p300 Alters Keratinocyte Cell Growth and Differentiation Waf1/CIP1 through Regulation of p21 Ping-Pui Wong, Adam Pickard, Dennis J. McCance* Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, United Kingdom Abstract Background: p300 functions as a transcriptional co-activator to regulate many cellular responses such as cell growth, transformation, development and differentiation. It has been shown to affect the transcriptional activity of p53 which regulates p21Waf1/CIP1 expression, however, the role of p300 in differentiation remains unclear. Methodology and Principal Findings: Knockdown of p300 protein with short hairpin RNA (shRNA) molecules delays human neonatal foreskin keratinocyte (HFKs) differentiation. Moreover, depletion of p300 increases the proliferative capacity of HFKs, extends the life span of cells and allows differentiated HFKs to re-enter the cell cycle. Studies indicate that depletion of p300 down-regulates the acetylation and expression of p53, and chromatin immunoprecipitation (ChIP) analysis shows that induction of p21Waf1/CIP1 in early differentiation is a result of p300 dependent activation of p53 and that depletion of p21Waf1/CIP1 results in the delay of differentiation and a phenotype similar to p300 depletion. Conclusions: p300 has a direct role in the control of cell growth and differentiation in primary epithelial cells, and p21Waf1/CIP1 is an important mediator of these p300 functions. Citation: Wong P-P, Pickard A, McCance DJ (2010) p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21Waf1/CIP1. PLoS ONE 5(1): e8369. doi:10.1371/journal.pone.0008369 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received September 1, 2009; Accepted November 20, 2009; Published January 13, 2010 Copyright: 2010 wong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Medical Research Council (MRC) grant G0700754, URL: http://www.mrc.ac.uk/index.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: d.mccance@qub.ac.uk Introduction p21Waf’1/CIP1 is a member of the Cip/Kip family of cyclin dependent kinase inhibitors that bind to and inhibit the cyclin dependent kinases, cyclinE/cdk2 and cyclinD/cdk4, which in turn prevents the phosphorylation of retinoblastoma protein (Rb) in order to induce cell cycle arrest, cell differentiation or senescence [1–5]. The family members (p21Waf’1/CIP1,p27Kip1 and p57Kip2) shares a high degree of sequence homology in their N-terminal domain, which allows them to recognize a wide range of cyclin/cdk targets [6]. In contrast, the C-terminal domain of p21Waf’1/CIP1, which can bind and inhibit the DNA replication function of proliferating nuclear antigen (PCNA) is unique [7,8]. Studies of p21Waf1/CIP1 mouse keratinocytes indicated that the induction of p21Waf1/CIP1 in early differentiation is required for initial commitment of keratinocytes to differentiate [9,10]. However, p21Waf1/CIP1 expression has to be down-regulated at a later stage of differentiation, as sustained over-expression of p21Waf1/CIP1 inhibits keratinocyte terminal differentiation independently of the cell cycle [11]. A tight regulation of p21Waf1/CIP1 expression is therefore required for the process of keratinocyte differentiation [12,13]. However, the mechanism of control of p21Waf1/CIP1 expression during early differentiation is unclear. One regulator is p300, which has been shown to modulate p21Waf1/CIP1 promoter activity during mouse keratinocyte differentiation [14] and has also been shown to regulate muscle cell differentiation in a MyoD dependent pathway [15,16]. p300 is a histone acetyltransferase which functions as a modulator of chromatin structure [17,18]. During gene transcription, it acetylates the N-terminal tails of the core histone to destabilize nucleosomes, thereby facilitating the binding of transcription factors to DNA [19]. p300 also functions as a transcriptional co-activator and is recruited to promoter regions via direct interaction with various transcription factors [20]. It has been shown to acetylate and regulate the transcriptional activity of p53 and p63 [21–23], both of which are upstream regulators of p21Waf1/CIP1 [24,25]. However, the mecha- nism by which p300 acts to affect cell growth and differentiation of normal human epithelial cells has not been elucidated. We report here that the induction of p21Waf1/CIP1 expression in early dif- ferentiation is regulated by p53 with p300 involved in p53 activation of p21Waf1/CIP1. Knockdown of endogenous p300 by shRNA causes a decrease in expression of differentiation markers of HFKs in organotypic raft culture. It also increases the proliferative capacity of HFKs and allows differentiated cells to re-enter cell cycle, which is also observed in p21Waf1/CIP1 deficient cells. Moreover, exogenous expression of p21Waf1CIP1 rescues the expression of differentiation marker in p300 depleted cells. Taken together, our results indicate that p300 has a direct role in the control of cell growth and differentiation in primary epithelial cells and that p21Waf1/CIP1 is an important mediator of these p300 functions. Results Knockdown of p300 Inhibits Early Keratinocyte Differentiation To study the role of p300 in HFK differentiation, we knocked down endogenous p300 by using two retrovirally expressed PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8369