Peroxisome Proliferator-Activated Receptor-α Is a Functional Target of p63 in Adult Human Keratinocytes  Silvia Pozzi, Michael Boergesen, Satrajit Sinha,

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Peroxisome Proliferator-Activated Receptor-α Is a Functional Target of p63 in Adult Human Keratinocytes  Silvia Pozzi, Michael Boergesen, Satrajit Sinha, Susanne Mandrup, Roberto Mantovani  Journal of Investigative Dermatology  Volume 129, Issue 10, Pages 2376-2385 (October 2009) DOI: 10.1038/jid.2009.92 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 p63 is a negative regulator of PPARα expression in adult human keratinocytes. (a) RT–PCR (left) and qRT–PCR (right) analyses of PPARα expression in HaCaT cells and KCs transiently (96hours) transfected with 3μg of either a plasmid encoding a short hairpin targeting GFP as a control (GFP RNAi) or one targeting all p63 isoforms (p63 RNAi). cDNAs were normalized versus GAPDH levels. Results are representative of at least three independent experiments. (b) Western blotting analysis of PPARα protein expression in HaCaT and KCs as described in (a). Protein loading was normalized versus vinculin. (c) Western blotting proving the specificity of the anti-PPARα antibody by ectopic expression of human PPAR subtypes (1.5μg each) in COS cells. (d) PPARα gene structure (adapted from Chew et al., 2003), splicing isoforms, and protein. PA, PB, PC, and PD represent the four characterized promoters. Journal of Investigative Dermatology 2009 129, 2376-2385DOI: (10.1038/jid.2009.92) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 PPARα is a p63 target. (a) Schematic representation of human PPARα genomic region. Transcription start sites (TSS) for each promoter and in silico identified consensus for TFs are shown. Double arrows represent the positions of amplicons generated by PCR after chromatin IP (ChIP) assay, identified by numbers on the bottom. Region 2 comprises a consensus for hepatocyte nuclear factor 4 (HNF4), which is a positive control for PPARα binding (Pineda-Torra et al., 2002). (b) Amplification of genomic regions, numbered as in (a), after ChIP, showing the in vivo binding of p63 and PPARα, as well as its heterodimeric partner RXR, to PPARα. The amplified FDXR promoter region is a positive control for p63 binding in human KCs (Viganò et al., 2006). Results are representative of three to five independent ChIP assays. (c) ChIPs performed on KCs. Journal of Investigative Dermatology 2009 129, 2376-2385DOI: (10.1038/jid.2009.92) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 p63 represses PPARα promoter B. (a) Schematic diagram of PPARα promoter A-luciferase construct -1664/+81, used in transient transfections. The position of amplicons 2 and 3 generated after ChIP are indicated by double arrows. (b) U2OS cells, devoid of endogenous p63, were exposed to increasing amounts (70–210ng) of p63 expression constructs for 24hours. (c) HaCaT cells, expressing high levels of ΔNp63α, were transfected with increasing amounts (50–100ng) of Tap63α or ΔNp63α, -β, and -γ encoding plasmids or (d) transiently knocked down (72hours) for GFP (GFP RNAi) as a control or p63 (p63 RNAi) before transfection with 1.2μg of the reporter construct for an additional 24hours. (e) Schematic diagram of PPARα promoter B-luciferase construct -1147/+34 used in transient transfections as described in (b). The position of amplicon 4 generated after ChIP is indicated by double arrows. (f, g, and h) Transient transfection of U2OS and HaCaT cells as described in (b, c and d) using the PPARα promoter B-luciferase -1147/+34 construct. (i) Left: schematic diagram of 5′ deletions of PPARα promoter B, -500/+34 and -200/+34, used in transient transfection in HaCaT cells as described above. The position of amplicon 4 generated after ChIP is indicated by double arrows. All results, shown as mean±SD of three independent experiments, each conducted in triplicate, are expressed as fold variation over the promoter-only construct, arbitrarily set as 1. Values were normalized by co-transfection with the 200ng CMV-βGal construct. Statistical significance was calculated using the Student's t-test (*P<0.5; **P<0.01). Journal of Investigative Dermatology 2009 129, 2376-2385DOI: (10.1038/jid.2009.92) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Negative regulation of PPARα by p63 overexpression. (a) Western blotting (top panels) proving the efficiency of ectopic p63 expression in HaCaT (left) and KCs (right) and semiquantitative RT–PCRs (bottom panels) showing PPARα-4 and -6 expression driven by promoter B. Results are representative of two independent experiments. Amplification of GAPDH ensured cDNA normalization. (b) qRT–PCR of global PPARα mRNA expression levels in HaCaT (left) and KCs (right) upon ectopic p63. (c) Western blotting showing PPARα protein levels in HaCaT after p63 ectopic expression. Protein loading was normalized versus laminin. Journal of Investigative Dermatology 2009 129, 2376-2385DOI: (10.1038/jid.2009.92) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 PPARα is a transcriptional regulator of ΔNp63α. (a) Stable knockdown of endogenous PPARα by lentiviral RNAi in HaCaT cells results in increased levels of ΔNp63α, as detected by qRT–PCR (left) and western blotting (right). HaCaT cells were infected with lentivirus expressing shRNA against LacZ or three different targets within the PPARα coding sequence. Cells were harvested after 10 days of puromycin selection and used for preparation of RNA and whole-cell lysates. (b) Left: schematic diagram of ΔNp63 regulatory regions, including the C40 enhancer in the fifth intron (Antonini et al., 2006). In silico identified consensus for the transcription factors is shown. Position of amplicons generated by PCR after ChIP assay are identified by double arrows and letters on the bottom. Right: p63, PPARα, and RXR in vivo binding to the ΔNp63 promoter and downstream regions, as detected by PCR after ChIP analysis. Results are representative of three to four independent ChIP experiments. (c) Schematic diagram of sequential 5′ deletions of p63 promoter constructs (left) used in transient transfections. The transcription start site (TSS), in silico-identified consensus for the transcription factors, and position of the amplicons, as in (b), are indicated. COS cells were transfected with increasing amounts (50–100ng) of PPARα-encoding plasmid, either in the absence or in the presence of RXRα (10ng), and 1.2μg of ΔNp63 promoter constructs (right). Results, shown as mean±SD of three to five independent experiments, each conducted in triplicate, are expressed as fold variation over the shorter promoter-only construct (-164/+69), arbitrarily set as 1. Values were normalized by co-transfection with the 200ng CMV-βGal construct. Statistical significance was calculated using the Student's t-test (*P<0.5; **P<0.02). Journal of Investigative Dermatology 2009 129, 2376-2385DOI: (10.1038/jid.2009.92) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions