Influence Of Histone Acetyltransferase P300 On Regulation Of P16～(INK4a) Gene Expression And Its Molecular Mechanisms

The regulation of expression of p16^INK4a gene has attracted a great deal of research attention, because this gene encodes a cyclin-dependent kinase inhibitor that plays a key role in cell cycle progression and cellular differentiation by negatively regulating the CDK4/6 activity. There has been increasing evidence that the expression of p16^INK4a gene is epigenetically controlled and is closely related with many cancers. Although previous biochemical studies revealed that epigenetic mechanisms such as DNA methylation and histone modifications may be involved in transcription regulation of this gene, a detailed description of the molecular basis for the roles of histone acetylation in p16^INK4a regulation has not been reported. In this study, we demonstrate that p300 was able to induce cell cycle arrest, and this process was reversed by p16^INK4a silencing by small interference RNA (siRNA) in HeLa cells. We also showed that p300 was involved in activation of p16^INK4a expression in 293T cells. Specifically, p300 cooperated with Sp1 to stimulate both the p16^INK4a promoter activity and the mRNA expression. The siRNA-induced partial silencing of Sp1 and p300 resulted in a significant inhibition of the p16^INK4a promoter activity. Co-immunoprecipitation (CoIP) and mammalian two-hybrid assays revealed that p300 and Sp1 formed a complex through interaction between the Q domain of p300 and the N-terminal domain of Sp1. The results of p16^INK4a promoter truncation mutants and point mutations linked to a luciferase reporter gene assays indicated that the proximal Sp1-binding site of p16^INK4a promoter played very important roles in its transcription activation by p300 and Sp1. The chromatin immunoprecipitation (ChIP) assays verified that p300 was recruited to p16^INK4a promoter, and the intrinsic histone acetyltransferase (HAT) activity of p300 was essential for p16^INK4a promoter activation through inducing the hyperacetylation of histone H4 at the p16^INK4a gene. These data will contribute to elucidating the unique mechanisms of p16^INK4a transcriptional control, which is crucial to the development of new therapeutic strategies for p16^INK4a -related gene therapy.