Histone deacetylase 3 represses p15 INK4b and p21 WAF1/cip1 transcription by interacting with Sp1 q Weifeng Huang, Dapeng Tan, Xiuli Wang, Songyan Han, Jiang Tan, Yanmei Zhao, Jun Lu * , Baiqu Huang Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China Received 30 October 2005 Available online 10 November 2005 Abstract Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the over- expression of HDAC3 repressed transcription of p15 INK4b and p21 WAF1/cip1 genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunoprecipitated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15 INK4b and p21 WAF1/cip1 transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15 INK4b , but not that of p21 WAF1/cip1 , implicating the different roles of HDAC3 in repression of p15 INK4b and p21 WAF1/cip1 transcription. Data from this study indicate that the inhibition of p15 INK4b and p21 WAF1/cip1 may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis. Ó 2005 Elsevier Inc. All rights reserved. Keywords: HDAC3; Sp1; Repression; p15 INK4b ; p21 WAF1/cip1 ; Cell cycle Introduction Both p15 INK4b and p21 WAF1/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 biolog- ical 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 epige- netic control of gene expression [3]. Recent evidence impli- cated 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 p21 WAF1/cip1 or p15 INK4b [4,5]. These studies highlight the importance of the HDACs in control of p15 INK4b and p21 WAF1/cip1 transcription in normal cell cycle regulation. The mammalian HDACs so far identified fall into three classes based on their structural and biochemical character- istics [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 involve- ment of HDAC3 in these processes have not been fully elucidated. In this study, we investigated the functional effect of HDAC3 on p15 INK4b and p21 WAF1/cip1 transcriptional reg- ulation. We show that HDAC3 was recruited to p15 INK4b and p21 WAF1/cip1 promoter regions to repress p15 INK4b and p21 WAF1/cip1 transcription, and this repression was 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.010 q 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. * Corresponding author. E-mail addresses: ycsuo@nenu.edu.cn, luj809@nenu.edu.cn (J. Lu). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 339 (2006) 165–171 BBRC