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. 2001 Jul;21(14):4684-99.
doi: 10.1128/MCB.21.14.4684-4699.2001.

Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis

S Ishida  1 , E Huang, H Zuzan, R Spang, G Leone, M West, J R Nevins
Affiliations

Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis

S Ishida et al. Mol Cell Biol. 2001 Jul.

Abstract

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G(1)/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G(1)/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G(1)/S, and the second group, which included the mitotic activities, was regulated at G(2). The activation of the G(2) genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.

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Figures

FIG. 1
FIG. 1
Analysis of cell cycle progression in MEFs. (A) MEF cells were synchronized either by serum starvation or by HU block and brought back to the cell cycle progression either by adding serum (left panel) or by adding the fresh medium containing serum without HU (right panel). Cells were harvested at the indicated time points, stained with propidium iodide, and processed for flow cytometry. Percentage of cells in S phase at each time point is plotted. (B) E2F DNA-binding activity in the samples described in panel A. Nuclear extracts prepared from the indicated samples were assayed for E2F DNA-binding activity by electrophoretic mobility shift assay as described in the text. The identity of the indicated E2F binding activities is based on relative gel mobility and identification with specific antibodies. (C) Cyclin E expression during the cell cycle progression. RNA was prepared from the indicated samples and analyzed by Northern blotting, using a cyclin E cDNA probe. An equal amount of mRNA was loaded in each lane. (D) Comparison of gene expression measurement by the Affymetrix GeneChip cyclin E array to that obtained by densitometric scanning of a Northern blot of the same RNA sample. The average difference values of cyclin E gene calculated by the Affymetrix GeneChip expression analysis algorithm were normalized across the two experiments and plotted (■). The intensity of the cyclin E bands in the Northern blot shown in panel C was measured by densitometric scanning. The values are normalized across each experiment and plotted (●).
FIG. 2
FIG. 2
Identification of patterns of gene expression following growth stimulation and during the mammalian cell cycle. Expression profile of individual genes in the serum stimulation and HU release experiments, clustered according to the methods described in the text. The expression level of each gene in the two experiments was displayed by a pseudo-color visualization matrix (6). In each experiment, a vertical column represents all of the clustered genes for a given time point. The intensity of expression, as determined from the average difference values calculated by GeneChip expression analysis (Affymetrix), is depicted by the intensity of red color.
FIG. 3
FIG. 3
Specific examples of genes regulated during the cell cycle. (A) Representative example of expression profile among each cluster is shown with its identification. The average difference value at each time point across the cell cycle experiments is plotted for each gene. NGF, nerve growth factor; RXR, retinoid X receptor. (B) Northern analysis for selected G2 cell cycle cluster genes. RNA samples prepared from the indicated times points of the HU release experiment were analyzed by Northern blotting, using probes for cdc2 and importin-α2. The cyclin E profile is shown for comparison. An equal amount of mRNA was loaded in each lane.
FIG. 3
FIG. 3
Specific examples of genes regulated during the cell cycle. (A) Representative example of expression profile among each cluster is shown with its identification. The average difference value at each time point across the cell cycle experiments is plotted for each gene. NGF, nerve growth factor; RXR, retinoid X receptor. (B) Northern analysis for selected G2 cell cycle cluster genes. RNA samples prepared from the indicated times points of the HU release experiment were analyzed by Northern blotting, using probes for cdc2 and importin-α2. The cyclin E profile is shown for comparison. An equal amount of mRNA was loaded in each lane.
FIG. 4
FIG. 4
Expression of E2F activities in quiescent fibroblasts. (A) Production of E2F binding activity in cells infected with recombinant adenoviruses containing either the E2F1 or E2F2 gene. Quiescent MEF cells were infected with either E2F1- or E2F2-expressing recombinant adenovirus or with GFP-expressing recombinant adenovirus as a control. E2F2 virus was infected at three different multiplicities to obtain the same E2F DNA-binding activity as with E2F1. E2F binding activities were analyzed by E2F DNA-binding assay with the nuclear extracts prepared from the infected cells. (B) Induction of cyclin E RNA accumulation in cells expressing E2F activities. Expression of cyclin E mRNA was analyzed by Northern analysis in E2F1- and E2F2-expressing cells. mRNA was prepared from the same cells analyzed for E2F DNA-binding activities in panel A and subjected to Northern analysis using a cyclin E probe.
FIG. 5
FIG. 5
Relationship of E2F-regulated genes to control in the cell cycle. (A) Genes identified as E2F-induced, plotted as a function of the number of experiments in which the gene was induced, are compared to the cell cycle-regulated clusters shown in Fig. 3. For this comparison, we have included those genes that were scored as induced by either E2F1 or E2F2 in at least three of the six assays that were performed. Although there is some probability that a gene induced in only three of six assays could represent a false-positive, the probability is very small. (B) Northern analysis of E2F gene induction. RNA samples prepared from cells infected with either Ad-Con, Ad-E2F1, or Ad-E2F2 virus were analyzed by Northern blotting using probes for RRM2, Cdc20, cyclin B1, and importin-α2. An equal amount of mRNA was loaded in each lane.
FIG. 6
FIG. 6
E2F gene regulatory pathway. Schematic representation of the pathway initiated upon stimulation of cell growth that leads to an induction of cyclin D/cdk4 activity. A primary target for D/cdk4 is the Rb protein, inactivating its ability to regulate the accumulation of E2F proteins. Previously identified E2F targets include replication enzymes, proteins that form the initiation complex, and activities that regulate the function of the origin complex. The data presented here now identify additional genes (indicated in bold) that fall within these previously defined groups as well as new groups as indicated. pol, polymerase; Enh, enhancer; Rib. red., ribonucleotide reductase; thy., thymidine.
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