(A) Chromatin immunoprecipitation assays were conducted with antibodies against the indicated proteins and chromatin isolated from exponentially proliferating, unsynchronized cultures of primary epidermal keratinocytes. The DNA immunoprecipitated was amplified with primers specific to the proximal promoter of the EBP1 gene. Controls included immunoprecipitation with an irrelevant IgG and amplification with 0.2% of the isolated chromatin, prior to immunoprecipitation (‘Input”). (B) Schematic diagram of the human EBP1 promoter fragment isolated and used in reporter assays. The positions relative to the transcription start site (set to +1) of putative binding sites identified in silico for the indicated transcription factors are shown. (C, D) The firefly luciferase reporter driven by the EBP1 promoter fragment shown in (B) (−980 Luc) was transiently transfected into primary human keratinocytes, together with vectors encoding the indicated E2F or pRb family proteins. All samples were cotransfected with a vector encoding Renilla luciferase. EBP1-luciferase activity was normalized to Renilla luciferase. The results are expressed as the mean +SEM (n = 3) relative to normalized luciferase activity in samples transfected with −980 Luc together with empty vector not encoding for any E2F or pRb protein (set to 1). * indicates p<0.05 (ANOVA).

(A) Schematic of EBP1 promoter constructs tested. The in silico identified E2F-binding site at position −374 is indicated with a star, and the numbers at right indicate the 5′-flanking sequences present in each construct. (B, C) The indicated constructs were transiently transfected together with a plasmid encoding Renilla luciferase, used to normalize for transfection efficiency. Normalized luciferase values are expressed as the mean + SEM (n = 3), relative to the basal activity of the −980 Luc construct (panel B) or the TK-81 vector (panel C), set to 1. * indicates p<0.05 (ANOVA). All luciferase values were significantly different from background activity obtained with the empty vector PXP2.

(A, B) Purified recombinant GST or GST-E2F1 was allowed to bind to [³²P]-labeled, double-stranded oligonucleotide probes corresponding to the DHFR E2F-binding site, or EBP1 promoter sequences containing the E2F binding site centered at position −158 or at position −113. Binding reactions were conducted in the presence of a 100-fold molar excess of unlabelled oligonucleotides corresponding to the wild type (wt) or mutant (mut) DHFR E2F-binding site (panel A), or wild type (wt) or mutant (mut) E2F-binding site in the TIMELESS promoter (panel B). Protein-DNA complexes were resolved by non-denaturing gel electrophoresis. GST-E2F1-containing complexes are shown. (C) Luciferase reporter constructs corresponding to positions −980 to +80 of the EBP1 promoter were transiently transfected into HaCaT cells, together with a vector or a plasmid encoding E2F1. The EBP1 constructs used were either wild type (wt), or mutants in which the E2F-binding sites centered at positions −158 (−158*) and/or −113 (−113*) were mutated. EBP1-directed luciferase activities were normalized to Renilla luciferase, and expressed relative to the activity of the wild type −980 Luc construct, which was set to 1. The results are expressed as the average + SEM (n = 3). * indicates p<0.05 (ANOVA) relative to −980 Luc in the absence of exogenous E2F1.

(A) Schematic of EBP1 promoter constructs tested. Putative E2F-binding sites at positions −759 and −374 are indicated with a star, and the numbers at right indicate the 5′-flanking sequences present in each construct. (B, C) The indicated EBP1 constructs were co-transfected with vectors encoding E2F1, E2F3 or pRb into primary human keratinocytes, together with a vector encoding Renilla luciferase, used to normalize for transfection efficiency. Normalized luciferase values are expressed as the mean + SEM (n = 3), relative to the basal activity of the −980 Luc construct (set to 1). * indicates p<0.05 (ANOVA).

(A) Purified recombinant GST or GST-E2F1 was allowed to bind to [³²P]-labeled, double-stranded oligonucleotide probes corresponding to the following sequences in the EBP1 promoter: −168 to −130 (−168), −155 to −120 (−155) and −136 to −100 (−136). Protein-DNA complexes were resolved by non-denaturing gel electrophoresis. The lower and the highest mobility signals in the gel correspond, respectively, to GST-E2F1-containing complexes and the free probe. A reaction with a probe corresponding to the E2F- binding site in the dihydrofolate reductase promoter (DHFR) was included as positive control. (B) Sequences of wild type and mutant probes used in EMSA experiments. Numbers on the left indicate the identification number for each probe. Bold, underlined A residues indicate mutations introduced in the probes. (C) EMSA using GST or GST-E2F1 together with a [³²P]-labeled, double-stranded oligonucleotide probe corresponding to the DHFR E2F-binding site, or one of the sequences shown in panel B, as indicated. (D) EMSA using GST or GST-E2F1 together with a [³²P]-labeled, double-stranded oligonucleotide probe corresponding to EBP1 promoter sequences from −166 to −104. The probes contained either wild type sequences (wt), or mutants in the GC-rich regions centered at positions −158, −113 (indicated with *), or both. The arrow indicates a lower mobility complex abundant in the binding reaction containing wild type probe. The highest mobility signals correspond to the free probe.

(A) Exponentially proliferating MCF-7 or HEK-293 cells were transiently transfected with an empty vector or an E2F1-encoding plasmid. Twenty-four hours after transfection, RNA was isolated from the cultures, reverse-transcribed and subjected to real-time PCR analysis using EBP1-specific primers. The data are normalized to control RPL 20, RPL 27 and RPL 30 RNA, and are expressed relative to vector-transfected cells, set to 1. Results are expressed as mean + SEM (n = 3). * indicates p<0.05 (Student's t test). (B) Triplicate cultures of exponentially proliferating, unsynchronized primary epidermal human keratinocytes were treated for 48 h in the presence or absence of TGF-β1 (10 ng/ml). Total RNA was isolated, fractionated on agarose gels and transferred to a membrane. Northern blot analysis was conducted with [³²P]-labelled probes corresponding to the EBP1 mRNA, or to 18S rRNA (as a loading control). The results are expressed as the mean + SEM of normalized EBP1 mRNA levels, with abundance in untreated cells set to 1 (n = 3). * indicates p<0.05 (Student's t test). (C) Primary human epidermal keratinocytes were co-transfected with −980 Luc and a vector encoding Renilla luciferase. Five hours after transfection, the cells were incubated with recombinant adenoviruses encoding β-galactosidase (β-gal) or HPV-16 E7 for 3 h. Following a 16-h incubation in normal growth medium, the cells were cultured for an additional 24-h period in the presence or absence of TGF-β1 (10 ng/ml). Luciferase activity in keratinocyte lysates was measured and normalized to Renilla luciferase. The results are expressed as the mean + SEM (n = 3), with luciferase activity in uninfected cells cultured without TGF-β1 set to 1. * indicates p<0.05 (ANOVA).