Id-1 destabilizes HBX protein through the proteasome-mediated protein degradation pathway

1742 Background/Aims: Kruppel-like factor 4 (KLF4) is a transcription factor that controls the expression of several genes that are known markers of the terminal differentiation of colonic stem cells, such as intestinal alkaline phosphatase and mucin. Furthermore, KLF4 is a tumor suppressor in the gut, inhibiting the expression of genes controlling the cell cycle, such as Cyclin E and Cyclin D1, and simultaneously induces expression of the cell cycle inhibitor p21Cip1/WAF1. Since KLF4 interacts with p300 and recruitment of co-activators to the transcriptional apparatus is a common method transcription factors use to activate gene expression, we decided to study what effect KLF4/p300 interaction would have upon expression of known KLF4 target genes. Results: Via GST-labeled pull-down assays, we confirmed that KLF4 and p300 do indeed interact. We then made several luciferase reporter constructs containing the full-length reporter sequence of various KLF4 target genes and tested the ability of p300 to synergize with KLF4 in transactivating these reporters. With some of these reporter constructs, p300 further increased KLF4-mediated transactivation, suggesting a synergistic interaction. To identify the KLF4 binding site, we made a panel of deletion mutants for each promoter. These binding sites were then confirmed using chromatin immunoprecipitation using a novel KLF4 antibody developed in our laboratory. Since KLF4 appears to recruit p300, a histone acetyltransferase, we decided to investigate the effect of KLF4 binding to its binding element on chromatin remodeling. We tested this using chromatin immunoprecipitation against acetylated histones. Conclusions: We confirmed that KLF4 and p300 interact and found that this interaction results in a synergistic activation of several known KLF4 target genes. Furthermore, we were able to identify the binding element for KLF4 on these target genes. Finally, we found that KLF4 plays a role in chromatin remodeling via recruiting p300 to the promoter, suggesting that acetylation is important for KLF4-mediated transactivation.