E2F Transcription Factors are Critical Regulators of the Cell Cycle, Apoptosis and Mitochondrial Function

The retinoblastoma (pRB) family of tumor suppressor proteins and the downstream E2F transcription factors that they regulate, play an instrumental role in both cell proliferation, and apoptosis. The importance of the Rb-E2F pathway is underscored by the obligatory inactivation of pRB control in most cancers. While the large number of E2F proteins presents an obstacle to genetic analysis in mammals, the highly conserved Rb-E2F families in Drosophila consist of only two E2F members, an activator, dE2f1, and a repressor, dE2f2. Previous studies have shown that proliferation of de2f1 mutant cells is severely reduced due to the unchecked activity of dE2f2. To better understand the mechanistic basis of this cell proliferation, our lab undertook a screen to identify suppressor genes, which when mutated, rescue the reduced cell proliferation of de2f1 mutants. In addition to identifying suppressors that compromise dE2f2-mediated repression, the screen also uncovered suppressors that leave this repression intact. One of the suppressors I investigated in greater detail was the gene belle which encodes an RNA helicase. Mutation of belle leads to a down-regulation of the p21/p27 Cyclin-Dependent Kinase Inhibitor homolog, Dacapo, and a downregulation in Hedgehog pathway activity, which is important for rescuing proliferation of de2f1 mutant cells. In addition to investigating the role of E2F in cell proliferation, my work revealed that E2f1 directly regulates mitochondrial function by regulating the expression of mitochondria associated genes, a novel class of E2F targets. In vivo, the genetic ablation of E2F leads to severe mitochondrial defects, which are phenocopied by targeted downregulation of several of these E2F target genes. These defects are due to the loss of the normal dE2f/dDP-dependent regulation of mitochondria associated targets. Furthermore, I identified a context, the normal induction of apoptosis in response to irradiation, in which this previously unappreciated role of E2F in the regulation of mitochondrial function is critical. Importantly, the function of E2f in regulating mitochondria associated gene expression, as well as the functional significance in respect to DNA damage-induced apoptosis, is conserved in mammalian cells

Loss of dE2F compromises mitochondrial function

E2F/DP transcription factors regulate cell proliferation and apoptosis. Here, we investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because we show that this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. We attribute this defect to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, our results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation

Loss of dE2F compromises mitochondrial function

E2F/DP transcription factors regulate cell proliferation and apoptosis. Here, we investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because we show that this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. We attribute this defect to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, our results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation

dE2F2-Independent Rescue of Proliferation in Cells Lacking an Activator dE2F1▿

In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor

Alternative polyadenylation factors link cell cycle to migration

Abstract Background In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration. Results RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3′ ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration. Conclusions Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration