Synergistic Activation of Human Involucrin Gene Expression by Fra-1 and p300— Evidence for the Presence of a Multiprotein Complex  James F. Crish, Richard L. Eckert  Journal of Investigative Dermatology  Volume 128, Issue 3, Pages 530-541 (March 2008) DOI: 10.1038/sj.jid.5701049 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Calcium treatment of p300 expressing keratinocytes produces a synergistic increase in hINV promoter activity. (a) Structure of hINV luciferase reporter constructs. pINV-2473 includes the full-length hINV promoter; pINV-41 is the hINV basal promoter (TATA box only). The locations of the proximal and distal regulatory regions are indicated (PRR, DRR), as are the positions of the AP1-1 and AP1-5 sites, the C/EBP site, and the Sp1 site. The black box represents the luciferase gene and the arrow indicates the site of transcription initiation and the direction of transcription. (b and c) Calcium treatment of p300 expressing cells results in synergistic activation of involucrin gene transcription. Keratinocytes (50% confluent) were transfected with 1μg of pINV-2473 or pINV-41 in the presence or absence of 0.5μg of p300 expression plasmid. After 24hours, the cultures were shifted to cell culture medium supplemented or not supplemented with 0.3mM calcium, for an additional 24hours. For TPA treatment, the cells were transfected as indicated above and then treated for 24hours in the presence or absence of 50ng TPA/ml. Cell extracts were then prepared for assay of luciferase activity. The values presented in this figure represent the averages of four separate experiments. As assessed by the Student's t-test, for the pINV-2473 construct, the difference between the control and all other groups is significant at the 95% confidence level. The difference between the 0.3mM calcium/TPA+p300 groups and all other groups is significant at the 95% confidence interval. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TPA-dependent hINV promoter activity requires p300. (a) Keratinocytes (50% confluent) were transfected with 1μg of pINV-2473, in the presence or absence of 0.5μg of HA-p300 or HA-d33 expression plasmids. The final DNA concentration was adjusted to a final concentration of 2μg with empty plasmid. At 24hours post-transfection, the cultures were treated for 24hours with or without 50ng TPA/ml. Cell extracts were then prepared for assay of luciferase activity. The results are the average values derived from three separate experiments. The difference between TPA treated and p300 groups and all other groups is significant at the 95% confidence interval. (b) Keratinocytes were transfected with pINV-2473, HA-p300, and HA-d33 plasmids as outlined above. At 24hours post-transfection the cells were harvested and total cell extracts were assayed for the presence of HA-p300 or HA-d33 by immunoblot using antibodies specific for p300 or HA epitope, followed by an appropriate secondary HRP-conjugated antibody and visualization of antibody binding using chemiluminescence reagents. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 p300 and Fra-1 act synergistically to increase hINV promoter activity. (a) Structure of hINV luciferase reporter constructs. pINV-2473 includes the full-length hINV promoter; pINV-41 is the hINV basal promoter (TATA box only). The conventions are exactly as outlined in Figure 1. In the DRR constructs, segments of the DRR region are fused adjacent the hINV basal promoter. The DRR(−2,473/−1,953) AP1-5mm has a mutation in the AP1-5 site that results in its inactivation (Welter et al., 1995). (b and c) Keratinocytes (50% confluent) were transfected with 1μg of the indicated DRR hINV promoter reporter plasmid in the presence of 0.5μg of HA-p300 or FLAG-hFra-1. At 24hours post-transfection, the cells were harvested and extracts prepared for assay of luciferase activity. The values are the mean of two separate experiments. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In vivo interaction of p300 and Fra-1 with the hINV promoter DRR. Normal human keratinocytes were treated with formaldehyde to crosslink intranuclear DNA–protein complexes. The nuclei were then prepared and lysed, and Fra-1 and p300 DNA complexes were immunoprecipitated using 5μg rabbit anti-Fra-1 and 5μg mouse monoclonal anti-p300 antibodies, respectively. Nonspecific precipitation was monitored by precipitation using normal mouse IgG (Santa Cruz, sc-2025). The DNA was released from the precipitated complex using 1% SDS/0.1M NaHCO3 and the released product was amplified by PCR and the amplified product was detected by ethidium staining of an agarose gel. The arrows indicate migration of the product and the primers. M=markers (nucleotides) and T=PCR product observed following amplification of 1% of material used for immunoprecipitation. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Evidence suggesting that p300 and Fra-1 interact. (a) Nuclear extracts were prepared from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-Fra-1) cell lines. Equal protein equivalents were separated by electrophoresis and immunoblotted with anti-Fra-1 or anti-FLAG. (b) FLAG-Fra-1 was precipitated from nuclear extract isolated from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-Fra-1) cell lines using 5μg of mouse monoclonal anti-FLAG M2. The precipitated protein was separated by electrophoresis and immunoblotted with anti-FLAG (top panel), anti-p300 (middle panel), and anti-P/CAF (bottom panel). Each group was incubated with the appropriate HRP-conjugated secondary antibody and the signal was visualized using chemiluminescence detection reagents. The asterisks indicate nonspecific bands. (c) FLAG-hFra-1 precipitation of other regulatory proteins. Proteins were immunoprecipitated using 5μg of mouse monoclonal anti-FLAG M2 as above and co-precipitated proteins were identified using antibodies specific for the indicated proteins. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 P11 column fractionation of nuclear extracts. Nuclear extract (NE, 2.5mg) was prepared from HeLa-Z (pLNCX2-FLAG-hFra1) cells and chromatographed on a 3ml P11 phosphocellulose column (Whatman) at a flow rate of 1 column volume per hour. The column, which was prepared in equilibration buffer containing 0.2M KCl (see Materials and Methods), was sequentially eluted with 1ml equilibration buffer containing 0.3, 0.4, 0.5, 0.85, and 1.2M KCl and 0.25ml fractions were collected. Each fraction was then dialyzed against the equilibration buffer and an equivalent quantity of protein (15μg) was separated by electrophoresis on a 10% (FLAG-hFra-1) or 5% (p300) polyacrylamide gel before immunoblotting. FLAG-hFra-1 was detected using peroxidase-conjugated anti-FLAG M2 mouse monoclonal antibody (Sigma F1804, 1:500), and JunB and p300 were detected using a 1:500 dilution of rabbit anti-JunB (Santa Cruz, sc-46X) and 2μg/ml mouse monoclonal anti-p300CT (Upstate, 05-257), respectively. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 p300 interaction with FLAG-hFra-1 requires additional protein factors. Nuclear extract was prepared from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-hFra-1) and 2mg was loaded onto a P11 column and eluted stepwise using the indicated level of KCl. (a) Protein (15μg) of the 0.4M KCl fraction was immunoprecipitated using 40μl of a 50% slurry of anti-FLAG M2 agarose (Sigma, A2220). The immunoprecipitated (IP) proteins were then immunoblotted (IB) with peroxidase-conjugated anti-FLAG M2 mAb (Sigma, F1804), rabbit anti-JunB or mouse monoclonal anti-p300. For the JunB and p300 blots, primary antibody binding was visualized using a 1:14,000 dilution of HRP-conjugated donkey anti-rabbit IgG (Amersham, NA 934) or HRP-conjugated sheep anti-mouse IgG (Amersham, NA 931V). Samples of total nuclear extract and 0.4M KCl fraction (15μg) were electrophoresed in parallel for immunoblot. (b) Seventy-five micrograms of total nuclear extract, prepared from HeLa-Z (pLNCX2-FLAG-Fra-1) cells was adjusted to the indicated KCl concentration and incubated for 8hours at 4°C before immunoprecipitation with anti-FLAG M2 agarose. The precipitated protein was electrophoresed and immunoblotted with anti-FLAG, anti-JunB, anti-JunD (Santa Cruz, sc-74x, 1:500 dilution) or anti-p300. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Fra-1 as a transcriptional activator. Normal human keratinocytes were transfected with 0.1μg of pFR-Luc in the presence of 0.1μg of the indicated Gal4-BD-FLAG-Fra-1 protein expression constructs. At 24hours after transfection, the cells were harvested and assayed for luciferase activity. (a) Regulation of pFR-Luc promoter activity by full-length Fra-1 and truncation mutants. Keratinocytes were transfected with 0.1μg of pFR-Luc in the present of 0.1μg of the indicated plasmids. After 24hours, the cells were harvested and extracts were assayed for luciferase activity. (b) Regulation of pFR-Luc promoter activity by the Fra-1AD and Fra-1AD truncation mutants. Keratinocytes were transfected and harvested for assay of luciferase activity as outlined above. The data is expressed as the mean±SEM, n=4. Immunoblot analysis reveals that all of the proteins (GAL4BD-FLAG-Fra-1, and so on) were expressed at comparable levels (not shown). The results are a representative experiment from two repeats. The values are expressed as sample means±SD. Journal of Investigative Dermatology 2008 128, 530-541DOI: (10.1038/sj.jid.5701049) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions