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. 2004 Nov 24;23(23):4615-26.
doi: 10.1038/sj.emboj.7600459. Epub 2004 Oct 28.

E2Fs link the control of G1/S and G2/M transcription

Wencheng Zhu  1 , Paloma H Giangrande, Joseph R Nevins
Affiliations

E2Fs link the control of G1/S and G2/M transcription

Wencheng Zhu et al. EMBO J. .

Abstract

Previous work has provided evidence for E2F-dependent transcription control of both G1/S- and G2/M-regulated genes. Analysis of the G2-regulated cdc2 and cyclin B1 genes reveals the presence of both positive- and negative-acting E2F promoter elements. Additional elements provide both positive (CCAAT and Myb) and negative (CHR) control. Chromatin immunoprecipitation assays identify multiple interactions of E2F proteins that include those previously shown to activate and repress transcription. We find that E2F1, E2F2, and E2F3 bind to the positive-acting E2F site in the cdc2 promoter, whereas E2F4 binds to the negative-acting site. We also find that binding of an activator E2F is dependent on an adjacent CCAAT site that is bound by the NF-Y transcription factor and binding of a repressor E2F is dependent on an adjacent CHR element, suggesting a role for cooperative interactions in determining both activation and repression. Finally, the kinetics of B-Myb interaction with the G2-regulated promoters coincides with the activation of the genes, and RNAi-mediated reduction of B-Myb inhibits expression of cyclin B1 and cdc2. The ability of B-Myb to interact with the cdc2 promoter is dependent on an intact E2F binding site. These results thus point to a role for E2Fs, together with B-Myb, which is an E2F-regulated gene expressed at G1/S, in linking the regulation of genes at G1/S and G2/M.

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Figures

Figure 1
Figure 1
Distinct patterns of G1/S and G2/M cell cycle regulation of E2F-dependent genes. (A) FACS analysis of REF52 cells that were synchronized by HU block, and then released into the cell cycle. (B) Northern analysis of G2/M gene and G1/S gene expression in HU-synchronized REF52 cells. Approximately 2 μg of mRNA was loaded in each lane. Expression of GADPH was evaluated to confirm equal loading.
Figure 2
Figure 2
Identification of positive and negative E2F elements controlling activities of G2/M promoters. (A) Schematic of human cdc2 and cyclin B1 promoters indicating E2F binding sites, MYB binding sites, CCAAT elements, and CHR elements. All elements are identified based on previous studies (Tommasi and Pfeifer, 1995; Zwicker et al, 1995a; Yun et al, 1999; Wasner et al, 2003), this study, and sequence alignment between human, mouse, and rat promoters in this study. All elements identified are conserved in all three species. (B) Positive and negative E2F elements are necessary for expression of the cdc2 gene. Luciferase activities assayed from REF52 cells transiently transfected with either wild-type (WT), the distal E2F site mutant (dE2Fm), or the proximal E2F site mutant (pE2Fm) human cdc2 promoter constructs. Left panel: mutation of the positive E2F element (dE2Fm) in human cdc2 promoter abolishes promoter activity. Right panel: mutation of the negative E2F element (pE2Fm) increases promoter activity. (C) Positive and negative E2F elements are necessary for expression of the cyclin B1 gene. Luciferase activities assayed from REF52 cells transiently transfected with either wild-type (WT), the distal E2F site mutant (−85), or the proximal E2F site mutant (+1) human cyclin B1 promoter constructs. Left panel: mutation of the positive E2F element (+1) in human cyclin B1 promoter abolishes promoter activity. Right panel: mutation of the negative E2F element (−85) increases promoter activity.
Figure 3
Figure 3
Multiple elements control human cdc2 promoter activity. (A) Schematic of human cdc2 wild-type (WT) and mutant promoters that contain mutations in the MYB binding site (MYBm), the distal CCAAT element (dCCAATm), the CHR element (CHRm), or the proximal E2F site and the CHR element (pE2Fm+CHRm) respectively. The arrows indicate the G1/S transition in the cell cycle. (B) Mutation of the MYB binding site abolishes human cdc2 promoter activity. Left panel: REF52 cells were synchronized by blocking with HU. Right panel: REF52 cells were synchronized by serum starvation. The arrows indicate the G1/S transition. (C) Mutation of the distal CCAAT element dramatically reduces human cdc2 promoter activity. Left panel: REF52 cells were synchronized by blocking with HU. Right panel: REF52 cells were synchronized by serum starvation. The arrows indicate the G1/S transition. (D) Mutation of the CHR element increases human cdc2 promoter activity in quiescence. REF52 cells containing transiently transfected reporter constructs were serum starved for 2 days, and then harvested for luciferase activities.
Figure 4
Figure 4
Interaction of E2F proteins with G2-regulated promoters. (A) Analysis of cell cycle gene expression in T98G cells. Left panel: FACS analysis of T98G cells that were synchronized by HU block and released into fresh 10% FBS containing DMEM for the indicated times. Additional sample was prepared from serum-starved cells (Q: quiescence). Right panel: Northern analysis of cdc2, cyclin B1, cyclin A2, and PCNA messages. (B) Both activator and repressor E2Fs bind to endogenous G2-regulated promoters. T98G cells at quiescence (Q), HU block (0 h), or release from HU block (6, 9, and 18 h) were harvested for ChIP analysis using the indicated anti-E2F antibodies. Immunoprecipitated chromatin was analyzed by semiquantitative PCR using primers in the cdc2, cyclin B1, cyclin A2, PCNA, and β-actin promoters. PCNA, an E2F-regulated G1/S gene, is used here as a positive control, while β-actin, a non-E2F-regulated gene, is used as a negative control. Input material represents 0.05% of the total chromatin material used for a given ChIP reaction.
Figure 5
Figure 5
Distinct interaction of E2F proteins with positive- and negative-acting E2F elements. (A) Schematic of wild-type (WT) and mutant human cdc2 promoters that contain mutations in the distal E2F site (dE2Fm), the proximal E2F site (pE2Fm), and both E2F sites (dbE2Fm). (B) Distinct interaction of E2F proteins with positive- and negative-acting E2F elements. REF52 cells containing transiently transfected reporter constructs were harvested at either HU block (0 h) or 6 h after release (6 h). Reporter plasmids containing chromatin material isolated as described in Materials and methods were immunoprecipitated using the indicated anti-E2F antibodies, and then analyzed by semiquantitative PCR using primers specific to transfected plasmids. Control reaction using the purified normal rabbit serum (NR) (Pierce) is shown. Primers specific to cotransfected plasmids harboring either β-gal (shown here) or renilla (data not shown) gene were used as negative controls. Input chromatin represents 0.1% of the total chromatin material in a given ChIP reaction.
Figure 6
Figure 6
A role for CCAAT and CHR elements in promoting the interaction of activator and repressor E2Fs. (A) NF-Y protein interacts with the endogenous cdc2, cyclin B1, cyclin A2, and PCNA promoters. Albumin promoter with no NF-Y binding was used as a negative control. T98G cells at quiescence (Q), HU block (0 h), or released from HU block (6, 9, and 18 h) were harvested for ChIP analysis using antibodies against NF-Y A and B subunits. (B) The binding of NF-Y to the distal CCAAT element is necessary for the binding of activator E2F3 to the distal E2F site. REF52 cells transiently transfected with wild-type (WT), the distal E2F site mutant (dE2Fm), and the distal CCAAT element mutant (dCCAATm) human cdc2 promoter reporter constructs were harvested at either HU block (0 h) or 6 h after release (6 h). The luciferase activities for these reporter constructs are shown in Figure 3C. The presence of the cdc2 promoter or control sequence (β-gal) from the transfected constructs in immunoprecipitates using anti-E2F3, anti-NF-YA, or control antibodies (NR) was detected by PCR as described in Figure 5. (C) An intact CHR element is necessary for E2F4 binding to the proximal E2F site. REF52 cells transiently transfected with the indicated reporter constructs were made quiescent by serum deprivation for 2 days. The luciferase activities for these constructs are shown in Figure 3D. Plasmid-based ChIP reaction was carried out using anti-E2F4 and control antibody (NR) and the samples were analyzed as described in Figure 5.
Figure 7
Figure 7
A role for B-Myb in the control of G2/M gene expression. (A) The binding of B-Myb to endogenous G2-regulated promoters (cdc2, CycA2, and cyclin B1) is delayed compared to the binding of the activator E2F (E2F3) to these promoters. T98G cells were released from serum starvation and samples were harvested at quiescence (Q), or 9, 12, 15, 18, 21, and 24 h after serum addition for ChIP analysis using the indicated antibodies. Two E2F-regulated G1/S genes (p68 and DHFR) and a non-E2F-regulated gene (albumin) were used as controls. (B) Relative kinetics of B-Myb and E2F3 protein accumulation. Western analysis of E2F3 and B-Myb using 40 μg of nuclear extract of T98G cells harvested at quiescence (Q) or at the indicated times following serum addition. NS: nonspecific band. (C) The binding of B-Myb to endogenous G2-regulated promoters (cdc2, cyclin B1, and cyclin A2) is prolonged (9 h time point) compared to the binding of activator E2F3 to these promoters. T98G cells were made quiescent (Q), blocked with HU (0 h), released from HU for 6, 9, and 18 h, and then harvested for ChIP analysis using the indicated antibodies (α-B-MybN: N-19; α-B-MybC: H115). Two E2F-regulated G1/S genes (cdc6 and PCNA) were used as controls. (D) Mutation in the MYB binding site abolishes the binding of B-Myb to human cdc2 promoter. REF52 cells transiently transfected with wild-type (WT), the distal E2F mutant (dE2Fm), and the MYB site mutant (MYBm) reporter constructs were made quiescent and then restimulated with serum for 18 h. The luciferase activities for these constructs are shown in Figure 3B. The presence of the cdc2 promoter or control sequence (Renilla) from the transfected constructs in immunoprecipitates using anti-E2F3, anti-B-Myb, or control antibodies (NR) was detected by PCR as described in Figure 5. (E) B-Myb is required for G2/M target gene expression. Western analysis of several G2/M genes (cdc2, cyclin A2, and cyclin B1) and G1/S genes (E2F3, DHFR, and cdc6) and C-Myb using cell extracts from asynchronously growing T98G cells with mock transfected as a control (con), or transfected with an effective B-MYB RNAi duplex 1 (#1) and a noneffective B-MYB RNAi duplex 2 (#2) as another control. Cell lysates were prepared 48 h after siRNA transfection. NS=nonspecific band.
Figure 8
Figure 8
C-Myb is not required for G2/M gene expression. (A) C-Myb does not bind to the endogenous G2-regulated promoters. T98G cells were released from serum starvation and samples were harvested at quiescence (Q), or at 9, 12, 15, 18, 21, and 24 h after serum addition for ChIP analysis using the indicated antibodies. One E2F-regulated G1/S gene (cdc6) and a non-E2F-regulated gene (albumin) were used as controls. (B) Western analysis of the above C-Myb ChIP samples. The mouse monoclonal C-Myb antibody (C-2; Santa Cruz) was used to probe the Western blot. (C) C-Myb is not required for G2/M gene expression. Western analysis of several G2/M genes (cdc2, cyclin A2, and cyclin B1), G1/S genes (E2F3, B-Myb, DHFR, and cdc6), and a noncycling gene SP1 using cell extracts from asynchronously growing T98G cells transfected with either a control luciferase siRNA or a C-Myb siRNA. Cell lysates were prepared 48 h after siRNA transfection. NS=nonspecific band.
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