Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription - PubMed (original) (raw)

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

Jesper Christensen et al. Nucleic Acids Res. 2005.

Abstract

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle.

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Figures

Figure 1

Analysis of the E2F8 ORF. (A) Alignment of the amino acid sequences predicted from the ORFs of human E2F8 and murine E2F8 using Vector NTI and a Clustal W algorithm. Identical residues are shaded in grey. A putative nuclear localization signal was identified using the ScanProsite program provided by the ExPASy World Wide Web molecular biology server of the Swiss Institute of Bioinformatics (SIB) (

) and is indicated by a black line. KEN boxes, which can mediate degradation through the ubiquitin-proteasome pathway, are indicated by asterisks. (B) Alignment of the amino acid sequences predicted from the ORFs of hE2F8 and hE2F7. Identical residues are shaded in grey. The highly conserved regions, which contain the two distinct DNA-binding domains aligned in (C) are boxed. (C) Alignment of the DNA-binding domains of E2F8 with the DNA-binding domains of the other E2F members. Identical and similar residues are shaded in grey. (D) Phylogenetic tree of E2F family based on NJ method of Saitou and Nei. The NJ method works on a matrix of distances between all pairs of sequence to be analysed. These distances are related to the degree of divergence between the sequences. The phylogenetic tree is calculated after the sequences are aligned. (E) Domain structure of E2F8 compared with the other members of the E2F family. Number of amino acids is indicated on the right. Shaded boxes indicate homologous regions. CycA, Cyclin A binding site; DB, DNA binding domain; Dim, dimerization domain; TA, transactivation domain; PB, pocket protein (pRB family) binding domain.

Figure 2

Expression of E2F8. Western blot analysis of HeLa cells transfected with different E2F8 expression constructs. HeLa cells were transiently transfected with expression vectors encoding untagged or N-terminally tagged versions of hE2F8 or mE2F8 as indicated. pCMV mediates expression of untagged hE2F8 or mE2F8, while pCMV-Ha, pCMV-Myc, pGal and pVP16 express E2F8 fused to a Ha-epitope tag, myc-epitope tag, yeast Gal4 DNA binding domain and herpes virus simplex VP16 transcriptional activation domain, respectively. Expression of E2F8 was detected using a rabbit polyclonal antibody against murine E2F8. The positions of a standard set of molecular weight markers are shown on the left.

Figure 3

Expression of E2F8 and E2F7 in various cell lines. The mRNA levels of E2F8 and E2F7 were analysed by real-time qPCR. RNA was extracted from exponentially growing cell lines. Origin of cell lines: C33A, cervix carcinoma; HCT116, adenocarcinoma; HeLa, cervix carcinoma; SAOS2, osteogenic sarcoma; U2OS, osteogenic sarcoma; BJ; MRC5; TIG3; and WI38 are all diploid lung fibroblasts. The E2F8 and E2F7 transcripts were quantified by qPCR (real-time PCR). The levels of E2F8 and E2F7 transcripts were normalized relative to β-actin. All data were further normalized to the E2F8 or E2F7 levels present in HeLa cells.

Figure 4

E2F8 is an E2F-target gene. (A) Real-time qPCR analysis of mRNA isolated from U2OS cells stably expressing Estrogen receptor ligand-binding domain fused to E2F1. The cells were grown in the presence or absence of OHT and CHX for 4 h as indicated in the figure. The levels of E2F8 and E2F7 transcripts were normalized to β-actin. All data were further normalized to the levels E2F8 or E2F7 in non-treated cells. (B) ChIP analysis of E2F1, E2F4 and E2F7 binding to the E2F8 promoter. Asynchronously growing U2OS cells were treated with formaldehyde and enrichment of the E2F8 promoter sequences was tested by ChIP using the indicated antibodies. β-Actin, CCNA2 (Cyclin A2) and E2F1 promoters were used, respectively, as negative and positive controls. The percentage of the bound promoter versus total promoter present in the cells is indicated. (C) Sequence of the 5′ region of the putative human E2F8 promoter. A broken arrow indicates the position of the putative transcription initiation site. Boxed sequences represent consensus E2F-binding sites. The arrows labelled pr1 and pr2 show the positions of the primer set used for detection of enriched E2F8 promoter sequences in ChIP assays.

Figure 5

E2F8 transcription is cell-cycle regulated. (A) HeLaS3 cells were synchronized to late G1/early S-phase by a double thymidine block. After release from the block, the cell-cycle profile was analysed by flow cytometry and total RNA was extracted for qPCR analysis at indicated time points. Top of the panel shows the histogram DNA profiles of propidium iodide stained cells. Asynchronous cells are labelled, AS, the time points for analysis are indicated below the figures. The E2F8, E2F7, Cyclin E1 (CycE1) and Cyclin B1 (CycB1) transcripts were quantified by qPCR (real-time PCR). The transcript levels were calculated relative to β-actin. All data were normalized to the transcript levels present in asynchronous HeLa cells. (B) U2OS cells were synchronized to late G2/early M-phase by a thymidine block followed by release into nocodazole-containing medium. After release from the block, the cell-cycle profile was analysed by flowcytometry and total RNA was extracted for qPCR analysis at indicated time points. Top of the panel shows the histogram DNA profiles of propidium iodide stained cells. The time points for analysis are indicated below the figures. After reverse transcription using random priming, the E2F8, E2F7, Cyclin E1 (CycE1) and Cyclin B1 (CycB1) transcripts were quantified by qPCR (real-time PCR). The transcript levels were calculated relative to β-actin. All data were normalized to the transcript levels present at the time of release.

Figure 6

Recombinant E2F8 binds the E2F DNA-binding consensus site. (A) SDS–PAGE analysis of recombinant his-tag purified E2F8 expressed in insect cells. Input is the cleared lysate used for chromatography. Fractions 1–5 denote the fractions eluted from chromatography column. An arrow at the right of the gel indicates the position of recombinant E2F8, and positions of a standard set of molecular weight markers are shown on the left. The gel was stained with Coomassie brilliant blue. (B) EMSA analysis of purified E2F8 containing fractions. One microlitre of each fraction (100–10 ng) shown in (A) were incubated with a 32P-labelled ds-oligonucleotide containing the consensus E2F-site derived from the Adenovirus E2 promoter. (C) Specificity of E2F8 binding by EMSA. Recombinant E2F8 (100 ng) was incubated in the presence of 32P-labelled ds-oligonucleotide containing the consensus E2F site and 3, 9 or 27 molar excess of unlabelled ds-oligonucleotide or a ds-oligonucleotide abolishing the core GCGC sequence of the Wt ds-oligonucleotide and analysed by an EMSA.

Figure 7

E2F8 is a repressor of E2F-responsive promoters. (A) U2OS cells were transfected with 100 ng of the reporter constructs indicated at the top of the panel. The pGAL-Luc reporter plasmid contains the luciferase gene driven by the adenovirus E1B minimal promoter (TATA) fused to five upstream GAL4-binding sites and was co-transfected with increasing amounts of pGAL-E2F8 or pGAL-E2F1 (10, 30 and 90 ng) of expression plasmid, respectively. pGL3-6xE2F contains six consensus E2F sites upstream of a TATA box. The pGL3 E2F1 (−242) contains 242 nt upstream of the transcription initiation site of the human E2F1 promoter linked to a luciferase reporter. pGL3 E2F1 (−242-E2F) is identical to pGL3 E2F1 (−242) except that the E2F-binding sites are mutated. These reporters were co-transfected with 30, 80 or 240 ng of pCMV-E2F8 or pCMV-E2F7b in the presence or absence of constant amounts (30 ng) of pCMV-E2F1 expression plasmid as indicated below the panel. The amount of expression plasmid was kept constant in all assays by addition of empty pCMV vector. For correction of transfection efficiency, 100 ng of pCMV-lacZ was included in all assays and luciferase activity was normalized to β-galactosidase activity. All experiments were performed in triplicate and reproduced at least three times. (B) U2OS cells were transfected with 100 ng of the reporter constructs indicated at the top of the panel. pGL3-CDC6 contains −1524 to + 225 bp of the human CDC6 promoter and pGL3-CyclinE1 contains −207 to 79 bp of the human CCNE1 (Cyclin E1) promoter, respectively. These reporters were co-transfected with 30, 80 or 240 ng of pCMV-E2F8. For normalization of transfection efficiency, 100 ng of pCMV-lacZ was included in all assays as described above.

Figure 8

Ectopic expression of E2F8 inhibits cell proliferation. (A) Ectopic expression of E2F8 in human TIG3 fibroblasts inhibits cell proliferation. Human TIG3-tert-ecoR fibroblasts were transduced with retroviruses encoding E2F8 (E2F8), an E2F8 mutant (E2Fmt) or virus without insert (puro). After puromycin selection, 50 000 or 12 500 of transduced cells were plated in selective medium and, after 3 weeks of incubation, the colonies were stained using crystal violet. (B) Ectopic expression of E2F8 in human TIG3 fibroblasts inhibits cell proliferation. Growth curve using a 3T3 protocol of TIG3 fibroblasts transduced with retroviruses encoding E2F8, an E2F8 mutant or a virus without insert (puro). (C) Ectopic expression of E2F8 in TIG3 fibroblasts inhibits DNA synthesis and causes S-phase accumulation. TIG3 fibroblast were transduced and selected as described above. 800 000 stably transduced cells were plated in 10 cm dishes and after 3 days of incubation the cells were pulsed with BrdU, stained with propidium iodide and analyzed by flowcytometry. The transduced gene is indicated in the top of each panel. The left panels are dot plot analysis of BrdU stained cells, while the right panels represent the corresponding histograms. (D) Ectopic expression of E2F8 represses E2F-target genes and Cyclin B1. TIG3 fibroblast were transduced and selected as described above. 800.000 stably transduced cells were plated in 10 cm dishes and, after 3 days of incubation the cells were harvested. Total RNA was extracted for qPCR analysis. After reverse transcription using random priming, the E2F8, Cyclin A2, Cyclin E1 and Cyclin B1 transcripts were quantified by qPCR (real-time PCR). The transcript levels were normalized relative to β-actin. Left panel shows relative Cyclin A2, Cyclin E1 and Cyclin B1 levels. The transduced gene is indicated below each bar in the histogram. Right panel shows the relative transcript levels of endogenous E2F8 compared with levels transduced by the retroviruses carrying E2F8 or E2Fmt, respectively.

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Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription - PubMed (original) (raw)