(Figure 4A). Also, real-time RT-PCR confirms that depletion of p300 significantly reduces p21Waf1/CIP1 mRNA during keratino- cyte differentiation (Figure 4B), indicating that p300 is important for transcriptional regulation of p21Waf1/CIP1 during differentia- tion. Others have shown that the two p53 binding sites are important for p21Waf1/CIP1 transcription and we have found the same requirement in differentiating keratinocytes (Figure S3). Since p300 can bind and acetylate p53 to activate p21Waf1/CIP1 expression during DNA damage and p300 and p53 are required for p21Waf1/CIP1 transcription, we studied the ability of p300 to bind p53 response elements in differentiating cells by using chromatin immunoprecipitations. Keratinocyte DNA was PCR amplified with primers specific for sequences that flank the two p53 response elements (RE1 and RE2). As a control for non- specific binding during immunoprecipitation, cross-linked lysates were immunoprecipitated with mouse or rabbit monoclonal IgG antibodies. ChIP analysis revealed that p53 bound strongly to RE1 and RE2 when cells were confluent (t = 0) and persisted during differentiation (Figure 4C). Unlike p53, p300 bound to both RE1 and RE2 only during differentiation (Figure 4C). To confirm that p300 requires p53 to bind to the p21Waf1/CIP1 promoter, we stably knocked down the expression of p53 in HFK by using retroviral vector (shp53) and Western blot analysis confirmed that the level of p53 was significantly reduced (,80%) (Fig. 4D). Interestingly, knock-down of p53 also reduced the expression of p21Waf1/CIP1 during early differentiation (Fig. 4D). ChIP analysis indicated that depleting p53 inhibited p300 binding to both RE1 and RE2 during differentiation (Figure 4C). These results suggest that p300 Role in Differentiation induction of p21Waf1/CIP1 in early differentiation is regulated by p300 dependent activation of p53. p21Waf1/CIP1 Has a Critical Role in Early Keratinocyte Differentiation Earlier work suggests that p21Waf1/Cip1 has a dual role in mouse keratinocyte proliferation and differentiation [12]. To determine the role in HFKs, cells were transfected with two siRNA molecules targeting p21Waf1/CIP1 and Western blotting of whole cell lysates with p21Waf’1/CIP1 antibody shows that both siRNA molecules, sip21Waf’1/CIP1A and sip21Waf’1/CIP1B, were able to knockdown p21Waf’1/CIP1 protein levels efficiently (,90%) (Figure 5A). Transfected cells were induced to differentiate at indicated times and harvested for immunoblotting analysis, which showed that the protein level of K1 was significantly down-regulated in the p21Waf’1/CIP1 depleted cells (Figure 5B). Also, p21Waf1/CIP1 depleted cells were more proliferative than control cells as measured by BrdU uptake and a significant number of these cells remained BrdU positive 48 hours after differentiation induced by calcium treatment (Figure 5C). HFKs depleted of p21Waf1/CIP1 were used to generate organotypic raft cultures and harvested 5 days after lifting to the air-liquid interface. Depletion of p21Waf1/CIP1 disrupts the formation of granular and cornified layers in the epidermis with reduced K1 expression and increased proliferation in the basal layer, resulting in a thicker epithelium (Figure 5D). Depletion of p21Waf1/CIP1 increased the number of BrdU positive cells by 70% in the basal layer compared to that of the control Figure 4. p300 regulates p21Waf1/CIP1 expression at transcriptional level. Stably expressing p300 targeted shRNAs cells were transfected with p21Waf1/CIP1 promoter luciferase construct. Transfected cells were either kept in low calcium (0.5 mM) or high calcium (1.5 mM) for the indicated times prior to the termination of the experiment (72 hours after transfection). Doxorubicin (0.1 mg/ml) was used as a positive control in this experiment. (A) Depletion of p300 down-regulates p21Waf1/CIP1 promoter activity after 16 hours of calcium treatment (Mean +/2 SE, three independent biological replicates; asterisk (*) p,0.01 relative to relevant control, Student’s t test). (B) Real time quantification confirms that depletion of p300 inhibits the transcription of p21Waf1/CIP1 during differentiation (Mean +/2 SE, two independent biological replicated; asterisk (*) p,0.05 relative to relevant control, Student’s t test). (C) ChIP assay for p53 and p300 binding to two p53 response elements located in the p21Waf1/CIP1 promoter in HFKs induced to differentiate by addition of calcium. PCR analysis of DNA precipitated by a p300 or p53 antibodies indicates that p300 only interacts with both p53 response elements within the p21Waf1/CIP1 promoter after 6 hours calcium treatment. Left panel: p53 binding to the p53 respond elements (RE1 and RE2) increases over a 16 hour periods. Right panel: Depletion of p53 results absence of p53 and p300 at both p53 response sites. Mouse (IgGM) and Rabbit IgG (IgGR) are included as negative control. (D) Western blot of the proteins from the same extracts as in (C). doi:10.1371/journal.pone.0008369.g004 PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8369