vector as a negative control and the cells induced to differentiate for 48 hours. Western blotting indicates that exogenous expression of p21Waf1/CIP1 restored the expression of K1 in p300 depleted cells (Figure 5F) and reduced proliferation to the level in control cells (Figure S4). Taken together, our results indicate that p300 may regulate keratinocyte cell growth and differentiation through regulation of p21Waf1/CIP1. Discussion We have shown that p300 is an important component for human keratinocyte differentiation and that the ability to regulate p21Waf1/CIP1 during the early phase of keratinocyte differentiation is one of the key roles. Depletion of p300 or p21Waf1/CIP1 has similar phenotypes with increased proliferation and reduced expression of differentiation markers. In cells depleted of p300, the level of p53 protein falls significantly, suggesting that p300 is required for p53 stability, since mRNA levels are the same as in control cells. This reduction in p53 protein levels was a consistent finding in primary keratinocytes depleted of p300. In cells depleted of CBP, levels of p53 remained similar to control cells, indicating that the effect on p53 levels was p300 specific. In depleted p300 cells, p21Waf1/CIP1 levels are also greatly reduced suggesting that p53 is responsible for the increase in p21Waf1/CIP1 observed early in differentiation. While previous work had shown that p300 plays a key role in C2C12 muscle cell proliferation and differentiation [26], no knockdown studies were carried out although surprisingly inhibiting p300 acetyltransferase activity did not have an effect on p21Waf1/CIP1 levels. In another study, micro-injection of p300 antibody inhibited C2C12 cell differentiation [15]. Since p300 has been shown to regulate G1/S phase transition [27], we asked whether a defect in cycle arrest due to p300 depletion could explain the observed inhibition of differentiation. BrdU incorpo- ration and replating experiments show that differentiated p300 knock-down cells were able to re-enter cell cycle while control cells could not. In addition, p300 depleted cells re-entered the cell cycle and proliferated when transferred from differentiation medium to growth medium. This suggests that p300 plays a critical role in differentiation associated cell cycle arrest. Similar findings were observed by Dotto et al. (1995), who showed that over-expression of p300 can overcome the negative effect of adenovirus E1A on p21Waf1/CIP1 promoter in differentiation. Since p300 is a transcriptional coactivator, it requires an interacting protein to bind the p21Waf1/CIP1 promoter [18]. We have shown that the p53 response elements of p21Waf1/CIP1 promoter were required for transcriptional activation during early differentiation and we also showed that knocking down p300 reduced the levels of p53 during differentiation. Since acetylation of p53 has been recently shown to regulate its stability and transcriptional activity [28,29], this suggests that acetylation of p53 by p300 may be important for its function in differentiation. However, since we did not show that the acetylation function of p300 is required for p21Waf1/CIP1 transcription and keratinocyte differentiation, the acetylation by p300 is yet to be resolved. However, our ChIP analysis result indicates that p300 interacts with both p53 response elements in differentiating cells, whereas this interaction was inhibited by stably knocking down p53. Also, knocking down p53 caused reduction in p21Waf1/CIP1 expression after calcium treatment. A similar finding was observed by Yugawa et al. (2007), who showed that inactivation of p53 by shRNA resulted in repression of keratinocyte differentiation marker and p21Waf1/CIP1 expression [30]. Taken together, these results suggest that p300 regulates p21Waf1/CIP1 promoter in a p53 dependent pathway. However, Missero et al. (1995) showed that induction of p21Waf1/CIP1 was p300 Role in Differentiation observed in p53 mouse keratinocyte cell during differentiation [14]. This may be explained by the chronic absence of p53 is somehow compensated for by other p53 family members, whereas acute depletion has a more immediate effect on p21Waf1/CIP1 levels. Moreover, we show that p63 has a dual role in keratinocyte growth and differentiation, partially via regulation of p21Waf1/CIP1 (unpublished data). This raises a possibility that p300 may regulate p63 transcriptional activity to control p21Waf1/CIP1 expression in keratinocyte differentiation [22]. In conclusion, our findings show that p300 and p21Waf1/CIP1 are required for differentiation of human keratinocytes and that p21Waf1/CIP1 is regulated by p53. Materials and Methods Cell Culture, Infections and Transfections Primary human foreskin keratinocytes (HFKs) were harvested from neonatal foreskins, cultured in low calcium in Epilife (Casade) and transduced with retrovirus produced in WNYX packaging cell line (ATCC) as previously described [31]. The retroviral vectors (scramble, shp300, and shp53) used in this study were purchased from Origene Technologies, U.S.A. For calcium induced differentiation, confluent monolayers of HFKs were induced to differentiate by withdrawal of growth factors and addition of 1.5 mM CaCl2. Differentiation of HFK cell lines in organotypic raft cultures was carried out as previously described for transduced lines [32]. The raft cultures were harvested, fixed in 4% PFA and then embedded in paraffin for subsequent sectioning and staining with haematoxylin and eosin (H&E). To label cells synthesizing DNA, cultures were pulsed with 10 mM bromodeox- yridine (BrdU) for 20 minutes prior to fixation. For the raft culture, 20 mM BrdU was added to the media 16 hours prior to harvest. For siRNA transfection, complexes are produced by combining 100 pmols of siRNA with 100 ml optimen (Invitrogen) followed by the addition of 3 ml FuGeneHD and incubated at room temperature for 15 minutes prior to addition to cells at a final concentration of 100 nM. Cells were incubated with siRNA/ FugeneHD complexes for 6 hours. The sequences of p21Waf1/CIP1 siRNA molecules were previously described [33,34]. siGENOME SMART pool p300 and CBP siRNA molecules were purchased from Dharmacon. Luciferase Assays HFKs were transiently transfected with the p21Waf1/Cip1 luciferase reporter constructs as previously described [14]. p21Waf1/Cip1 promoter deletion luciferase reporter constructs (0-luc to 4-luc) were kindly provided by Dr. Wafik S. El-Deiry [35]. All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3- basic. p21Waf1/Cip1-mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu [36]. pMT5-p21Waf1/CIP1-Flag over-expression vector was purchased from Addgene. All transfec- tions were performed with Fugene 6 and cells were harvested to 72 hours after transfection. Luciferase measurements were per- formed by using the dual-luciferase kit (Promega). Real-Time RT-PCR Analysis RNA extraction was carried out with High Pure RNA isolation kit (Roche) according to manufacturer’s instructions. RNA (1 mg) was treated with RQ1 RNAase free DNase (Promega) prior to first stand cDNA synthesis using random primers with transcriptor high fidelity cDNA synthesis Kit (Roche) according to manufacturers instructions. Amplification of PCR products was quantified using FastStart SYBR Green Master (Roche) according to manufacturers instruction and fluorescence montoried on a DNA Engine Peltier PLoS ONE | www.plosone.org 6 January 2010 | Volume 5 | Issue 1 | e8369