The E2F family of transcription factors are key regulators of the cell cycle in all metazoans. While they are primarily known for their role in cell cycle progression, E2Fs also play broader roles in cellular physiology, including the maintenance of exocrine tissue homeostasis. However, the underlying mechanisms that render exocrine cells particularly sensitive to E2F deregulation remain poorly understood. The Drosophila larval salivary gland, like its mammalian counterpart, is an exocrine tissue that produces large quantities of "glue proteins" in the endoplasmic reticulum. Here, we show that E2F activity is important for the exocrine function of the Drosophila salivary gland. The loss of de2f1b, an alternatively spliced isoform of Drosophila E2F1, leads to elevated DNA damage and accumulation of cytoplasmic DNA (cytoDNA) in the salivary glands. Surprisingly, we found that IRE1, a key sensor of the unfolded protein response, is required for endoplasmic reticulum homeostasis during development that is critical for preventing cytoDNA accumulation in the salivary gland. Importantly, we found evidence demonstrating that IRE1 activity is attenuated in de2f1b-deficient salivary glands, contributing to endoplasmic reticulum dysfunction and cytoDNA accumulation. Together, these findings reveal an unanticipated link between endoplasmic reticulum homeostasis and cytoDNA processing and offer mechanistic insights into why exocrine tissues are particularly vulnerable to E2F deregulation.

The E2F family of transcription factors are key regulators of the cell cycle in all metazoans. While they are primarily known for their role in cell cycle progression, E2Fs also play broader roles in cellular physiology, including the maintenance of exocrine tissue homeostasis. However, the underlying mechanisms that render exocrine cells particularly sensitive to E2F deregulation remain poorly understood. The Drosophila larval salivary gland (SG), like its mammalian counterpart, is an exocrine tissue that produces large quantities of "glue proteins" in the endoplasmic reticulum (ER). Here, we show that E2F activity is important for the exocrine function of the Drosophila SG. The loss of de2f1b , an alternatively spliced isoform of Drosophila E2F1 , leads to elevated DNA damage and accumulation of cytoplasmic DNA (cytoDNA) in the SGs. Surprisingly, we found that IRE1, a key sensor of the unfolded protein response, is required not only for ER homeostasis but also for preventing cytoDNA accumulation in the SG. Importantly, we found evidence demonstrating that IRE1 activity is attenuated in de2f1b -deficient SGs, contributing to both ER dysfunction and cytoDNA accumulation. Together, these findings reveal an unanticipated link between ER homeostasis and cytoDNA processing and offer mechanistic insight into why exocrine tissues are particularly vulnerable to E2F deregulation. Author Summary We discovered an unexpected consequence of E2F deregulation in ER homeostasis. The loss of de2f1b in the Drosophila salivary gland impedes physiological activation of an ER-resident protein, IRE1, a central component of the unfolded protein response. We show that IRE1 is required not only for ER homeostasis but also for preventing DNA accumulation in the cytoplasm, a phenotype observed in de2f1b deficient salivary glands. Our study reveals a previously uncharacterized role of IRE1-dependent ER function and offers new perspectives on why exocrine tissues are particularly vulnerable to the loss of E2Fs. ### Competing Interest Statement The authors have declared no competing interest.

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1→S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 5'UTR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression.

Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)-Cdk2 activity, which is known to be largely regulated at the level of transcription. In this study, we report an additional level of E2F-dependent control of CycE-Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). We provide evidence showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE-Cdk2 activity. Furthermore, we demonstrate that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, our study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.

While a plethora of genetic techniques have been developed over the past century, modifying specific sequences of the fruit fly genome has been a difficult, if not impossible task. clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 truly redefined molecular genetics and provided new tools to model human diseases in Drosophila melanogaster. This is particularly true for genes whose protein sequences are highly conserved. Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism whose missense mutations are found in several neurological disorders, including Arts syndrome. In addition, PRPS is deregulated in cancer, particularly those that become resistant to cancer therapy. Notably, Drosophila PRPS shares about 90% protein sequence identity with its human orthologs, making it an ideal gene to study via CRISPR/Cas9. In this review, we will summarize recent findings on PRPS mutations in human diseases including cancer and on the molecular mechanisms by which PRPS activity is regulated. We will also discuss potential applications of Drosophila CRISPR/Cas9 to model PRPS-dependent disorders and other metabolic diseases that are associated with nucleotide metabolism.

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.

Polyploidy is an integral part of development and is associated with cellular stress, aging and pathological conditions. The endoreplication cycle, comprised of successive alternations of G and S phases without cell division, is widely employed to produce polyploid cells. The endocycle is driven by continuous oscillations of Cyclin E/Cdk2 activity, which is governed by E2F transcription factors. In this study, we provide mechanistic insight on how E2F-dependent Cdk oscillations during endocycles are maintained in Drosophila salivary glands. Genetic experiments revealed that an alternative splicing isoform of E2F1, E2F1b, regulates the circuitry of timely S phase entry and exit by activating a subset of E2F target genes. E2F1b regulates the Drosophila ortholog of p27 CIP/KIP -like Cdk inhibitor Dacapo to precisely time S phase entry by controlling the CycE/Cdk2 activity threshold. Upon entry to S phase, E2F1b-dependent PCNA expression establishes a negative feedback loop through the PIP box-mediated degradation of E2F1. Overall, our study uncovers a network of E2F-dependent genetic oscillators that are critical for the periodic transition between G and S phases during endoreplication.

Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders.

Increasing evidence indicates that ribosomes are heterogeneous and perhaps dynamic, in contrast to the classical view of them as constitutive machinery for protein synthesis. In Drosophila melanogaster, nine ribosomal protein genes are each present in two paralogs, and in many of these cases one of the paralogs is primarily expressed in germline tissues. Among these are two genes encoding ribosomal protein S5, called RpS5a and RpS5b. We demonstrate that RpS5b is specifically required for oogenesis. Females lacking RpS5b produce ovaries with polarity defects, fused egg chambers, and overproliferation of posterior follicle cells. Oogenesis ceases at stage 8-9, and widespread apoptosis follows. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting RpS5a in an RpS5b mutant background worsens the RpS5b phenotype and causes complete germline lethality. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, proteome comparison of wildtype and RpS5b ovaries revealed that RpS5b mitochondrial fractions were depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration. As observed by immunohistochemical staining and transmission electron microscopy, RpS5b nurse cell mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We also observed that RpS5b ovaries have elevated ROS, consistent with mitochondrial dysfunction. We conclude that RpS5b-containing ribosomes preferentially associate with mRNAs encoding mitochondrial components and serve an essential function in oogenesis.

Across metazoans, cell cycle progression is regulated by E2F family transcription factors that can function as either transcriptional activators or repressors. For decades, the Drosophila E2F family has been viewed as a streamlined RB/E2F network, consisting of one activator (dE2F1) and one repressor (dE2F2). Here, we report that an uncharacterized isoform of dE2F1, hereon called dE2F1b, plays an important function during development and is functionally distinct from the widely-studied dE2F1 isoform, dE2F1a. dE2F1b contains an additional exon that inserts 16 amino acids to the evolutionarily conserved Marked Box domain. Analysis of de2f1b-specific mutants generated via CRISPR/Cas9 indicates that dE2F1b is a critical regulator of the cell cycle during development. This is particularly evident in endocycling salivary glands in which a tight control of dE2F1 activity is required. Interestingly, close examination of mitotic tissues such as eye and wing imaginal discs suggests that dE2F1b plays a repressive function as cells exit from the cell cycle. We also provide evidence demonstrating that dE2F1b differentially interacts with RBF1 and alters the recruitment of RBF1 and dE2F1 to promoters. Collectively, our data suggest that dE2F1b is a novel member of the E2F family, revealing a previously unappreciated complexity in the Drosophila RB/E2F network.

AbstractPhosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism. While missense mutations ofPRPS1have been identified in neurological disorders such as Arts syndrome, little is known on how they contribute to pathogenesis. We engineeredDrosophila PRPS(dPRPS) alleles that carry patient-derived PRPS missense mutations. AlthoughdPRPSmutant flies develop normally, they have profound defects in autophagy induction and lysosome function. Consequently,dPRPSflies are sensitive to nutrient deprivation as they are unable to break down lipid storage by macroautophagy. In addition, we provide evidence showing thatdRPPSis required for proper cellular response to oxidative stress, providing a possible mechanism by which PRPS1 dysfunction contributes to neurological disorders.

Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism. While missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, little is known on how they contribute to pathogenesis. We engineered Drosophila PRPS (dPRPS) alleles that carry patient-derived PRPS missense mutations. Although dPRPS mutant flies develop normally, they have profound defects in autophagy induction and lysosome function. Consequently, dPRPS flies are sensitive to nutrient deprivation as they are unable to break down lipid storage by macroautophagy. In addition, we provide evidence showing that dRPPS is required for proper cellular response to oxidative stress, providing a possible mechanism by which PRPS1 dysfunction contributes to neurological disorders.

The E2F family of transcription factors are evolutionarily conserved regulators of the cell cycle that can be divided into two groups based on their ability to either activate or repress transcription. In Drosophila, there is only one "activator" E2F, dE2F1, which provides all of the pro-proliferative activity of E2F during development. Interestingly, the de2f1 gene can be transcribed from multiple promoters resulting in six alternate transcripts. In this study, we sought to investigate the biological significance of the alternate transcriptional start sites. We focused on the de2f1 promoter region where tissue and cell-type specific enhancer activities were observed at the larval stage. While a genomic deletion of this region, de2f1(ΔRA), decreased the overall expression level of dE2F1, flies developed normally with no obvious proliferation defects. However, a detailed analysis of the de2f1(ΔRA) mutant eye imaginal discs revealed that dE2F1 is needed for proper cell cycle exit. We discovered that dE2F1 expression during G1 arrest prior to the differentiation process of the developing eye is important for maintaining cell cycle arrest at a later stage of the eye development. Overall, our study suggests that specific alternate transcripts of "activator" E2F, dE2F1, may have a dual function on cell cycle progression and cannot simply be viewed as a pro-proliferative transcription factor.

A Polycomb group protein, Posterior sex combs (Psc), was identified in a genetic screen designed to find factors that can specifically induce morphological defects in rbf1 mutant eyes. We discovered that rbf1 mutations enhance developmental phenotypes caused by Psc overexpression such as ectopic cell death and disorganized ommatidia. Our genetic analysis revealed that Psc-induced developmental defects are strongly influenced by CAF1p55, which is a shared component of several chromatin-associated complexes including a histone chaperone complex, chromatin assembly factor-1 (dCAF-1). Interestingly, the expression levels of dCAF-1 components, CAF1p105 and CAF1p180, are increased in rbf1 mutants, whereas the expression level of CAF1p55 itself remains relatively unchanged. We demonstrated that the increased levels of CAF1p105 and CAF1p180 are required for the hypersensitivity of rbf1 mutant cells to Psc-induced cell death and for the developmentally regulated cell death normally observed in rbf1 mutant eyes. We propose that Caf1p105 and Caf1p180 are important determinants of cell death sensitivity in rbf1 mutant cells and contribute to the genetic interaction between Psc and rbf1.

Summary Mutations in rbf1, the Drosophila homologue of the RB tumour suppressor gene, generate defects in cell cycle control, cell death, and differentiation during development. Previous studies have established that EGFR/Ras activity is an important determinant of proliferation and survival in rbf1 mutant cells. Here, we report that Capicua (Cic), an HMG box transcription factor whose activity is regulated by the EGFR/Ras pathway, regulates both proliferation and survival of RB-deficient cells in Drosophila. We demonstrate that cic mutations allow rbf1 mutant cells to bypass developmentally controlled cell cycle arrest and apoptotic pressure. The cooperative effect between Cic and RBF1 in promoting G1 arrest is mediated, at least in part, by limiting Cyclin E expression. Surprisingly, we also found evidence to suggest that cic mutant cells have decreased levels of reactive oxygen species (ROS), and that the survival of rbf1 mutant cells is affected by changes in ROS levels. Collectively, our results elucidate the importance of the crosstalk between EGFR/Ras and RBF1 in coordinating cell cycle progression and survival.

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.

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes.

Retinoblastoma tumor suppressor protein (pRb) regulates various biological processes during development and tumorigenesis. Although the molecular mechanism by which pRb controls cell cycle progression is well characterized, how pRb promotes cell-type specification and differentiation is less understood. Here, we report that Extra Macrochaetae (EMC), the Drosophila homolog of inhibitor of DNA binding/differentiation (ID), is an important protein contributing to the developmental defects caused by Rb deficiency. An emc allele was identified from a genetic screen designed to identify factors that, when overexpressed, cooperate with mutations in rbf1, which encodes one of the two Rb proteins found in Drosophila. EMC overexpression in an rbf1 hypomorphic mutant background induces cone cell and photoreceptor defects but has negligible effects in the wild-type background. Interestingly, a substantial fraction of the rbf1-null ommatidia normally exhibit similar cone cell and photoreceptor defects in the absence of ectopic EMC expression. Detailed EMC expression analyses revealed that RBF1 suppresses expression of both endogenous and ectopic EMC protein in photoreceptors, thus explaining the synergistic effect between EMC overexpression and rbf1 mutations, and the developmental defect observed in rbf1-null ommatidia. Our findings demonstrate that ID family proteins are an evolutionarily conserved determinant of Rb-deficient cells, and play an important role during development.

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement.

Previous studies in Drosophila melanogaster have demonstrated that many tumor suppressor pathways impinge on Rb/E2F to regulate proliferation and survival. Here, we report that Tuberous Sclerosis Complex 1 (TSC1), a well-established tumor suppressor that regulates cell size, is an important regulator of dE2F1 during development. In eye imaginal discs, the loss of tsc1 cooperates with rbf1 mutations to promote ectopic S-phase and cell death. This cooperative effect between tsc1 and rbf1 mutations can be explained, at least in part, by the observation that TSC1 post-transcriptionally regulates dE2F1 expression. Clonal analysis revealed that the protein level of dE2F1 is increased in tsc1 or tsc2 mutant cells and conversely decreased in rheb or dTor mutant cells. Interestingly, while s6k mutations have no effect on dE2F1 expression in the wildtype background, S6k is absolutely required for the increase of dE2F1 expression in tsc2 mutant cells. The canonical TSC/Rheb/Tor/S6k pathway is also an important determinant of dE2F1-dependent cell death, since rheb or s6k mutations suppress the developmentally regulated cell death observed in rbf1 mutant eye discs. Our results provide evidence to suggest that dE2F1 is an important cell cycle regulator that translates the growth-promoting signal downstream of the TSC/Rheb/Tor/S6k pathway.