P63 Maintains Keratinocyte Proliferative Capacity Through Regulation of Skp2–P130 Levels

Research Article 1635 p63 maintains keratinocyte proliferative capacity through regulation of Skp2–p130 levels

Simon S. McDade, Daksha Patel and Dennis J. McCance* Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen’s University Belfast, Belfast BT9 7BL, UK *Author for correspondence ([email protected])

Summary p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the a and b isoforms of p63 result from p53–p21-mediated accumulation of retinoblastoma (Rb) family member p130. Hypoproliferation in p63- depleted HFKs can be rescued by depletion of p53, p21CIP1 or p130. Furthermore, we identified the gene encoding S-phase kinase- associated protein 2 (Skp2), the recognition component of the SCFSkp2 E3 ubiquitin ligase, as a novel target of p63, potentially influencing p130 levels. Expression of Skp2 is maintained by p63 binding to a site in intron 2 and mRNA levels are downregulated in p63-depleted cells. Hypoproliferation in p63-depleted cells can be restored by re-expression of Skp2. Taken together, these results indicate that p63 plays a multifaceted role in maintaining proliferation in the mature regenerating epidermis, in addition to being required for differentiation.

Key words: Keratinocyte, Proliferation, p63

Introduction al., 1998) is DNp63a, which contains a sterile alpha motif (SAM The p53 family member p63 is crucial to the development of domain) and transcriptional inhibitory domain in its extended C- stratifying epithelia in human (Rinne et al., 2007), mouse (Mills et terminus, which can facilitate transcriptional repression in cis or al., 1999; Yang et al., 1999) and zebrafish (Lee and Kimelman, trans. Regulation of p63 levels is further complicated by post- 2002). p63–/– mice exhibit severe developmental defects in tissues transcriptional targeting by microRNAs such as mir-203 (McKenna

Journal of Cell Science derived from stratifying epithelia and die perinatally because of an et al., 2010; Yi et al., 2008) or by being targeted for degradation almost complete absence of mature epidermis (Mills et al., 1999; by E3 ligases such as ITCH and FBW7 (Galli et al., 2010; Rossi Yang et al., 1999). These phenotypes are mirrored in human et al., 2006). syndromes caused by mutations of the gene encoding p63 (TP63) Despite a large body of literature, the exact function of DNp63a (Rinne et al., 2007). in the post-developmental epidermis and whether this is influenced Unlike p53, p63 is rarely mutated in tumours (Irwin and Kaelin, by other p63 isoforms or, indeed, p53 and p73 remains unclear. In 2001); however, p63 expression is frequently increased in squamous the mature regenerating epidermis, depletion of all p63 isoforms in cell carcinomas (reviewed in Deyoung and Ellisen, 2007), where organotypic cultures results in hypoproliferation and loss of the DN isoforms of p63 are thought to oppose the activity and anti- differentiation (Truong et al., 2006), similar to defects observed in proliferative response of p53 family members. In addition, loss of knockout mice. Impaired proliferation can be rescued by expression of TA p63 isoforms has been shown in bladder simultaneous knockdown of p53; however, this is insufficient to carcinomas (Park et al., 2000) and recent data from TAp63-specific induce differentiation. In addition, DNp63a and p53 have been knockout mice indicates that TAp63 behaves as a tumour suppressor shown to inversely regulate target genes such as p21CIP1 (p21) in (Guo et al., 2009; Su et al., 2010), although the effects are dependent the context of DNA damage (Schavolt and Pietenpol, 2007). on tissue type and the expression of other p53 family members (Su Whether DNp63a-mediated inhibition of p53-induced target genes et al., 2010). Taken together, these data suggest that p63 might act is important for normal epidermal morphogenesis remains unclear, as a proto-oncogene or a tumour suppressor depending on the as the p53 knockout mouse develops normal skin (Donehower et cellular context, isoforms and other p53 family members expressed. al., 1992) and is only altered in its ability to undergo differentiation This is perhaps not surprising, given the complicated nature of as a protective mechanism following insult, such as UV-B treatment the TP63 gene, which encodes at least six isoforms (supplementary (Tron et al., 1998). material Fig. S1A), a complexity mirrored in the other p53 family In addition to maintaining proliferation by opposing p53 functions, members. The six p63 isoforms are a result of alternative promoter DNp63a has also been shown to inhibit notch signalling in the usage producing transactivation domain (TA) or DN N-termini epidermis and the subsequent activation of p21 (Nguyen et al., coupled with three C-terminal splice variants (a, b, g), generating 2006), which is required for cell cycle exit (Missero et al., 1995) isoforms with differing as well as overlapping functions (reviewed during commitment to terminal differentiation. However, DNp63a in McDade and McCance, 2010). The predominant isoform also synergises with notch in the activation of keratin 1, a marker of expressed in basal keratinocytes and stratifying epithelia (Yang et early differentiation (Nguyen et al., 2006). Other evidence for a role 1636 Journal of Cell Science 124 (10)

in differentiation is the fact that DNp63a has been shown to induce resulted in hypoproliferation, as measured by the number of 5- expression of a number of genes involved in differentiation, such as bromodeoxyuridine (BrDU)-incorporating cells (Fig. 1A; IKKa, PERP, p57 and the notch ligand Jag-1 (Beretta et al., 2005; supplementary material Fig. S1B,D), and loss of differentiation, as Candi et al., 2007; Candi et al., 2006; Ihrie et al., 2005). assessed by morphology and indirect immunofluorescence of early Recent data from our laboratory using neonatal human foreskin (keratin 1) and intermediate (loricrin) markers of differentiation keratinocytes (HFKs) indicates that p21 induction is important for (Fig. 1A,B; supplementary material Fig. S1C,E). We further proliferation and induction of differentiation (Wong et al., 2010). confirmed that, similar to findings from Truong et al. (Truong et Here, we demonstrate that the predominant DNp63a isoform is al., 2006), simultaneous p53 depletion fully restored proliferation important for regulation of p21 during differentiation and that p63 with no rescue of differentiation (data not shown). Because we depletion results in p53-dependent aberrant activation of p21 and consistently observed increased p21 protein levels in p63-depleted hypoproliferation. In addition, we identify accumulation of the cells (Fig. 1A), we further examined p21 expression in 3D retinoblastoma (Rb) family member p130 as a key event in the organotypic raft cultures depleted for p63 by indirect hypoproliferation observed in p63-depleted HFKs and demonstrate immunofluorescent staining. This revealed not only increased levels a novel role for p63 in regulating transcription of the S-phase of p21, but also altered localisation (Fig. 1B; supplementary kinase-associated protein (Skp2), which has been shown to material Fig. S1C). In scrambled control rafts, p21 is detected in modulate p130 levels and subsequent proliferation. the basal and immediately suprabasal layers, where cells are exiting the cells cycle, whereas p21 expression was detected throughout Results the p63-depleted rafts (Fig. 1B; supplementary material Fig. S1C). Dynamic regulation of p21 is required for efficient The altered localisation and levels of p21 could be rescued by epidermal differentiation adenoviral re-expression of the DNp63a isoform (Fig. 1A,B). We Organotypic raft cultures of neonatal HFKs represent an ideal re-expressed DNp63a because it is expressed >20-fold more than model system to investigate the role of p63 isoforms in proliferation the b and >100-fold more than the g isoforms in cycling HFKs and differentiation. Therefore, we established stable and transient (supplementary material Fig. S1D). Expression of the DNp63a RNA interference (RNAi) models targeting a subset of p63 isoforms was sufficient to restore differentiation, as measured by isoforms, with small interfering (si)RNA directed to either the 3Ј- expression of early (keratin 1) and intermediate (loricrin) markers UTR of the a and b isoforms (UTRi) or the DNA-binding domain (Fig. 1B). Exogenous re-expression of DNp63a in p63-depleted of all isoforms (Pan) (supplementary material Fig. S1A). Initial cells also significantly increased proliferation to levels observed in experiments revealed that p63 depletion with UTRi and Pan RNAi control cells transduced with DNp63a (Fig. 1C). Journal of Cell Science

Fig. 1. Re-expression of DNp63a rescues loss of differentiation and proliferation in p63-UTRi knockdowns. (A)Western blot analysis of HFKs transfected with siRNA targeting p63 UTR (UTRi) or scrambled control (Scr). The depleted cells were infected with adenovirus (MOI50) expressing DNp63a with an HA tag (DNa) or GFP control virus 4 days post infection. (B)Sections of organotypic raft cultures generated from the same cells stained for H&E and indirect immunofluorescent staining for early [keratin 1 (K1)] and intermediate [loricrin (Lor)] markers of differentiation and p21. Scale bar: 100mM. (C)Number of BrDU-incorporating cells assessed by immunofluorescent staining. The graph represents the average number of BrDU-positive cells in the basal epithelial layer from organotypic raft cultures cells per 1000mM, expressed as a percentage of the scrambled control (mean ± s.e. for at least 10 counts each of three independent biological replicates; P-value from paired t-test, *P<0.05, ***P<0.001). p63 regulates Skp2–p130 levels 1637

p63 depletion results in aberrant p53-mediated activation striking contrast, p21 induction in response to DNA damage induced of p21 expression by adriamycin treatment is completely abrogated in p53-depleted The DNp63a isoform has previously been shown to repress cells (Fig. 2C). Furthermore, we detected p21 expression in transcription of p21 by binding to two p53 response elements in organotypic raft cultures of p53 depleted by shRNA (Fig. 2D). the p21 promoter (Westfall et al., 2003). Therefore, the increase in To determine whether the observed hypoproliferation in p63- p21 levels observed in p63-depleted cells probably represents depleted HFKs is p21 dependent, we simultaneously depleted aberrant de-repression of p53-mediated activation of the p21 HFKs with siRNAs targeting p63 (UTR or pan), p21 or scrambled promoter. We examined p21 levels in HFKs stably transduced with control (Fig. 3A). Organotypic raft cultures revealed that p21 short hairpin RNA (shRNA) targeting p53. Although there was a depletion resulted in partial rescue of proliferation in keratinocyte greater than 90% reduction in mRNA levels of p53 in the cells, rafts depleted of the a and b p63 isoforms (UTR), whereas a levels of expression of mRNA encoding p21, although reduced, smaller effect was observed in pan p63-depleted rafts that was not were clearly not as significantly decreased as those of p53, statistically significant (Fig. 3B). Similar to p53, p21 depletion did indicating a p53-independent mechanism of expression (Fig. 2A). not restore expression of markers of differentiation or morphology Furthermore, the transient induction observed during calcium- (Fig. 3C). In contrast to p53 knockdown, p21 depletion alone induced differentiation seems to be, in part, p53 independent, resulted in decreased expression of markers of differentiation (Fig. because p21 is still induced, albeit at lower levels (Fig. 2B). In 3C), as we have previously shown (Wong et al., 2010). Taken Journal of Cell Science

Fig. 2. p21 is transiently induced during differentiation in a p53-independent manner. (A)Quantitative PCR analysis of levels of mRNAs encoding p53 and p21 in HFKs depleted of p53 by shRNA. Each sample was analysed in triplicate and normalised to RPLPO as control, and expressed as a percentage of the scrambled control (Scr) (mean ± s.e. of three independent biological replicates). (B)Western blot analysis of expression of p63, keratin 1 (K1), p21, p53 and actin loading control in HFKs expressing p53shRNA and scrambled control HFKs following calcium treatment to induce differentiation for the indicated times. (C)Western blot analysis of expression of p63, p21, p53 and actin loading control in HFKs expressing p53shRNA and scrambled control HFKs following adriamycin treatment to induce DNA damage for the indicated times. (D)Sections of organotypic raft cultures generated from HFKs expressing p53shRNA and scrambled control HFKs stained for H&E and indirect immunofluorescent staining for early (K1), intermediate (loricrin) and late (filaggrin) markers of differentiation, p21 and p53. Scale bar: 100mM. 1638 Journal of Cell Science 124 (10)

Fig. 3. Hypoproliferation in p63-depleted HFKs is rescued by simultaneous depletion of p21. (A)Western blot analysis of expression of p63, p21, p53 and actin loading control in HFKs doubly transfected with p63-targeting siRNA (UTRi or Pani) and scrambled control (Scr) or p21-targeting siRNA (p21i). (B)Number of BrDU-incorporating cells in organotypic raft culture assessed by immunofluorescent staining. The graph represents the average number of BrDU-positive cells in the basal epithelial layer from organotypic raft cultures per 1000mM, expressed as a percentage of the scrambled control (mean ± s.e. for at least 10 counts each of three independent biological replicates; P-value for paired t-test, *P<0.05, **P<0.01; ns, not significant). (C)Sections of organotypic raft cultures generated from the same cells stained for H&E and indirect immunofluorescent staining for early (K1) and late filaggrin (Fil) markers of differentiation (scale bar100mM).

together, these data suggest that p21 induction is mediated by a that p130 is the major pocket protein accumulated downstream of

Journal of Cell Science p53-independent mechanism in HFKs in response to differentiation p53 and p21 in replicative and DNA-damage-induced senescence signals. (Helmbold et al., 2009), whereas Rb and p107 levels are consistently decreased. Therefore, because of the p130–p53–p21 Hypoproliferation in p63-depleted cells is dependent on association and because we only observed an increase in p130 the Rb family member p130 protein levels in the p63-depleted cells and p130 depletion resulted Cyclin-dependent kinase inhibitors such as p21 mediate their in greater and more significant rescue of proliferation in p63- inhibitory effects on the cell cycle through the Rb family depleted cells, we examined the effect of p130 depletion in (DeGregori, 2004). We therefore examined levels of the Rb family organotypic raft cultures. Depletion of p130 fully rescued members in p63-depleted cells and found that p63 depletion hypoproliferation induced in rafts depleted of p63 by UTR siRNA consistently resulted in increased p130 levels, whereas p107 and (Fig. 5A,B). Similar to p53 and p21 depletion, p130 knockdown Rb levels were not consistently altered, as measured by western did not rescue expression of markers of differentiation (Fig. 5C). blot (Fig. 4A). Quantitative PCR analysis revealed that the increase Taken together, these results indicate that p63 depletion results in in p130 levels was at the protein level, because mRNA levels of cell cycle arrest dependent on p21 and Rb family members, in p130 are not significantly altered in p63-depleted cells (Fig. 4B). particular p130. To determine whether p130, p107 or Rb family members are required for hypoproliferation in p63-depleted cells, we SKP2 is a novel p63-regulated gene simultaneously depleted HFKs for p63 a and b isoforms using the Next, we looked for potential regulators of p130 stability and UTR-targeting siRNA and individual Rb family members (Fig. identified SKP2 as a potential p63-regulated gene, because it was 4C), and quantified the number of BrDU-incorporating cells in 2D recently shown to be regulated by p53 in response to DNA damage culture (Fig. 4D). Depletion of p130 alone resulted in an increase (Barre and Perkins, 2010). We wanted to determine whether Skp2 in the number of BrDU-positive cells and rescued proliferation of expression was positively regulated by p63, because Skp2 can p63-UTR-depleted cells to ~110% of the number of BrDU-positive target p130 and p21 for ubiquitylation and degradation as part of cells observed in scrambled control (Fig. 4D). By contrast, p107 the SCFSkp2 complex (Bhattacharya et al., 2003; Yu et al., 1998). depletion did not significantly affect proliferation alone or in p63- In addition, we also identified SKP2 as a potential p63 target gene depleted cells, whereas Rb knockdown results in a smaller increase in a p63 chromatin immunoprecipitation (ChIP)-seq experiment in in proliferation and partial rescue of proliferation to ~70% that of HFKs (S. S. McD. and D. J. McC., unpublished). Furthermore, scrambled control. This is interesting, because recent data indicate examination of an independent p63 genome tiling ChIP-chip p63 regulates Skp2–p130 levels 1639

Fig. 4. p130 is the major Rb family member required for hypoproliferation in p63-depleted HFKs. (A)Western blot analysis of p63, p130, Rb and p107 protein levels and actin loading control in HFKs depleted of a and b isoforms of p63 (UTRi). (B)Quantitative PCR analysis of levels of mRNA encoding p130, Rb, p107 and p63 in p63-depleted HFKs. Each sample was analysed in triplicate and normalised to RPLPO as control and expressed as a percentage of the scrambled control (mean ± s.e. of three independent biological replicates). (C)Western blot analysis of p63, p130, Rb and p107 protein levels and actin loading control in HFKs simultaneously depleted for a and b isoforms of p63 (UTRi) or all isoforms of p63 (Pani) and individual Rb family members. (D)The number of BrDU-incorporating cells in HFKs simultaneously depleted for p63 and p130, Rb or p107, or scrambled control. The graph represents the percentage of BrDU- positive cells, expressed relative to scrambled control (mean ± s.e. of at least 500 cells, from three independent biological replicates; P-value for paired t-test, *P<0.05, **P<0.01; ns, not significant).

approach in ME180 cervical carcinoma cells (Yang et al., 2006) (Fig. 6E). Taken together, these data identify SKP2 as a novel p63

Journal of Cell Science and ChIP-seq in normal human epidermal keratinocytes target gene. (Kouwenhoven et al., 2010) revealed the same, but less finely mapped, predicted p63-binding site ~4 kb into intron 2 of the Depletion of Skp2 in HFKs results in p130 accumulation SKP2 gene (Fig. 6A). To determine whether modulation of Skp2 levels in HFKs leads to To establish whether Skp2 is transcriptionally regulated by p63, increased p130 levels, we depleted Skp2 with RNAi in HFKs and we first examined Skp2 expression in p63-depleted HFKs and observed increased levels of p130 by western blot; in addition, we showed that expression is decreased at both protein and mRNA also observed a consistent but modest increase in p21 levels (Fig. levels (Fig. 6B,C). To validate p63 binding to regulatory regions 7A). This was concomitant with a 40% decrease in the number of of the SKP2 gene, ChIP assays with a pan-p63 antibody and control BrDU-incorporating cells in 2D culture (Fig. 7B). To determine IgG were carried out with chromatin isolated from cycling cells. whether expression of exogenous Skp2 in p63-depleted cells Binding of p63 to the characterised p53-binding site in the SKP2 rescued proliferation, we transiently transfected HFKs depleted for promoter (–1254) and to the intron 2 binding site identified in the p63 with GFP alone or with Skp2 and 10% GFP (as a marker) and ChIP-seq study (transcription start site +4 kb) of the SKP2 gene pulsed with BrDU. We then stained cells for BrDU and GFP, and was quantified by real-time PCR (Fig. 6D). The primer sets used counted the number of GFP-positive cells that were also positive flank the p53-binding site in the SKP2 promoter (Barre and Perkins) for BrDU. The results indicate that expression of Skp2 in p63- and the p63-binding site in intron 2 identified by ChIP-Seq and a depleted cells restores proliferation (Fig. 7C). negative control region (an NFkB-binding site at –243 in the SKP2 promoter). An approximately sixfold enrichment (corresponds to Discussion 1% input) for the intron 2 p63-binding site compared with the The p63 transcription factor has been proposed as a master regulator control was observed (Fig. 6D). Binding of p63 to the p53-binding of proliferation and differentiation of stratifying epithelia. Its role in site in the SKP2 promoter (Prom) was also observed; however, maintaining proliferation and differentiation is somewhat enrichment was only 2–3 fold at this site in p63 ChIP compared controversial, because of subtle differences in phenotypes of different with IgG control. Examination of an NFkB-binding site in the knockout mice (Mills et al., 1999; Yang et al., 1999). The complex SKP2 promoter (–234) as a negative control indicated that there nature of the TP63 gene and its clear role in development make it was no enrichment in p63 ChIP assays compared with IgG control difficult to explore the functions of p63 in the mature epidermis of assays. The specificity of these results was further confirmed by the knockout mouse. Using human organotypic raft cultures of separating products of ChIP-PCR by agarose gel electrophoresis normal human epidermal keratinocytes, it has recently been shown 1640 Journal of Cell Science 124 (10)

Fig. 5. p130 accumulation in p63-depleted HFKs is required for hypoproliferation. (A)Western blot analysis of p63 and p130 levels and actin loading control in HFKs doubly transfected with UTRi and scrambled control or p130-targeting siRNA. (B)Number of BrDU-incorporating cells in organotypic raft cultures assessed by immunofluorescent staining. The graph represents the average number of BrDU- positive cells in the basal epithelial layer from organotypic raft cultures per 1000mM, expressed as a percentage of the scrambled control (mean ± s.e. for at least 10 counts each of three independent biological replicates). (C)Sections of organotypic raft cultures generated from the same cells stained for H&E and indirect immunofluorescent staining for early (K1) and intermediate (Lor) markers of differentiation. Scale bar: 100mM.

that depletion of p63 results in p53-dependent hypoproliferation, is a change in the distribution of p21, from expression in just the which can be fully rescued by p53 depletion; however, this is basal cell to expression throughout the epithelial layers. This insufficient to restore differentiation (Truong et al., 2006). change in distribution was rescued by exogenous expression of

Journal of Cell Science Using organotypic raft cultures with neonatal HFKs, we DNp63a. Furthermore, we observed p21 induction in p53-depleted demonstrate that RNAi-mediated depletion specifically of the a cells following calcium treatment to induce differentiation, in and b p63 isoforms results in loss of differentiation and contrast to DNA damage in these p53-depleted HFKs, in which hypoproliferation, indicating that these isoforms are required for induction of p21 is completely abrogated. The results suggest that both differentiation and maintenance of proliferative capacity. As expression of the predominant DNp63a isoform is required for the described previously for depletion of all p63 isoforms (Truong et regulation of p21 expression. al., 2006), the hypoproliferation observed in a and b knockdowns To determine the role of p21 in hypoproliferation in p63-depleted could be fully rescued upon p53 depletion. Similar to depletion of cells, we investigated downstream targets of p21. The Rb family all isoforms (Truong et al., 2006), in p63-UTR-depleted HFKs, we members are important in regulating epidermal growth and observed a p53-dependent increase in p21 levels. differentiation. This is exemplified by the human papillomaviruses, When we depleted p21 levels in p63 knockdowns, we observed which deregulate keratinocyte growth control through degradation a partial rescue of proliferation in p63-depleted HFKs in four-day- of Rb, p107 and p130 mediated by the E7 protein (Ueno et al., old organotypic raft cultures This increase in proliferation was 2006). We found that p130 is increased at the protein level in p63- statistically significant in the raft depleted for the a and b isoforms, depleted cells, suggesting stabilisation of the protein in the absence but not when all isoforms were depleted. Truong et al. (Truong et of p63. Importantly, the increase in p130 in these cells probably al., 2006) had previously reported that p21 depletion delayed contributes to the hypoproliferative phenotype, because depletion hypoproliferation in p63-depleted cells, but in their model system of p130 leads to restoration of proliferation. It is interesting to note of human epidermal keratinocytes this was insufficient to rescue that depletion of Rb resulted in a smaller but significant increase proliferation in four-day-old rafts using an siRNA that targeted all in proliferation, indicating that it also plays a role in controlling p63 isoforms. The differences might be due to the different isoforms proliferation. Furthermore, the Rb family member p130 has been depleted (the g isoforms remaining in the cells depleted of a and shown recently to be the major pocket protein required for b) and the models utilised (neonatal foreskin keratinocytes versus replicative and DNA-damage-induced senescence downstream of adult epidermal keratinocytes). The neonatal model might contain p53–p21 and p16INK4A (Helmbold et al., 2009). Taken together, a greater proportion of basal progenitors and p53-mediated this suggests that accumulation of p130 is one of the mechanisms activation of p21 has been shown to play a role dampening down underlying the hypoproliferation observed in HFKs acutely depleted proliferation in these cells (Gatza et al., 2007; Topley et al., 1999). for p63. In addition, p130 has been shown to be a possible effector Also, we show that, together with an increase in p21 levels, there of cell cycle arrest induced by transforming growth factor b (TGFb) p63 regulates Skp2–p130 levels 1641

Fig. 6. Skp2 expression is transcriptionally regulated by p63. (A)Schematic representation of the p63-binding region of Skp2 identified from ChIP-seq and ChIP-ChIP experiments (S. S. McD. and D. J. McC., unpublished) (Kouwenhoven et al., 2010; Yang et al., 2006). (B)Western blot analysis of p63 and Skp2 levels in HFKs depleted for p63 (UTRi). (C)Quantitative PCR analysis of mRNA levels of Skp2 and p63 in HFKs depleted by p63-UTRi siRNA. Each sample was analysed in triplicate, normalised to RPLPO as control and expressed as a percentage of the scrambled control (mean ± s.e. of three independent biological replicates). (D)Quantitative PCR analysis of DNA from ChIP of cycling HFKs treated with p63 antibody and matched IgG control. PCR with primers directed to p63-binding sites in Skp2 promoter (–1254 bp), intron 2 (+4 kb) and NFkB-binding site in proximal promoter (–243 bp) as negative control. Expressed as fold enrichment compared to IgG (mean ± s.e. of two independent biological replicates). (E)Agarose gel analysis of ChIP-PCR products.

(Choi et al., 2002), which is an important regulator of epithelial (Skp2–Rbx1–Cul1–Skp1), which plays an important role in S-

Journal of Cell Science cell cycle exit (Herzinger et al., 1995). phase progression, has recently been shown to be cooperatively Because levels of the mRNA encoding p130 are unchanged in regulated by p53 and NFkb in response to DNA damage (Barre p63-depleted cells, it is clear that the protein stability is regulated and Perkins, 2010). Skp2 promotes S-phase progression by post-transcriptionally. Interestingly, Skp2, which is the substrate degrading negative cell cycle regulators, including p27, p21 and recognition component of the SCFSkp2 E3 ubiquitin ligase complex p130 among others, and can repress p53 activation by p300

Fig. 7. Skp2 depletion results in increased p130 levels. (A)Western blot analysis of p63, Skp2, p130, Rb, p21 and actin loading control in HFKs depleted by siRNA targeting Skp2. (B)The number of BrDU-incorporating cells in HFKs depleted for Skp2 or scrambled control. The graph represents the percentage of BrDU-positive cells, expressed relative to scrambled control (mean ± s.e. of at least 500 cells, from three independent biological replicates). (C)The number of BrDU-incorporating cells in HFKs depleted for Skp2 or scrambled control and transfected with GFP alone or Skp2 plus 10% GFP. The graph represents the percentage of BrDU-GFP- positive cells, expressed relative to scrambled control (mean ± s.e. of at least 250 cells, from three independent biological replicates; P-value for paired t-test, *P<0.05). 1642 Journal of Cell Science 124 (10)

(reviewed in Frescas and Pagano, 2008). Results from a p63 ChIP- Adenovirus expressing DNp63a including an HA tag was produced by amplifying DNp63a with primers incorporating an HA tag at the C-terminus, cloned into seq experiment in HFKs (S. S. McD. and D. J. McC., unpublished), pENT11 (Invitrogen) and recombined using LR Clonase into the pADCMV as well as a similar ChIP-seq experiment in normal human (Invitrogen) destination adenovirus backbone according to the manufacturer’s epidermal keratinocytes (NHEKs) and a genome tiling ChIP-chip instructions (Invitrogen). experiment in ME180 cells, identified a common p63-binding site Real-time reverse-transcriptase PCR analysis in intron 2 of the SKP2 gene (Kouwenhoven et al., 2010; Yang et RNA extraction was carried out with a High Pure RNA isolation kit (Roche), al., 2006). Our results indicate that binding of p63 to the intron 2 according to manufacturer instructions. RNA (1 mg) was treated with RQ1 RNase- binding site is responsible for promoting SKP2 transcription in Free DNase (Promega) prior to first strand cDNA synthesis using random primers with the Transcriptor High-Fidelity cDNA Synthesis Kit (Roche) according to keratinocytes. We also show that exogenous expression of Skp2 in manufacturer instructions. Amplification of PCR products was quantified using p63-depleted cells is sufficient to rescue proliferation in p63- FastStart SYBR Green Master (Roche) according to manufacturer instruction and depleted HFKs. Taken together, we identify SKP2 as a novel pro- fluorescence monitored on a DNA Engine Peltier Thermal Cycler (Bio-Rad) equipped proliferative p63 target gene that probably exerts its function with a Chromo4 Real-Time PCR Detection System (Bio-Rad). Melting curve analysis was also performed. In brief, cDNA samples were diluted 1:10 and quantified by through targeted degradation of p130 and p21 to maintain cell amplification against serial dilutions of appropriate control cDNA with the following proliferation (Bhattacharya et al., 2003; Yu et al., 1998). Supporting cycling conditions: initial denaturation 95°C for 10 minutes and 40 cycles of 95°C our findings, recent data indicate a positive role for p63 in for 15 seconds, 58°C for 15 seconds, 60°C for 60 seconds. Specific primer sets were utilised for a, b and g p63, p53 (Wang and Seed, 2003), p21Cip1 and RPLPO (large maintaining expression of pro-proliferative cell cycle target genes ribosomal protein) (Minner and Poumay, 2009) (for primer sequences, see and opposing p53-mediated repression (Lefkimmiatis et al., 2009). supplementary material Table S2). Expression levels were assessed in triplicate, Interestingly, Skp2 expression has recently been shown to be normalised to RPLPO levels and graphs represent the combined results of at least required for spontaneous tumorigenesis in Rb- (Lin et al., 2010) three independent biological replicates. and PTEN-deficient mice (Wang et al., 2010), and is required for ChIP assays Ras-mediated transformation (Wang et al., 2010). Furthermore, Chromatin immunoprecipitations were carried out as described previously (Wong et inhibition of activation of the SCFSkp2 complex with a neddylation al., 2010). An anti-pan-p63 monoclonal (clone 4A4; Santa Cruz) antibody or control mouse immunoglobulin G (IgG) were used for ChIP. The recovered DNA was inhibitor was sufficient to suppress tumorigenesis (Wang et al., subjected to PCR amplification using quantitative PCR as described above. For ChIP 2010). This is intriguing as several lines of evidence implicate p63 primer sequences, see supplementary material Table S2. in preventing senescence (Guo et al., 2009; Keyes et al., 2006). Western blot analysis Therefore, it will be important to determine the precise role played Protein lysate concentrations were 40 mg for western blots as previously described by p63 in regulating Skp2 expression, because increased expression (Westbrook et al., 2002). Primary antibodies used are described in supplementary of p63 and Skp2 is frequently observed in squamous cell material Table S3. Secondary antibodies used in this study were goat anti-mouse and anti-rabbit HRP (Santa Cruz). Luminescence was revealed by incubation with either carcinomas (Gstaiger et al., 2001; Sniezek et al., 2004). Western Lightning ECL (Perkin-Elmer) or West Femto substrate (Pierce), and signal Our data indicate that p63 plays a multifaceted role in the detected on an Alpha Innotech FluorChem SP imaging system. mature epidermis. It maintains proliferation by repressing p53- mediated activation of p21 and inhibition of p130, and by inducing Immunofluorescent staining of organotypic raft sections Tissue embedding and H&E staining were carried out as previously described expression of pro-proliferative genes such as SKP2. The (McKenna et al., 2010). Images were taken on an Olympus BH-2 microscope with identification of SKP2 as a p63 target gene is novel and particularly an Olympus D25 camera using Cell B software (Olympus). 5 mm thick paraffin-

Journal of Cell Science interesting, considering its recently identified essential role in embedded sections were deparaffinised with xylene, rehydrated with step-down concentrations of ethanol and submitted to antigen retrieval with boiling citrate several models of tumorigenesis and potential use as a therapeutic buffer (DAKO). Indirect immunofluorescent staining was performed using the target (Bauzon and Zhu, 2010; Lin et al., 2010; Wang et al., 2010; indicated primary antibodies and Alexa-Fluor-488-conjugated secondary reagents Zhu, 2010). (Molecular Probes). Briefly, sections were permeabilised with 10% fetal calf serum (FCS) and 0.2% TRITON X-100 for 30 minutes at room temperature, rinsed and incubated with primary antibody overnight at 4°C in 10% FCS, washed and incubated Materials and Methods with secondary goat anti-mouse or anti-rabbit antibodies conjugated to Alexa-Fluor- Cell culture, infections and transfections 488 or Alexa-Fluor-594 (Molecular Probes) at room temperature for 1 hour, washed Isolation of primary neonatal HFKs and generation of shRNAs were carried out as and mounted with Prolong Gold antifade reagent plus DAPI (Molecular Probes). previously described (Incassati et al., 2006). Differentiation of HFK cell lines in Primary antibodies used are described in supplementary material Table S3. BrDU- organotypic raft cultures was carried out as previously described (McCance et al., pulsed cells on cover slips were fixed for 10 minutes with 4% PFA, washed three 1988). The raft cultures were harvested, fixed in 4% paraformaldehyde (PFA), and times with PBS, submitted to antigen retrieval and stained as described above. For then embedded in paraffin for subsequent sectioning and staining with haematoxylin raft sections, the number of BrDU-positive cells was counted for a minimum of 10 and eosin (H&E). In order to label DNA-synthesizing cells, 20 mM BrDU was added fields of view (1000 mM). For cover slips, a minimum of 10 fields of view and 500 to the raft culture 16 hours prior to harvest. Adenovirus expressing DNp63a including cells were counted for each slide and expressed as the percentage of BrDU-positive an HA tag was generated in the pADCMV destination adenovirus backbone, cells (BrDU-DAPI). Graphs represent the mean of three independent biological amplified, purified and titred on HEK293 cells according to the manufacturer’s replicates. In all cases, immunofluorescent images were captured on a Leica AF6000 instructions (Invitrogen). For adenoviral infection, cells were incubated with a inverted fluorescence microscope and Leica AF imaging software. Exposure times multiplicity of infection (MOI) of 50 for 6 hours in culture media. siRNA transfection were kept constant within an experiment; images were pseudocoloured and processed of HFK cells was carried out with 200 nm siRNA using FuGENE HD transfection identically in all cases. reagent (Roche) according to the manufacturer’s instructions. siRNAs were manually designed for p63 or Rb-family-targeting sequences were taken from pre-designed Ambion Silencer Select siRNAs, and are described in supplementary material Table This work was supported by grants from the National Institutes of S1. For calcium-induced differentiation, confluent monolayers of HFKs were induced Health (AI030798) and the Medical Research Council (G0700754). to differentiate by withdrawal of growth factors and addition of 1.5 mM CaCl2. We would like to thank Neil Perkins for siRNA targeting Skp2 and primer details. Deposited in PMC for release after 6 months. Plasmids and constructs pSuper-retro constructs expressing shRNAs against the 3Ј-UTR of p63 (p63-UTR), Supplementary material available online at the p63 DNA-binding domain (p63-pan) and p53 (p53) were generated by legating http://jcs.biologists.org/cgi/content/full/124/10/1635/DC1 annealed oligonucleotides (supplementary material Table S1) containing 21-mer targeting sequences into pSuper (Puro or Neo) according to manufacturers’ References instructions (Oligoengine); a scrambled control targeting no annotated gene was Barre, B. and Perkins, N. D. (2010). The Skp2 promoter integrates signaling through the described previously (shSCRAM) (Incassati et al., 2006). All retroviral plasmid NF-kappaB, p53, and Akt/GSK3beta pathways to regulate autophagy and apoptosis. constructs were sequenced prior to transfection into FNYX-GP packaging cells. Mol. Cell 38, 524-538. p63 regulates Skp2–p130 levels 1643

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