The identification of E2F1-specific target genes

Abstract

The E2F family of transcriptional regulators consists of six different members. Analysis of E2F-regulated promoters by using cultured cells suggests that E2Fs may have redundant functions. However, animal studies have shown that loss of individual E2Fs can have distinct biological consequences. Such seemingly conflicting results could be due to a difference in E2F-mediated regulation in cell culture vs. animals. Alternatively, there may be genes that are specifically regulated by an individual E2F which have not yet been identified. To investigate this possibility further, we have analyzed gene expression in E2F1 nullizygous mice. We found that loss of E2F1 did not cause changes in expression of known E2F target genes, suggesting that perhaps E2F1-specific promoters are distinct from known E2F target promoters. Therefore, we used oligonucleotide microarrays to identify mRNAs whose expression is altered on loss of E2F1. We demonstrate by chromatin immunoprecipitation that several of the promoters that drive expression of the deregulated mRNAs selectively recruit E2F1, but not other E2Fs, and this recruitment is via an element distinct from a consensus E2F binding site. To our knowledge, these are as yet undocumented examples of promoters being occupied in asynchronously growing cells by a single E2F family member. Interestingly, the E2F1-specific target genes that we identified encode proteins having functions quite different from the function of known E2F target genes. Thus, whereas E2F1 may share redundant functions in cell growth control with other E2F family members, it may also play an important biological role distinct from the other E2Fs.

Figure 1

Loss of E2F1 has minor effects on expression of known target genes. (A) Reverse transcription–PCR was used to analyze the levels of dhfr, TK, cyclin E, and Rb mRNA in RNA prepared from the livers of C3H/HeJ and C57BL/6J wt vs. E2F1 nullizygous newborn mice. (B) Chromatin immunoprecipitations were performed from C57BL/6J wt and nullizygous E2F1 embryos using antibodies to E2F1 to -6, and a no antibody control. The chromatin was then analyzed by PCR using primers specific for the dhfr, TK, cyclin E, and Rb promoters. For this experiment, as well as those shown in Figs. 3 and 4, the amount of chromatin used in the PCR reaction of the input lane represents 0.4% of the starting material, and the amount of sample used in the PCR reaction of the immunoprecipitated lane represents 6.7% of the precipitated chromatin. Therefore, approximately equal signals in the sample and input lanes suggest that about 6% of the starting material was precipitated by the antibody.

Figure 2

Loss of E2F1 has minor effects on expression of all genes. (A) mRNA was prepared from quiescent or regenerating livers from 6-week-old C3H/HeJ mice and used to probe murine Affymetrix DNA microarrays. The colored boxes represent the difference call for each gene in the Mu6500 tetraset made by the Affymetrix genechip software algorithm (green, no change; red, mRNAs that are higher in regenerating liver; blue, mRNAs that are higher in quiescent liver). (B) mRNA was prepared from the livers of newborn wt or E2F1 nullizygous C3H/HeJ mice and used to probe murine Affymetrix DNA microarrays. The colored boxes represent the difference call for each gene in the Mu6500 tetraset made by the Affymetrix genechip software algorithm (green, no change; red, mRNAs that are higher in wt liver; blue, mRNAs that are higher in null liver). A complete data set for this experiment can be found at our web site.

Figure 3

The identification of E2F1-specific target promoters. For both A and B, the controls and amounts of chromatin used are as described in Fig. 1; the chromatin was analyzed by using primers shown in C. (A) A chromatin immunoprecipitation experiment was performed from liver isolated from adult C3H/HeJ wt or E2F1 nullizygous mice using antibodies to the E2F family members, as well as a no antibody control. (B) A chromatin immunoprecipitation experiment was performed from liver isolated from adult C3H/HeJ wt and E2F1 nullizygous mice using antibodies to C/EBPα and C/EBPβ, as well as a no antibody control. (C) Shown is a schematic of the E2F1-specific promoters and the approximate location of the primers (arrows) used in the chromatin immunoprecipitation experiments. The numbers are relative to +1 being the 5′ end of the mRNA (indicated by a bent arrow).

Figure 4

Analysis of the binding of E2F1 to target promoters in different tissues. A chromatin immunoprecipitation experiment was performed using the indicated tissues and antibodies to the different E2Fs. The chromatin was analyzed by using primers specific for the indicated promoters. The controls and amounts of chromatin used are as described in Fig. 1.