Cell cycle-dependent and cell cycle-independent control of transcription by the Drosophila E2F/RB pathway

Abstract

To determine which E2F/RB-family members are functionally important at E2F-dependent promoters, we used RNA interference (RNAi) to selectively remove each component of the dE2F/dDP/RBF pathway, and we examined the genome-wide changes in gene expression that occur when each element is missing. The results reveal a remarkable division of labor between family members. Classic E2F targets, encoding functions needed for cell cycle progression, are expressed in cycling cells and are primarily dependent on dE2F1and RBF1 for regulation. Unexpectedly, there is a second program of dE2F/RBF-dependent transcription, in which dE2F2/RBF1or dE2F2/RBF2 complexes repress gene expression in actively proliferating cells. These new E2F target genes encode differentiation factors that are transcribed in developmentally regulated and gender-specific patterns and not in a cell cycle-regulated manner. We propose that dE2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, dE2F/RBF-mediated repression is exerted on genes that encode an assortment of cellular functions, and these effects are reversed on sets of functionally related genes in particular developmental contexts. As a result, dE2F/RBF regulation is used to link gene expression with cell cycle progression at some targets while simultaneously providing stable repression at others.

Figure 1.

Specific depletion of dE2F/RBF pathway components by RNA interference. (A) Western blot analysis of whole-cell extracts following treatment with luciferase (Luc), dE2F1 (E1), dE2F2 (E2), dDP (DP), RBF1 (R1), RBF2 (R2), or RBF1 and RBF2 (R1&2). (B) Northern blot analysis of total RNA isolated from depleted cells. (C) FACS profile of cells treated for 4 d with luciferase (Luc), RBF1 (R1), RBF2 (R2), dE2F1 (E1), or dDP (DP) dsRNA; and for 8 d with luciferase (Luc), dE2F2 (E2), or both RBFs (R1&2) dsRNA.

Figure 2.

Distinct patterns of transcriptional changes upon loss of individual components of the dE2F/RBF network. Fold-change is expressed as a log₂ of the difference (C) in expression level between control cells and cells depleted for dE2F1 (E1), dE2F2 (E2), dDP (DP), RBF1 (R1), RBF2 (R2), or RBF1 and RBF2 (R1&2). Color code: log₂C ≤ -1.0, dark green; -1.0 ≥ log₂C ≥ -0.6, light green; no change, pale yellow; 0.6 ≤ log₂C ≤ 1.0, orange; log₂C ≥ 1.0, red. Data set is organized by the changes in dE2F1-, dE2F2-, or dDP-treated cells. (A) Down in dE2F1 and in dDP. (B) Down in dE2F1, no change in dDP. (C) Down in dE2F1 and up in dE2F2. (D) Up in dE2F2, no change in dDP. (E) Up in dE2F2 and in dDP.

Figure 3.

Unique and redundant functions for RBFs, and opposing roles for dE2Fs, at individual E2F-regulated promoters. (A) Northern blot analysis using probes to multiple transcripts from each group of targets illustrates differential requirements for RBF1 and RBF2. Total RNA was isolated from luciferase (Luc), dE2F2 (E2), RBF1 (R1), RBF2 (R2), RBF1 and RBF2 (R1&2). Cells were treated for 8 d with dsRNA. (B) Northern blot analysis using probes to Group A and Group E genes shows two extreme modes of E2F regulation. Total RNA was isolated from cells treated for 4 d with luciferase (Luc), dE2F1 (E1), dE2F2 (E2), dDP (DP); and for 8 d with luciferase (Luc) or with dE2F1 and dE2F2 (E1&2).

Figure 4.

Identification of the dE2F/RBF family members present at the promoters of each group of E2F target genes. (A) Antibodies against dE2F1, dE2F2, dDP, RBF1, RBF2, and rabbit anti-mouse serum (NS Ab) were used to enrich for the promoter regions of several E2F target genes. The promoters of Group A, B, C, and D genes associate with all proteins. Group E genes and CG8316 are not bound by dE2F1. The asterisks denote genes that were unaffected in RBF depletions. (B) dE2F1 does not bind to the promoter of CG3105, even in the absence of dE2F2. ChIP was performed with chromatin isolated from cells treated for 8 d with control (Luc) or dE2F2 (E2) dsRNA.

Figure 5.

dE2F/RBF regulation of Group E genes is independent of cell cycle position. In situ hybridization of third instar eye discs (A) or stage 13 embryos (B) from w¹¹¹⁸ (wild type) and de2f2^-/- mutant animals with probes to PCNA or RNR2 (Group A) and Arp53D (Group E). Arp53D does not exhibit the cell cycle-regulated patterns of expression seen with PCNA and RNR2. (C) Binding analysis of dE2F/RBF proteins in S-phase cells at the promoters of DNA polymerase α (Group A) and Arp53D (Group E). dE2F2/RBF repressor complexes are not disrupted in S phase at promoters of Group E genes. (D) dE2F2/RBF complexes repress transcription of Group E genes in S phase. Northern blot analysis of total RNA isolated from S-phase cells treated with control (Luc), dE2F2 (E2), or RBF1 and RBF2 (R1&2) dsRNA.

Figure 6.

dE2F2 is required to repress the transcription of genes with tissue- and/or gender-specific expression patterns. Total RNA isolated from w¹¹¹⁸ (wild-type) male and female adults (w) and from de2f2 mutant male and female adults (e2^-) was analyzed by Northern blot for the expression levels of several Group E and D genes and for the nebbish transcript (a Group C gene).

Figure 7.

A novel picture of E2F transcriptional regulation. (A) The genes regulated by dE2F1 activation and dE2F2 repression are required for the regulation of distinct cellular functions. dE2F1 is required to activate the expression of genes involved in cell cycle progression. dE2F2, on the other hand, is required to repress a variety of tissue-specific genes involved in differentiation. The previously reported gain-of-function of dE2F2 at cell cycle promoters in the absence of dE2F1 is represented by the dotted arrow. (B) The mechanism of differential E2F regulation. The expression of dE2F1-dependent transcripts is coupled with a burst of dE2F1 expression at the G1/S transition of the cell cycle and with high G1/S Cdk activity. dE2F2-dependent transcripts are not activated by dE2F1, and dE2F2/RBF complexes present at their promoters are not disrupted by G1/S Cdks. Their expression requires tissue-specific transcription factors (X) and/or developmentally regulated signals.