BIOLOGICAL OVERVIEW

E2f and E2f2 are the only two representatives of the E2F transcription factor family in Drosophila. The proteins control the expression of genes that regulate the G1-S transition of the cell cycle. Interestingly, E2f2 can bind to E2F binding sites, but the function of E2f2 appears to be distinct from that of E2f. Cotransfection of E2f2 represses the expression from the PCNA gene promoter while cotransfection of E2f activates the expression (Sawado, 1998). Similar findings are also observed in transfection experiments in which E2f strongly activates transcription, while E2f2 fails to activate a reporter with E2F binding sites. These results suggest that E2f2 may function mainly to repress transcription while E2f functions mainly to activate transcription (Du, 2000). Indeed, while loss of E2f1 function compromises cell proliferation, these defects are due in large part to the unchecked activity of E2f2, since they can be suppressed by mutation of E2f2. Examination of eye discs from E2f1;E2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins (Frolov, 2001). Similarly, during oogenesis E2f2 functions to inhibit widespread genomic DNA synthesis in late stage follicle cells, and may do so by repressing the expression of specific components of the replication machinery (Cayirlioglu, 2001). These studies show how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions (Frolov, 2001).

Studies in mammalian cells strongly indicate that several E2F family members share overlapping functions. If the Drosophila E2F proteins perform functions in vivo that are similar and overlapping, then E2f1;E2f2 double mutants would be predicted to be more severely affected than either the E2f1 or E2f2 single mutants. However, if dE2F1 and E2f2 act antagonistically then this result is unlikely. Although several outcomes are possible, the simplest possibility to consider is that the double mutant might be less severely affected than the single E2f1 or E2f2 mutants (Frolov, 2001).

To approach this problem, animals trans-heterozygous for null alleles of E2f2 (de2f2^76Q1 and de2f2^G5.1) and dE2F1 (de2f1⁹¹ and de2f1^rm729) were generated. Trans-heterozygous combinations of alleles were used to minimize the possible effects of any additional mutations that might be linked in cis. Embryos trans-heterozygous for E2f1 mutations lack the G₁/S transcriptional program, show a dramatic reduction in DNA synthesis after stage 13, and hatch to give severely abnormal larvae. Five days after egg laying (AEL) E2f1 mutant larvae are much smaller than wild type. These animals fail to develop to the third larval instar and contain severely underdeveloped imaginal discs and CNS (Frolov, 2001).

Strikingly, the slow growth and abnormal larval development of the E2f1 mutant are almost completely suppressed by removing the E2f2 activity. Trans-heterozygous double mutants, de2f2^76Q1/de2f2^G5.1; de2f1⁹¹/de2f1^rm729, develop normally without any significant delay in larval growth, reach pupal stage, and finally die as mid- or late-pupae. The imaginal discs and CNS of de2f1;de2f2 double-mutant larvae are similar in development to wild-type primordia (Frolov, 2001).

E2f1 has previously been shown to be required for normal cell proliferation and DNA replication at several stages of Drosophila development. E2f1 mutant clones fail to proliferate in eye and wing imaginal discs. Surprisingly, in eye discs that lack both E2f1 and E2f2, the pattern of DNA synthesis is largely normal. In wild-type or in mutant discs, BrdU staining is localized primarily to the large area of asynchronously dividing cells in the first mitotic wave and in the synchronous second mitotic wave. Similar patterns are observed in E2f2 mutant discs. No BrdU incorporation is detected in the morphogenetic furrow, and even relatively long pulses of BrdU incorporation (2 h) fails to reveal a significant number of ectopic S-phases posterior to the second mitotic wave in the double-mutant discs. Staining for ELAV, a marker of committed neuronal precursors, shows that the onset of neuronal differentiation is not severely perturbed by the absence of E2F proteins (Frolov, 2001).

In examining large numbers of eye discs for these genotypes it was observed that the intensity of label incorporated into the most intensely labeled nuclei during long-term BrdU pulses tended to be slightly reduced in de2f1;de2f2 double mutants relative to wild-type discs. However this reduction is considerably less than the normal variation that occurs between discs of a single genotype. To further examine cell cycle progression in the absence of E2F regulation, de2f2;de2f1 double-mutant discs were double stained with anti-cyclin A and anti-phosphorylated histone H3 (phosH3) antibodies. Cyclin A is commonly used as a G₂ marker because it is expressed in cells that have passed the G₁/S transition and is degraded when cells enter mitosis. The phosH3 antibody preferentially stains M-phase cells. The patterns of Cyclin A and phosH3 staining in de2f2;de2f1 double-mutant eye discs were indistinguishable from wild type. Thus, cells of the eye imaginal disc can proliferate asynchronously in the first mitotic wave, arrest synchronously in the morphogenetic furrow, re-enter the cell cycle synchronously in the second mitotic wave, permanently exit the cell cycle, and differentiate, all in the absence of both dE2F1 and E2f2 (Frolov, 2001).

It is concluded that the loss of E2f2 suppresses the larval growth, cell proliferation, and DNA-replication defects that are caused by the mutation of E2f1. This suppression confirms that dE2F1 and E2f2 have antagonistic functions, at least during larval development. In theory, the effects of E2f2 that are evident in the E2f1 mutant could be caused by an unusual E2f2 activity that is independent of DP transcription factor (dDP). However, the loss of dDP function behaves in a similar way to loss of E2f2 and suppresses the developmental defects of E2f1 mutant larvae. Thus it is most likely that the severe defects seen in E2f1 mutant larvae require the action of E2f2/dDP heterodimers (Frolov, 2001).

The relatively normal patterns of cell proliferation in de2f1;de2f2 mutants are, at first glance, difficult to reconcile with the idea that E2F is a critical regulator of gene expression and cell proliferation. The expression of E2f1-regulated genes was examined in E2f2 and de2f1; de2f2-mutant animals. Third instar eye discs were chosen for this analysis to avoid the possible contribution of maternally supplied products. Initially expression of PCNA, one of the best-characterized E2F-regulated genes, was monitored by in situ hybridization. In wild-type eye discs, the pattern of PCNA expression is tightly coupled to the pattern of DNA replication. High levels of PCNA transcripts are present anterior to the furrow and within a narrow stripe that overlaps with the second mitotic wave. No PCNA expression is present in the furrow and in the cells just anterior to the furrow. In de2f1;de2f2 double-mutant eye discs, PCNA expression is abnormal. In these animals, a weak staining is observed in mutant eye discs without any specific pattern. PCNA transcripts appear to be present at a low level in the anterior portion of the disc, including the morphogenetic furrow and the second mitotic wave. In E2f2 mutant animals, PCNA expression is not downregulated as cells enter the furrow, and this results in an expanded region of PCNA transcription that encompasses both the first and second mitotic waves and the morphogenetic furrow (Frolov, 2001).

To compare the overall levels of E2F-target genes expression, Northern blot analysis was performed using total RNA from third instar eye imaginal discs dissected from E2f2 single and de2f1;de2f2 double-mutant larvae. The steady state level of PCNA transcripts is elevated in the E2f2 mutant and declines in the double mutant to a level that is lower than the wild-type control. These results suggest that E2f2 represses PCNA expression and that the elevated level of PCNA expression in the E2f2 mutant is due to the activity of dE2F1. The expression of MCM3, another proposed target of dE2F1, increases in the E2f2 single mutant and this effect is suppressed in the double mutant, but in this case the total amount of MCM3 RNA in the double-mutant discs is indistinguishable from wild type. Even though the pattern of RNR2 expression has been shown to be dependent on dE2F1, little change was seen in the total level of RNR2 transcripts in E2f1 mutant larvae or in de2f2, or de2f1;de2f2 mutant eye discs (Frolov, 2001).

It is concluded that both E2f1 and E2f2 are required for the normal pattern of PCNA expression. E2f2 is needed to repress PCNA expression in the morphogenetic furrow, whereas E2f1 is required for the high levels of expression in the first and second mitotic waves. Although the pattern of PCNA, MCM3, and RNR2 transcripts depends on E2f1 and/or E2f2, significant levels of each of these transcripts can be detected in the absence of both E2f1 and E2f2 (Frolov, 2001).

Because Cyclin E is one of the best-known targets of E2F and is rate limiting for S-phase entry the pattern of Cyclin E transcription was examined in the de2f2;de2f1 double-mutant animals. The normal pattern of Cyclin E expression is not altered in E2f2 mutant discs and a similar pattern is also evident in de2f2;de2f1 double mutants. However in the absence of both E2f1 and E2f2, the variations in Cyclin E expression are reduced and the pattern of expression is less distinct. Northern analysis shows that the steady-state level of Cyclin E transcripts is not decreased in the absence of E2F proteins. These results are consistent with evidence that E2f1 contributes to the pattern of Cyclin E expression but is not required for Cyclin E transcription. The finding that E2F target genes are expressed in de2f2;de2f1 double mutants may explain, at least in part, why normal cell proliferation is possible in the absence of E2F proteins (Frolov, 2001).

E2f1 provides an essential function in vivo. E2f1 mutants are defective during embryogenesis, show a significant delay in larval growth, and fail to complete larval development. E2f1 mutant embryos lack a G₁/S transcriptional program that normally accompanies S-phase entry and loss of E2f1 leads to an almost complete cessation of DNA synthesis by stage 13 of embryogenesis. Analysis of E2f1 mutant clones in imaginal discs confirms that dE2F1 is required for normal cell proliferation and suggests that E2F also acts in postmitotic cells. Studies of partial loss-of-function alleles in the ovary have implicated E2F in the shut off of DNA synthesis in follicle cells and have shown that E2f1 is required in this cell type for amplification of chorion gene clusters (Frolov, 2001).

dDP mutant embryos resemble E2f1 mutants in lacking a G₁/S transcriptional program, but the effects of dDP mutation on the expression of genes that are normally expressed at G₁/S varies and depends on the target gene examined. Examination of dDP mutant clones and specific alleles of dDP shows that dDP is required during oogenesis and that it is required for the shut off of DNA synthesis in follicle cells. However, the patterns of DNA synthesis and cell proliferation are not severely affected in dDP mutant embryos or dDP mutant larvae, indicating that the functions of dE2F1 and dDP are not equivalent (Frolov, 2001).

The differences between the E2f1 and dDP mutant phenotypes have led to speculation that dE2F1 might have functions that are independent of dDP, or alternatively that dDP might have functions that are independent of dE2F1. One gene that is likely to have an impact on these phenotypes is E2f2, a E2f1-related gene that was uncovered by direct sequencing of transcription units within the 39B-D cytological region, in a two-hybrid screen using RBF1 as bait, and in the Drosophila genome project. Biochemical experiments have shown that E2f2 can cooperate with dDP to generate specific DNA-binding activity on a consensus E2F-binding site and that a E2f2 expression plasmid weakly repressed the transcription of an E2F-reporter construct when transfected into the Drosophila Kc cell line. Nothing is known about the normal function of E2f2. This study shows that E2f2 is a physiological partner for dDP and RBF and that E2f2 acts antagonistically to dE2F1 during Drosophila development. Mutations in E2f2 relieve the block to DNA synthesis and larval development that is caused by mutation of E2f1. These results show that E2F-control of cell proliferation results from the interplay between two types of E2F complexes that have opposing activities and antagonistic functions (Frolov, 2001).

GENE STRUCTURE

cDNA clone length - 1456

Bases in 5' UTR - 160

Exons - 5

Bases in 3' UTR - 160

PROTEIN STRUCTURE

Amino Acids - 370

Structural Domains

Mammalian E2F transcription factors comprise a family of proteins encoded by distinct genes which function in the form of heterodimers with DP proteins. In Drosophila, only a single E2F-related transcription factor, dE2F, has been reported. A cDNA has been identified and characterized encoding another E2F family member in Drosophila, termed E2f2. The predicted amino acid sequence shares 38.8% identity with dE2F, including the QKRRIYDITNVLEGI motif, which is highly conserved in mammalian E2F family members and dE2F. The 18 amino acids, located in the carboxy-terminal region of the mammalian E2F family, sufficient for binding to pRb are also conserved in E2f2. Band mobility shift analyses with glutathione S-transferase fusion proteins reveal E2f2 binding to E2F-recognition sites to be dependent on the presence of dDP protein, in apparent contrast to dE2F. Furthermore, cotransfection experiments in Kc cells demonstrate E2f2 repression of the PCNA gene promoter activity, while E2f causes activation, the target site for the repression being identical to the dE2F-recognition site (Sawado, 1998).

date revised: 1 January 2024

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