The acetylation of transcription factor HBP 1 by p 300 / CBP enhances p 16 INK 4 A expression

HBP1 is a sequence-specific DNA-binding transcription factor with many important biological roles. It activates or represses the expression of some specific genes during cell growth and differentiation. Previous studies have exhibited that HBP1 binds to p16 promoter and activates p16 expression. We found that trichostatin A (TSA), an inhibitor of HDAC (histone deacetylase), induces p16 expression in an HBP1-dependent manner. This result was drawn from a transactivation experiment by measuring relative luciferase activities of p16 promoter with HBP1-binding site in comparison with that of the wild-type p16 promoter by transient cotransfection with HBP1 into HEK293T cells and 2BS cells. HBP1 acetylation after TSA treatment was confirmed by immunoprecipitation assay. Our data showed that HBP1 interacted with histone acetyltransferase p300 and CREB-binding protein (CBP) and also recruited p300/CBP to p16 promoter. HBP1 was acetylated by p300/CBP in two regions: repression domain (K297/305/307) and P domain (K171/419). Acetylation of Repression domain was not required for HBP1 transactivation on p16. However, luciferase assay and western blotting results indicate that acetylation of P domain, especially K419 acetylation is essential for HBP1 transactivation on p16. As assayed by SA-beta-gal staining, the acetylation of HBP1 at K419 enhanced HBP1-induced premature senescence in 2BS cells. In addition, HDAC4 repressed HBP1-induced premature senescence through permanently deacetylating HBP1. We conclude that our data suggest that HBP1 acetylation at K419 plays an important role in HBP1-induced p16 expression. INTRODUCTION HBP1 is homologous to the sequence-specific high mobility group (HMG) family of transcription factors (1,2). We have previously isolated HBP1 as a retinoblastoma (RB) partner and have determined that it functions as a proliferation regulator by inhibiting oncogenic pathways as a transcriptional repressor (3–8). Recently, the HBP1 transcriptional repressor has been reported as a putative substrate for the p38 MAPK in cell-cycle arrest (9). Mechanistically, p38 MAPK-mediated phosphorylation of the HBP1 leads to increased protein stability and G1 arrest. A specific p38 MAPK phosphorylation site (serine 401) has been identified in the HBP1 protein. Moreover, our previous work demonstrated that HBP1 is necessary for premature senescence induced by Ras-p38 MAPK (10). Furthermore, HBP1 itself also induces premature senescence through upregulating p16 expression in primary cells (18). p16 knockdown can abolish HBP1and Ras-induced premature senescence. Together, our previous data support a model in which HBP1 is a downstream effector of Ras, and p38 MAPK, and provide new insights into the signaling mechanisms that lead to premature senescence. All of the data indicate that HBP1 modulation is a complex process, and the biological consequences of HBP1 activation induced by certain stimuli may depend on HBP1 post-translational modification at multiple sites. There is no report about the roles of the acetylated HBP1. Investigating whether or not HBP1 is acetylated by acetyltransferase and if the acetylated HBP1 increases its DNA binding as well as downstream transcriptional activity are the objectives of this report. Histone deacetylase (HDAC) inhibitors have been extensively studied in basic biological research to gain an understanding of basic chromatin structure and transcriptional control and have recently been introduced as potential clinical treatment for cancer (11–15). Generally, HDAC inhibitors induce accumulation of hyperacetylated nucleosome core histones and cause transcriptional *To whom correspondence should be addressed. Tel: +8610 828