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J. Biol. Chem., Vol. 280, Issue 18, 18211-18220, May 6, 2005
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From the Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, and Department of Molecular Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
Received for publication, February 7, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because the identification of the founder E2F family member, E2F1, six additional family members have been identified. Structurally, E2F1–6 share a highly conserved DNA-binding and dimerization domain. E2F1–6 bind DNA-target sequences as heterodimers with DP1 or DP2. Although heterodimerization is required for high affinity sequence-specific DNA binding, the specificity of the dimer is determined by the E2F component (10, 12). The most recently identified E2F family member, E2F7, is unique in having a duplicated conserved E2F-like DNA-binding domain and in lacking a DP-dimerization domain. E2F1–5 possess conserved transactivation and pocket protein-binding domains at the C terminus that are absent in E2F6 and E2F7 (10, 13–16). Based on structural and functional considerations, E2Fs have been classified as either "activator" (E2F1, E2F2, and E2F3a) or "repressor" E2Fs (E2F3b, E2F4, E2F5, E2F6, and E2F7). Activator E2Fs are maximally expressed late in G1 and can be found in association with E2F-regulated promoters during the G1-S transition, co-inciding with the activation of many E2F-target genes. Mouse embryonic fibroblasts ablated for all of the three activator E2Fs are severely compromised in E2F-target gene expression as well as the capacity to proliferate, underscoring the importance of the activator subclass in cell cycle progression (28). In contrast to the activator E2Fs, E2F3b, E2F4, and E2F5 are expressed in quiescent cells and can be found associated with E2F-binding elements on E2F-target promoters during G0 phase (12, 13, 17–19). Among the repressor subclass, E2F6 mediates repression via recruitment of the Polycomb group of proteins or complexes containing Mga and Max proteins (20, 21). Although the most recently identified E2F7 protein can function as a repressor independent of DP interaction, its exact mechanism of mediating repression is not understood (14–16).
E2Fs regulate the transcription of a multitude of genes involved in DNA replication, cell cycle regulation, chromatin assembly and condensation, chromosome segregation, DNA repair, and checkpoint control (10–13). Consistent with its role in various important cellular processes, E2Fs are evolutionarily conserved among plants and animals with the exception of yeast. Drosophila melanogaster has two E2F genes named dE2F1, which acts as an activator E2F, and dE2F2, which represents a repressor E2F. Each of these E2Fs heterodimerize with a single DP protein found in Drosophila called dDP (22). E2F-like activities have also been found in Xenopus laevis and Caenorhabditis elegans (23, 24). In the plant, Arabidopsis thaliana, six E2Fs (AtE2Fa-f) and two DPs (AtDPa-b) have been described. Of these, AtE2Fa-c are reminiscent of the mammalian activator E2F1–3a subclass and E2Fd-f resemble the recently discovered E2F7 in that they possess a duplicated DNA-binding domain and exhibit repressor function. Besides human and mouse, Arabidopsis is the only other species where E2Fs with duplicated DNA-binding domain have been described (25, 26).
Here we report the identification and characterization of yet another mouse E2F family member that we call E2F8, which closely resembles E2F7. E2F8 is expressed in a cell cycle-regulated fashion, has duplicated DNA-binding domains that are essential for binding to consensus E2F-binding DNA elements, represses E2F-target genes, and negatively influences cellular proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Mouse Embryonic Fibroblasts and in Vitro Cell Culture—Primary mouse embryonic fibroblasts (MEFs) were generated using standard techniques as described before (28). All of the cell lines were grown in DMEM with 15% FBS. Cells were starved in DMEM containing 0.2% FBS when they were 
70% confluent. After starving for 48–60 h, they were stimulated to grow with DMEM containing 15% FBS. Cells were harvested at different time points after serum stimulation for BrdUrd incorporation assay, Northern blot assay, and real-time RT-PCR.
BrdUrd Incorporation Assay—BrdUrd was added to the medium, and cells were incubated with BrdUrd for 2 h being harvested and fixed in methanol/acetone 1:1. We stained cells in 35-mm dishes with anti-BrdUrd primary antibody (NA-61 from Oncogene) and anti-mouse rhodamine secondary antibody and counterstained with 4',6-diamidino-2-phenylindole (DAPI). At least 400 cells/35-mm plate were counted.
Northern Blot Assay—For the cell cycle Northern Blot analysis, total RNA was isolated from MEFs using TRIzol (Invitrogen) and mRNA was subsequently purified using PolyATract mRNA isolation system (Promega). Poly(A) mRNA was separated on a 1% agarose gel containing 6% formaldehyde and transferred onto a GeneScreen membrane (PerkinElmer Life Sciences). The mouse tissue Northern blot was purchased from Origene. The 3' 1850 bp of the E2F8 cDNA including the 3'-untranslated region (UTR) was used as a probe for both Northern blot analyses. The probe was radiolabeled with 50 µCi of [
-32P]dCTP using Prime-It RmT (Stratagene). Hybridization was carried on overnight under high stringency conditions (5x saline/sodium phosphate/EDTA, 50% formamide, 5x Denhardt's solution, 1% SDS at 42 °C) and washed several times (0.2x SSC, 0.2% SDS at 65 °C) before autoradiography.
Real-time RT-PCR—Approximately 1 x 106 cells were harvested at the indicated time point, and total RNA was isolated using the Qiagen RNA Miniprep column as described by the manufacturer, including a DNase treatment before elution from the column. Reverse transcription of 2 µg of total RNA was performed by combining 1 µl of Superscript III reverse transcriptase (Invitrogen), 4 µl of 10x buffer, 0.5 µl of 100 mM oligo(dT) primer, 0.5 µl of 25 mM dNTPs, 1.0 µl of 0.1 M dithiothreitol, 1.0 µl of RNase inhibitor (Roche Applied Science), and water up to a volume of 20 µl. Reactions were incubated at 50 °C for 60 min and then diluted 5-fold with 80 µl of water. Real-time RT-PCR was performed using the Bio-Rad iCycler PCR machine. Each PCR reaction contained 0.5 µl of cDNA template and primers at a concentration of 100 nM in a final volume of 25 µl of SYBR Green reaction mixture (Bio-Rad). Each PCR reaction generated only the expected amplicon as shown by the melting-temperature profiles of the final products and by gel electrophoresis. Standard curves were performed using cDNA to determine the linear range and PCR efficiency of each primer pair. Reactions were done in triplicate, and relative amounts of cDNA were normalized to GAPDH. The sequences of the primer pair used for the E2F8 cDNA were 5'-CCGGCACAACCTCACCAAAAC-3' and 5'-TCCCCCGCGTAGAGAAGAGG-3'. Primer sequences of the E2F-target genes are available upon request.
5'-RACE PCR—Total RNA was isolated from wild-type primary MEFs and mouse thymus, and the mouse testis cDNA was purchased in the Marathon-Ready cDNA kit (Clontech). cDNA was prepared, and 5'-RACE PCR was performed using the BD SMART RACE cDNA amplification kit (BD Biosciences) following the manufacturer's protocol. The reverse primer used for 5'-RACE PCR were 5'-TCACGCGTAAGGACTTGTCTTTGC-3' mapping to the exon 6 of E2F8 gene, and the nested primer was 5'-CGTCCCGAGGGTTTTGGTGAGGTT-3' located in the exon 5 of the E2F8 gene. The products of 5'-RACE PCR were cloned using the TOPO TA cloning kit for sequencing (Invitrogen). At least 20 colonies from each tissue type were sequenced.
Luciferase Reporter Assay—A 3.5-kb promoter fragment was isolated from the BAC clone RPCI 24-294G9 and subcloned into pBluescript vector. The primer pair used for amplifying the long promoter (LP) fragment was 5'-GAGAGAGGTACCGTCCTCCAACCCCTCGTTTG-3' and 5'-AGAGAGAAGCTTGCTGAAGTTTCTCGCCTGACAC-3' and that for amplifying the short promoter (SP) fragment was 5'-GAGAGAGGTACCAGCTCTGAAGGAGGATTGACAGG-3' and 5'-AGAGAGAAGCTTGCTGAAGTTTCTCGCCTGACAC-3'. The amplified fragments were subcloned into the pGL2Basic vector using the HindIII and KpnI restriction sites.
The two E2F-binding consensus sites in the E2F8 promoter fragment were mutated using the QuikChange site-directed mutagenesis kit as described by the manufacturer. The mutations introduced are depicted in Fig. 4A. All of the constructs were confirmed by sequencing.
Subconfluent REF52 cells grown in triplicates were transfected using the Superfect reagent (Qiagen). The cells were transfected with the firefly luciferase expression vectors and thymidine kinase (TK) Renilla luciferase as internal control. The cells then were serum-starved in DMEM containing 0.2% FBS for 48 h and stimulated to enter the cell cycle by DMEM containing 15% FBS. Cells were harvested at the indicated time points, and luciferase was detected by the Dual-Luciferase reporter assay system (Promega).
Western Blot Analysis—Cell protein lysates were separated in SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Blots were first incubated in blocking buffer (10% skim milk in Tris-buffered saline plus Tween 20) for 1 h and subsequently incubated overnight in blocking buffer containing the antibody specific for Myc tag 9E10 (Santa Cruz Biotechnology, SC-40). The primary antibody was then detected using horseradish-peroxidase-conjugated secondary antibody and ECL reagent as described by the manufacturer (Amersham Biosciences).
Immunofluorescence—MEFs were grown in 35-mm dishes and transfected with His-Myc-tagged E2F8 or control vector. The cells were fixed with 4% paraformaldehyde and methanol/acetone (1:1). The Myc antibody 9E10 was the primary antibody used, and rhodamine-conjugated anti-mouse IgG (Vector Laboratories) was used as the secondary. The cells were counterstained with DAPI. The procedure for the staining was same as described before (14).
Electromobility Shift Assay for DNA Binding—The probe used for the DNA binding assay was a fragment of the adenoviral E2 promoter containing two E2F-binding sites. The complimentary strands of the probes were biotinylated on the 5' end and were annealed together to make a double-stranded probe end-labeled with biotin. The sequence of the wild-type probe was 5'-TCGAGACGTAGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTAGTCCTTAACTCGA-3', and that of the mutated probe was 5'-TCGAGACGTAGTTTTAAGGCTTAAATTTGAGAAAGGGCTTGAAACTAGTCCTTAACTCGA-3'. The binding reaction was carried out in a 20-µl volume using 40 fmol of biotinylated probe and 8 pmol of non-biotinylated wild-type or mutated probe as and when required. The binding conditions were the same as described before (16). The Supershift analysis was carried out as previously described using an antibody specific against Myc (Santa Cruz Biotechnology, SC-40). Proteins were translated using the TNT Quick Coupled transcription/translation system (Promega). After carrying out the binding reaction at 30 °C for 30 min, Ficoll was added to it to a final concentration of 4% and it was separated on a 4% polyacrylamide gel. After running the gel, it was transferred to Hybond N+ membrane (Amersham Biosciences) and UV-cross-linked. It then was probed using the LightShiftTM chemiluminescent EMSA kit (Pierce) following the manufacturer's protocol. The chemiluminescence was detected on Hyperfilm (Amersham Biosciences).
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Structural Analysis—Sequence alignments of the E2F8 DNA-binding domains by the ClustalW method were used to generate models for E2F8 DNA binding (19). Modeling requests were submitted to the SWISS-MODEL protein modeling server using the previously solved E2F4/DP2 crystal structure Protein Data Bank file (1CF7 [PDB] ) as the template.
Co-immunoprecipitation—Transiently transfected 293 cells were harvested in cold phosphate-buffered saline, and cell pellets were lysed in 10x volume of lysis buffer (0.05 M sodium phosphate, pH 7.3, 0.3 M NaCl, 0.1% Nonidet P-40, 10% glycerol with protease inhibitor mixture). Lysates were incubated with Protein G Plus/protein A-agarose beads (Calbiochem) at 4 °C for 1 h to preclear. The precleared lysates were incubated with appropriate antibody overnight. Protein G Plus/protein A-agarose beads were added and incubated for 1 h at 4 °C. Protein binding to the beads were released and resolved by SDS-PAGE followed by immunoblotting. Immunoprecipitation and Immunoblotting were performed using M2 monoclonal anti-FLAG (Sigma), anti-HA (Roche Applied Science), anti-Myc 9E10 antibodies.
Retroviral Infection—Full-length cDNAs for His-Myc-tagged E2F8 was subcloned into the pBABE retroviral vector containing a hygromycin-resistance gene. High-titer retroviruses were produced by transient transfection of retroviral constructs into the Phoenix-Eco packaging cell line as described previously (33). MEFs were infected with the retrovirus using standard methods and were selected in the presence of 200 µg ml-1 hygromycin.
Proliferation Assay—MEFs were plated at 4 x 104 cells/60-mm plate and grown in DMEM with 15% FBS. Duplicate plates were counted daily using a BD Biosciences Coulter counter and were replated every 72 h at the same density of the initial plating.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis using the University of California Southern California Genome Browser-BLAT tool (29) revealed that the mouse E2F8 gene is located on chromosome 7 and contains 13 exons separated by 12 introns (Fig. 1A). We sequenced a BAC clone containing the E2F8 gene to determine the sequences across the splice junctions. From 5' to 3' end of the gene, the 12 splice sites have the universal consensus splice junction dinucleotides GT/AG.
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E2F8 Protein Characteristics and Homology with the Other E2Fs—The E2F8 protein consists of 860 amino acids with a predicted molecular mass of 95 kDa. It has two E2F-like DBDs and three putative nuclear localization signals (Fig. 1, C and D). The presence of two E2F-like DBDs is reminiscent of the mammalian E2F7 and Arabidopsis E2Fd-f. The alignment of the DBDs of mouse E2F1–8 and Arabidopsis E2Fd using the ClustalW program shows high homology with notable conservation of the RRXYDI DNA recognition motif (Fig. 2A). Despite significant homology in the duplicated DBD, E2F8 is devoid of any other E2F-like domains including the pocket protein-binding, transactivation, and DP dimerization domains, a characteristic shared by mammalian E2F7 and Arabidopsis E2Fd-f proteins (Fig. 2C).
Phylogenetically, the DBDs of Arabidopsis E2Fd-f and mouse E2F7 and E2F8 cluster together (Fig. 2B). Significantly, when the full-length mouse E2F proteins were analyzed for evolutionary relationship on the basis of their primary structure, we also obtained a segregation pattern that is reflective of their known functional characteristics. The acquisition of additional E2Fs may stem from a developmental requirement for E2F activity in multiple different tissues as organisms evolve to be structurally and functionally more complex down the path of evolution (Fig. 2D).
E2F8 Expression Pattern in Tissues and over the Cell Cycle—As a first step toward the understanding of E2F8 function, we investigated its tissue and cell cycle expression patterns. A 3' 2500-bp fragment of E2F8 cDNA, which lacked any significant sequence overlap with any of the other known E2Fs, was used to probe tissue-specific Northern blots. We found that E2F8 is highly expressed in the liver, skin, thymus, and testis but not in the brain, muscle, and stomach (Fig. 3A). Interestingly, this pattern of expression is almost identical to that previously found for E2F7, giving rise to the possibility that E2F7 and E2F8 may have overlapping or complementary functions in these organs.
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2.9-kb genomic fragment containing the -1993 to +855 region of the E2F8 gene into a firefly-luciferase transcriptional reporter plasmid and called this construct LP. A second E2F8 genomic fragment extending from +166 to +855 bp, which represents the most proximal sequence near exon 1c (Fig. 4A), was also cloned into the firefly-luciferase reporter plasmid, which we called the SP. We tested the promoter activity of these constructs in REF52 cells using the TK promoter-driven Renilla luciferase plasmid as an internal control for transfection efficiencies. Since both our reporter constructs contained the E2F DNA-binding elements a and b, we first sought to determine whether these constructs were responsive to the overexpression of E2F1, an activator of E2F. As expected, E2F1 overexpression led to a dose-dependent activation of luciferase activity from either the LP or SP reporter construct (Fig. 4B). Thus, we hypothesized that the observed growth-dependent expression of E2F8 could be the result of an E2F autoregulatory loop mediated via these putative E2F-binding elements. To test this hypothesis, we analyzed the transcriptional activity of these reporter constructs in synchronized REF52 cell populations. We transfected our reporter constructs in REF52 cells and serum-starved them in medium containing 0.2% FBS for 60 h in order to synchronize them in G0 and then re-stimulated the cells with medium containing 15% FBS. As expected, the expression from both the SP and LP constructs was significantly increased in the18-h re-stimulated samples, indicating that the cell growth regulation of E2F8 is, at least in part, transcriptional in nature (Fig. 4C). To assess the role of the two consensus E2F-binding elements in regulating the expression of E2F8, we mutated both the E2F-binding elements, a and b, in the SP reporter construct (SP*ab) and re-assessed their promoter activity in E2F1-overexpressing cells. As shown in Fig. 4E, the mutation of the two E2F DNA recognition sites reduced but did not completely eliminate the E2F responsiveness of these reporters, indicating that additional non-consensus E2F-target sites must also mediate their E2F responsiveness. To determine whether these consensus E2F sites played a role in the growth regulation of E2F8, we analyzed the activity of both the wild-type (SP) and mutant SP reporter constructs (SP*b) in quiescent REF52 cells. Mutation of site b (SP*b) led to a small but reproducible 2-fold increase in reporter expression, indicating that E2F8 expression in quiescent cells is likely repressed through an E2F-dependent mechanism (Fig. 4D). Although the expression of E2F8 is likely to be complex, the above data suggest that the E2F-binding sites contribute to the positive and negative regulation of its expression during the cell cycle.
Subcellular Localization and DNA Binding Activity of E2F8 —To gain insight into the possible function of the E2F8 protein product, we overexpressed a Myc-tagged version of the murine E2F8 protein (Myc-E2F8) in MEFs and assessed its effect on cellular proliferation. Western blot analysis of Myc-E2F8-transfected cell lysates using Myc epitope-specific antibodies (9E10) revealed a protein product that migrated in SDS-PAGE with a mobility of 115 kDa. Two additional Myc-E2F8-specific products of 110 and 82 kDa were also evident in these lysates and probably represent degradation cleavage products of the full-length protein (Fig. 5A). Consistent with the presence of three potential nuclear localization signals, immunofluorescence microscopy using anti-Myc epitope antibodies revealed Myc-E2F8 to be completely localized to the nucleus (Fig. 5B).
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Similar to Arabidopsis E2Fd-f and mammalian E2F7, E2F8 contains two DBDs (referred to as DBD1 and DBD2 from N to the C terminus). This domain arrangement is identical to the recently described E2F7 protein, which binds DNA independently of DP (16). Given this similarity and the high degree of homology between E2F and DP DNA-binding domains, we tested whether modeling of E2F8 would allow the DNA-binding domains to adopt a structure homologous to E2F/DP binding as determined from the crystal structure of the E2F4/DP2 heterodimer (31). Sequence alignments by the ClustalW method and through the SWISS-MODEL server showed that DBD2 has a higher sequence homology to the DP DNA-binding domain than DBD1. Based upon conservation of the residues involved in heterodimerization and the binding of the E2F DNA consensus sequence, we were able to model E2F8 binding to DNA with DBD2 adopting the structure of the DP binding partner. Our in silico analysis and model for DNA binding suggest that E2F8 binds DNA with DBD1 in the position of the E2F binding partner and DBD2 in the position of the DP binding partner (Fig. 5E).
To determine whether each domain directly contributes to E2F8 DNA binding activity, we have introduced point mutations in DBD1, DBD2, or both DBD1 and DBD2 (DBD1–2) that are predicted to disrupt DNA binding activity. The conserved leucine 118 and glycine 119 in DBD1 and leucine 266 and arginine 267 in DBD2 were replaced with glutamate and phenylalanine, respectively (Fig. 5E). These leucines contribute to the dimerization interface of the DNA-binding domains and are conserved across all of the E2F family members as shown in Fig. 2A. In E2F1, the corresponding conserved leucine at position 132 is thought to make important heterodimerization contacts with DP and its mutation abrogates DNA binding activity (31, 32). Disruption of these two amino acids in DBD1 or DBD2 of E2F8 completely abrogated its DNA binding capacity, indicating that the integrity of both of the DBDs of E2F8 is important for its DNA binding function (Fig. 5D). These observations are consistent with our modeling of E2F8, which indicates that DNA binding is dependent upon dimerization interactions at both interfaces of DBD1 and DBD2 (Fig. 5E) (16, 26).
E2F8 Forms Homodimers—Previous work from LaThangue's group demonstrated that the related E2F7 family member can form homodimers (16). To test the possibility that E2F8 could also oligomerize, we co-expressed FLAG-tagged E2F8 and HA-tagged E2F8 in the 293 cells and assessed their ability to interact with each other by immunoprecipitation and immunoblotting using anti-FLAG or anti-HA antibodies. To rule out any nonspecific antibody interactions, singly transfected cells were also immunoprecipitated with either FLAG-E2F8 or HA-E2F8 as controls (Fig. 6A). In this analysis, HA-tagged E2F8 could be detected in the anti-FLAG immunoprecipitates derived from the doubly transfected cells but not from the singly transfected HA-tagged E2F8 samples. Likewise, FLAG-tagged E2F8 could be detected in the HA immunoprecipitates from doubly but not singly transfected samples. These data indicate that E2F8 can indeed form oligomers.
These findings raise the possibility that the inability of the E2F8 DBD mutants to bind to E2F consensus sites could be due to the disruption of E2F8 oligomerization. Co-immunoprecipitation assays demonstrated that the single (DBD1 or DBD2) or double (DBD1–2) DBD mutants of E2F8 still retained the capacity to oligomerize (Fig. 6B and data not shown). These results demonstrate that oligomerization of E2F8 is independent of its DNA binding activity.
E2F8 Overexpression Blocks Cellular Proliferation—E2Fs are thought to be critical players in orchestrating the control of cellular proliferation. To determine the potential role of E2F8 in the control of cellular proliferation, E2F8 was overexpressed in MEFs and proliferation was monitored over a period of 7 days. To this end, primary MEFs were infected with retroviruses expressing Myc-E2F8 and transduced cells were selected for 48 h in hygromycin. Cells were then plated in medium containing 15% FBS, and viable cells were harvested and counted every 24 h over a period of 7 days. Relative to control-treated cells, MEFs overexpressing Myc-E2F8 proliferated considerably slower (Fig. 7A). Consistent with the observed growth retardation, the expression of E2F-target genes in synchronized populations of Myc-E2F8-overexpressing cells was significantly reduced (Fig. 7B). Whether the inhibition of E2F-target gene expression is a direct or indirect effect of E2F8 overexpression remains to be determined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E2F8 is an E2F member based on the presence of conserved DNA-binding domains and its ability to bind to E2F consensus sites found in many E2F-regulated promoters. Structure modeling predicts that the duplicated DBDs of E2F8 can interact with each other to form a functional DNA binding unit, thus alleviating the requirement to interact with DP. Interestingly, this modeling predicts that the key conserved leucine residues, which are important for the interaction between the DBDs of the E2F and DP heterodimers, are also important for the intramolecular interactions between DBD1 and DBD2. This prediction is supported by our data demonstrating that introduction of point mutations at this conserved leucine residue in either of the E2F8 DBDs abrogates DNA binding activity. Co-immunoprecipitation assays demonstrate that E2F8, like E2F7, can form homodimers, suggesting that these two unique E2Fs could potentially form contacts with multiple consensus E2F sites at once.
E2F7 and E2F8 share a number of characteristics that could reflect their unique function. Each is expressed in a cell growth-dependent manner with peak levels found during S phase and is expressed in the same adult tissues of mice. Both have the ability to homodimerize and to repress E2F-dependent gene expression. Importantly, their overexpression can lead to a pronounced decrease in the proliferative capacity of cells. These observations have led us to suggest that these two E2F family members may have overlapping and/or synergistic functions in the control of cellular proliferation. Although the identification of E2F8 adds further complexity to the E2F family of transcription factors, our findings begin to place E2F members into distinct subclasses that have general structural and functional themes that might be used to differentially regulate cellular proliferation.
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FOOTNOTES |
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Supported by T32 National Institutes of Health postdoctoral fellowship.
To whom correspondence should be addressed: Dept. of Molecular Virology, Immunology and Medical Genetics, and Dept. of Molecular Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210. Tel.: 614-688-4567; Fax: 614-688-4245; E-mail: Leone-1{at}medctr.osu.edu.
1 The abbreviations used are: Rb, retinoblastoma; DBD, DNA-binding domain; RACE, rapid amplification of cDNA ends; MEFs, mouse embryonic fibroblasts; LP, long promoter; SP, short promoter; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; UTR, untranslated region; ORF, open reading frame; BrdUrd, 5-bromo-2'-deoxyuridine; HA, hemagglutinin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin probe as a loading control. B, primary MEFs were brought to quiescence by serum starvation and stimulated to proliferate by the addition of fresh medium with 15% serum. Cells were harvested for RNA at 0, 12, 18, and 24 h after serum stimulation as indicated, and Northern blot analysis was performed as described under "Materials and Methods." The membrane was probed with E2F8-specific probe or with GAPDH probe as a loading control. C, primary MEFs or p53-/- MEFs were brought to quiescence by serum starvation and stimulated to proliferate by the addition of 15% FBS containing medium. Cells were harvested for RNA at 0, 9, 12, 15, 18, 21, and 24 h after serum stimulation, and real-time RT-PCR was performed with primers for E2F8 and Cdc6 as described under "Materials and Methods." Real-time RT-PCR for GAPDH was done to standardize the amount of cDNA in each sample. Cells treated identically were harvested for BrdUrd incorporation. BrdUrd was added to the medium 3 h before harvesting. Cells were stained with anti-BrdUrd antibody with DAPI counterstaining, and BrdUrd-positive cells were counted as described under "Materials and Methods."
