Biochemical and Biophysical Research Communications
Histone deacetylase 3 represses p15INK4b and p21WAF1/cip1 transcription by interacting with Sp1☆
Introduction
Both p15INK4b and p21WAF1/cip1 are the cyclin kinase inhibitors (CKIs), which can bind to and inhibit the cyclin-dependent kinases (CDKs) [1]. They play significant roles in checkpoint transition of cell cycle progression and cellular differentiation, and therefore subject to different levels of regulation in different cellular settings and biological phenomena [2]. Both histone acetylases (HATs) and histone deacetylases (HDACs) are the key enzymes that catalyze the reversible acetylation/deacetylation of core histone tails, which is an essential mechanism of the epigenetic control of gene expression [3]. Recent evidence implicated that some histone deacetylase inhibitors may arrest human tumor cells at the G1 phase of the cell cycle by increasing the mRNA and protein levels of p21WAF1/cip1 or p15INK4b[4], [5]. These studies highlight the importance of the HDACs in control of p15INK4b and p21WAF1/cip1 transcription in normal cell cycle regulation.
The mammalian HDACs so far identified fall into three classes based on their structural and biochemical characteristics [6]. The most extensively studied HDACs are those of the mammalian Class I HDAC enzymes that are highly homologous to yeast Rpd3, comprising HDAC1, HDAC2, HDAC3, HDAC8, and HDAC11. Although there is clear evidence for the role of HDAC3 in the regulation of cell cycle progression [7], the molecular mechanisms of the involvement of HDAC3 in these processes have not been fully elucidated.
In this study, we investigated the functional effect of HDAC3 on p15INK4b and p21WAF1/cip1 transcriptional regulation. We show that HDAC3 was recruited to p15INK4b and p21WAF1/cip1 promoter regions to repress p15INK4b and p21WAF1/cip1 transcription, and this repression was mediated, at least in part, through the interaction of HDAC3 with Sp1, a transcription factor of p15INK4b and p21WAF1/cip1 promoters. Also, knockdown of HDAC3 expression by siRNA targeting HDAC3 up-regulated the transcriptional expression of p15INK4b, but not that of p21WAF1/cip1. Data presented in this report implicate that the down-regulation of p15INK4b and p21WAF1/cip1 may contribute to the potential of HDAC3 to promote cell proliferation and oncogenesis.
Section snippets
Materials and methods
Plasmids and cell culture. Plasmids of CMV-Sp1 and pSG4+Sp1 were kindly provided by Dr. Robert Tjian (University of California at Berkeley). Plasmids of PCMx-GAL4DBD, PMH100-TK-luc, p15(−2500/+160), p15(−1040/+160), p15(−750/+160), p15-luc(−113/+160), and p21-luc(−297/+8) were described previously [8], [9], [10]. The plasmids of FLAG-tagged HDAC1 and HDAC3 were gifts from Dr. Wolfgang Fischle (Gladstone institute of Virology and Immunology). The plasmid of p21-luc(−2400/+11) was a gift from Dr.
HDAC3 inhibited the p15INK4b and p21WAF1/cip1 promoter activity
It has been implicated that HDACs contribute to the regulation of p15INK4b and p21WAF1/cip1 transcription. We used both p15INK4b (−2500/+160) and p21WAF1/cip1 (−2400/+11) reporter constructs to investigate the effect of HDAC1/3 on these promoters. The results of luciferase activity analyses indicated that HDAC3 repressed both p15INK4b (−2500/+160) and p21WAF1/cip1 (−2400/+11) promoter-driven luciferase activities by 46% and 56%, respectively (Fig. 1A). Meanwhile, HDAC1 significantly inhibited
Discussion
Previous studies demonstrated that histone deacetylase inhibitors could induce the arrest of cells at the G1/G2 phase, implicating the specific roles of HDACs in control of cell cycle progression and cell differentiation [16]. One of the important molecular bases underlying these effects is the induction and activation of CKI proteins such as p15INK4b and p21WAF1/cip1. HDAC3 has been demonstrated to contribute to the control of cell cycle. In this report, we showed that overexpression of HDAC3
Acknowledgments
We thank Drs. Robert Tjian (University of California at Berkeley), Ronald M. Evans (The Salk Institute for Biological Studies), Martin Eilers (Institute of Molecular Biology and Tumor Research, Marburg, Germany), Keji Zhao (National Institutes of Health, Bethesda, MD), Wolfgang Fischle (Gladstone Institute of Virology and Immunology), and Bert Vogelstein (The Johns Hopkins University School of Medicine at Maryland) for providing plasmid constructs. This work was supported by grants from The
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Abbreviations: HDAC, histone deacetylase; HAT, histone acetylases; CKI, cyclin kinase inhibitor; CDKs, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; Co-IP, co-immunoprecipitation; RNAi, RNA interference.