A comprehensive ChIP-chip analysis of E2F1, E2F4, and E2F6 in normal and tumor cells reveals interchangeable roles of E2F family members

Abstract

Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F. This very close relationship between E2F binding sites and binding sites for general transcription factors in both normal and tumor cells suggests that a chromatin-bound E2F may be a signpost for active transcription initiation complexes. In general, we found that several E2Fs bind to a given promoter and that there is only modest cell type specificity of the E2F family. Thus, it is difficult to assess the role of any particular E2F in transcriptional regulation, due to extreme redundancy of target promoters. However, Ntera2 carcinoma cells were exceptional in that a large set of promoters were bound by E2F6, but not by E2F1 or E2F4. It has been proposed that E2F6 contributes to gene silencing by recruiting enzymes involved in methylating histone H3. To test this hypothesis, we created Ntera2 cell lines harboring shRNAs to E2F6. We found that reduction of E2F6 only induced minimal alteration of the transcriptome of Ntera2 transcriptome. Our results support the concept of functional redundancy in the E2F family and suggest that E2F6 is not critical for histone methylation.

Figure 1.

The E2F family. Shown is a schematic comparing the domain structure of E2Fs 1–8; not shown are the lower abundance isoforms of E2F6 (Kherrouche et al. 2004). We have chosen E2F1 as a representative of the class of E2F family members that contains the N-terminal cyclin binding domain and the C-terminal RB1 family binding domain, E2F4 as a representative of the class of E2F family members that lacks the cyclin binding domain but contains the RB1 family member binding domain, and E2F6 as a representative of the class of E2F family members that are thought to function as repressors in an RB1-independent manner.

Figure 2.

E2F family members bind near start sites in both normal and tumor cells. ChIP–chip assays were performed using three independent cultures for each of three different cell types (HeLa, GM06990, or Ntera2 cells) and antibodies to E2F1, E2F4, or E2F6. The samples were analyzed on ENCODE arrays (see Methods) and peaks were called for each array using the L1 category of peaks called by the Tamalpais program (Bieda et al. 2006). A binding site was identified if a site was enriched in at least two of the biological replicate experiments for a particular antibody/cell type combination. (A) Shown are the number of E2F1, E2F4, or E2F6 binding sites identified in the ENCODE regions for the three different cell types. Because the E2Fs bind to many of the same regions, the total number of E2F binding sites for a given cell type is less than the sum of the E2F1, E2F4, and E2F6 sites; there were 270 distinct E2F binding sites for HeLa cells, 187 for GM06990 cells, and 232 for Ntera2 cells (see Supplemental Table S2). (B) Shown is the % of each set of E2F1, E2F4, and E2F6 binding sites for each cell type that is within 2 kb of a transcription start site. (C) ChIP–chip binding patterns for a region of chromosome 1 are shown for RNA polymerase II (POLII), TAF1 (TAF), E2F1, E2F4, and E2F6 in GM06990 (GM) cells. The Y-axis indicates fold enrichment of the ChIP sample.

Figure 3.

E2Fs bind to a similar set of target genes in a given cell type. Pairwise comparisons of the percentage of promoters in common in the sets of top 2000 ranked promoters in E2F1, E2F4, and E2F6 ChIP–chip assays in HeLa (A), Ntera2 (B), MCF7 (C), GM06990 (D), and MCF10A (E) cells. In A–C the overlap in top-ranked promoters after comparison of E2F arrays to IgG arrays is also shown. In A–E, the number expected by random chance to be in common when comparing the data sets is also shown. In F, the percentages of promoters in common in two independent E2F6 ChIP–chip assays in the set of the top 25, 50, 100, 500, 1500, and 2000 ranked promoters is shown for MCF10A cells. Also shown in F is the number expected by random chance to be in common when comparing the various numbers of ranked promoters.

Figure 4.

E2F family members show little cell type specificity in binding. (A) Shown are the five cell types used in this study, with an indication of how they are organized into different comparison sets. (B) A comparison of the top 100 E2F1, E2F4, or E2F6 target promoters in both normal cells (GM06990 vs. MCF10A), in the normal vs. tumor breast cells (MCF10A vs. MCF7), and in all pairwise comparisons of the three tumor cell lines is shown; a combined list of the names for the top 100 E2F target promoters in the five cell types is provided as Supplemental Table S4. (C) A comparison of the top 2000 E2F1, E2F4, or E2F6 target promoters in both normal cells (GM06990 vs. MCF10A), in the normal vs. tumor breast cells (MCF10A vs. MCF7), and in all three tumor cell lines is shown.

Figure 5.

E2F6 has a unique set of target genes in Ntera2 cells. The top 2000 ranked promoters in the E2F1, E2F4, and E2F6 ChIP–chip assays from Ntera2 cells were analyzed using the DAVID program. The set of promoters that were in the top 2000 on both E2F6 arrays but not in the top 2000 on either of the E2F1 or E2F4 arrays was also analyzed (E2F6-specific targets). All categories shown are significantly enriched as compared with a random list of genes (see Supplemental Table S6).

Figure 6.

Comparison of RNA levels of E2F target genes. The expression levels of the RNA of the top 2000 E2F1, E2F4, and E2F6 target genes were determined by analyzing RNA from Ntera2 cells using Illumina arrays. For comparison, the set of E2F6-specific genes was also analyzed. Values <300 are considered to represent genes that are not expressed, whereas values >300 represent expressed genes.

Figure 7.

E2F6 binds to a small percentage of the H3me3K9 or H3me3K27 targets. (A) The top 2000 E2F1, E2F4, E2F6, H3me3K9 and H3me3K27 promoters were compared. As a negative control, the top 2000 promoters from a nonspecific IgG ChIP–chip assay were also compared with the E2F target promoters. (B) DAVID analysis of the set of promoters bound by both E2F6 plus H3me3K9 and by E2F6 plus H3me3K27.

Figure 8.

Reduction of E2F6 does not affect histone modifications at HOX promoters. ChIP analysis was performed using antibodies to E2F6 and H3me3K27 in control Ntera2 cells (C) and Ntera2 cells harboring a plasmid that produces shRNAs against E2F6 (KD).

Figure 9.

Categorizing the universe of promoters that bind E2F. Combining the lists of highly enriched (>1.0 on the array) promoters from one array from each of the 15 conditions (3 E2Fs × 5 cell types), we found a total of exactly 8000 unique promoters. Then, we classified each promoter into the displayed categories. Almost half of the 8000 promoters were found in multiple cell types and were bound by multiple E2Fs. A large fraction of the 1 E2F in 1 Cell Type category is comprised of promoters that were only found as Ntera2 E2F6 promoters, consistent with pairwise promoter array analyses across E2Fs and cell types (see Figs. 3 and 4).