Abstract
TGF-β is a powerful pleotrophic cytokine that regulates numerous cellular processes through the modulation of gene expression. Well-characterized alterations in the TGF-β signaling pathway are frequently observed in pancreatic cancer. However new data suggests that transcriptional silencing of the type II TGF-β receptor may play a role. The mechanism underlying this transcriptional control remains elusive. Here we present data on a novel TGF-β-inducible mediated mechanism of transcriptional repression through the Sp/KLF family of transcription factors. Using a combination of bioinformatics and gene expression analysis we identified several Sp/KLF's that are inducible by TGF-β1 which have not been previously reported. Using cloned expression vectors and a type II TGF-β receptor luciferase reporter, we identified KLF14, a previously uncharacterized Sp/KLF transcription factor, as a novel TGF-β1 inducible repressor of the type II TGF-β receptor. KLF14 showed marked transcriptional repression of the TGFβRII promoter at a wide range of concentrations. Deletion and site-direct mutagenesis revealed that KLF14 represses the TGFβRII promoter through Sp1-like sites located on the proximal promoter. Moreover, KLF14 antagonizes Sp1 transcriptional activation by competition for this site to determine the relative transcriptional activity. Furthermore, KLF14 is able to repress the promoters of other downstream target genes of the TGF-β cascade, such as p21, in reporter assays. Using chromatin immunoprecipitation assays we confirmed that KLF14 binds to the endogenous type II TGF-β receptor promoter and this binding is enhanced by TGF-β1 in pancreatic cancer cells. Analysis of the transcription mechanism, show HDAC and mSin3a are recruited by KLF14 to this promoter. Further studies showed that KLF14 is able to induce specific chromatin modifications. Looking at the chromatin landscape we found an increase in methylation markers of repression and a decrease in acetylation markers of activation. In summary, KLF14 is a novel TGF-β-inducible mSin3a-dependent transcriptional repressor of the type II TGF-β receptor in pancreatic cancer that leads to repressive chromatin modifications consistent with gene silencing.
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