG1 by AP4 was performed by EMSA and ChIP analysis. Our data showed that AP4 binding was significantly lower in Tsc2−/− and Tsc2+/− cells compared with Tsc2+/+ cells. In addition, incubation of nuclear extracts with AP4-specific antibodies was competed the protein–DNA complex demonstrate that the Tsc2+/+ cells contain a protein, AP4, that binds specifically to a region in the OGG1 promoter. In addition, mutations of oligonucleotide (CAGaTG and aAGaTG) at −163/−168 nt region of AP4 show significant decrease in the protein–DNA complex. ChIP analysis further provided confirmation that Ap4 binds the OGG1 promoter at sites located at −163/−168 nt region. Collectively, the data indicate decreased binding of AP4 to the OGG1 promoter in tuberin-deficient cells. In addition, the mutations in consensus sequence of AP4 motif (CAGCTG to CAGATG) significantly inhibited OGG1 promoter activity strongly suggest that CAGCTG, at which AP4 is purported to bind to OGG1 promoter, is important for the OGG1 promoter activity.
In summary, these data indicate that tuberin regulates the OGG1 and this effect is at least partially through the transcription factor AP4. The transcription factor AP4 is an important regulator of the OGG1 promoter in the cell cultured and kidney tissue. Accumulation of significant levels of 8-oxodG in tumour kidney of Eker rat indicate that tuberin plays a significant role in protecting the cells from oxidative DNA damage. It is probably that loss of tuberin results in loss of AP4 and OGG1 expression and function in that may expose the cells to further genetic alterations and leading to accumulation of mismatched DNA base lesions, a form of genomic instability that if not repaired could accelerate further genetic alterations leading to the full-blown tumour phenotype in kidneys. These data indicate a novel role for tuberin in the regulation of DNA repair pathway through the transcription factor AP4 and provide a potential mechanism of Tsc2 and OGG1 in progression of kidney tumours.