BIOLOGICAL OVERVIEW

The E2F protein is a critical component for normal cell cycle regulation. As a transcription factor, it positively regulates many of the genes required for initiation of S phase (the DNA synthetic phase). The complexity of E2F regulation is fairly well understood in human biology, while the availability of mutations makes it a prime target for investigation in Drosophila.

Drosophila E2F mutants complete early cell cycles, using maternal gene products, but DNA synthesis fails in cycle 17. Instead of undergoing their normal proliferation during a rapid cycle lasting about 40 minutes, cell cycles in the CNS of E2F mutants gradually decrease DNA synthesis after stage 12. Abdominal histoblasts also cease DNA replication in E2F mutants. Messenger RNAs coding for proteins required for DNA synthesis disappear in E2F mutants (Duronio, 1995a).

Interactions with cyclin E are especially complex. Cyclin E, which is the regulatory subunit of the cyclin E/cdc2c kinase heterodimer, regulates entry in S by phosphorylation of target substrates. Cyclin E expression at G1-S requires E2F. Activation of E2F without cyclin E is not sufficient for S phase. Early in G1, ectopic expression of cyclin E alone can bypass E2F and induce S phase. Thus cyclin E is a downstream target of E2F, coupling E2F activity to G1 control (Duronio, 1995b).

How is E2F regulated? In mammalian systems E2F is held inactive by the retinoblastoma (Rb) family of pocket proteins (See Drosophila Retinoblastoma-family protein). Hypophosphorylated Rb can interact with E2F during G1. This complex can bind to DNA and repress transcription of E2F target genes. A chain of events leads first to sequestration and ultimately the release of E2F. The sequestering of E2F is actively carried out by Rb family members until Rb is inactivated by hyperphosphorylation, carried out by cyclin dependent kinases. At this point, E2F is released allowing it to activate genes required for DNA synthesis and entry into S (Zhu, 1995 a and b).

An additional factor conspires to hold E2F inactive in G1 phase: the association of E2F with p53. As a consequence of its ability to physically associate with E2F, the expression of wild-type p53 can inhibit transcriptional activation by E2F. The expression of both E2F1 and DP1 (E2F's dimerization partner) can also downregulate p53-dependent transcriptional activation (O'Conner, 1995).

Equally important as the G1 phase regulation of E2F is the inactivation of E2F during S phase, when it is no longer required for gene activation. In mammalian systems the inactivation of E2F is again carried out by cyclin A dependent kinase activity. Just as there is a physical association of E2F with the Retinoblastoma protein in G1, there is an association of cyclin A with E2F in S phase (Kitagawa, 1995). These G1 and S phase interactions of E2B have yet to be documented in Drosophila. In Drosophila cyclin A is not synthesized until G2, too late to inactivate E2F in S phase.

E2F is capable of driving the G1-S transition of the cell cycle. However, mice in which the E2F-1 gene has been disrupted develop tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that disrupt G1-S transcription late in Drosophila embryogenesis and five mutations in the dDP gene (DP transcription factor) were identified. Sequencing of dDP reveals the presence of several important motifs, including the DNA-binding region, the DEF box that is predicted to be required for DP/E2F heterodimerization, and three other highly homologous regions named DP-conserved box 1 (DCB1), DCB2, and negatively charged box (NCB). Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The mutant phenotypes reveal that both genes promote progression of the cell cycle (Royzman, 1997).

Although the dE2F mutant animals survive through larval life, a dramatic delay is observed in larval growth. It takes between 288 and 432 hr for the dE2F mutant larvae to pupate, compared to 120 hr for heterozygous sibling controls. Five days after egg laying (AEL) the dE2F mutant larvae are very sluggish and much smaller in size than their wild-type counterparts. The polytene salivary gland and diploid imaginal discs can not be identified in the 5-day-old dE2F mutant larvae, presumably because they are so small. The brains are also greatly reduced in size as compared to wild type. The size of the dE2F mutant larvae increases over time, and the internal tissues approached wild-type size. Therefore, DNA replication can occur during this larval period, but it is slow. Replication in the absence of dE2F is further evidenced by the formation of banded polytene salivary gland chromosomes in some of the 12- to 18-day larvae. Although the polytene chromosomes from the dE2F mutant larvae are smaller and more fragile than normal they are clearly visible. Thus, it is concluded that S phase occurs in the absence of dE2F, but dE2F is necessary for timely replication and growth. In addition to the growth delay, the dE2F mutant larvae had another striking phenotype: melanotic pseudotumors are formed. Melanotic tumors are groups of cells within the larvae that are recognized by the immune system and encapsulated in melanized cuticle. They are referred to as pseudotumors to emphasize that they are not necessarily the consequence of hyperproliferation but can be abnormal cells recognized by the immune cells. Small pseudotumors were first observed in the dE2F mutants 7 days AEL, and these early pseudotumors grow and darken as the larvae age. In the dE2F mutants that initiate pupation, numerous additional small pseudotumors form (Royzman, 1997).

Approximately half of the dDP mutant pupae reach adulthood in the pupal case. These adults struggled to eclose but ultimately die. Organisms dissected from the pupal case have essentially normal heads and thoraxes. However, their abdominal defects are severe. This is informative as the head and thorax are derived from imaginal discs, whereas the abdomen arises from the abdominal histoblast nests. The imaginal discs proliferate during larval stages, but the abdominal histoblast nests proliferate during pupal development. Thus, pupal lethality may result from a defect in abdomen formation that occurs during pupal development. Having shown that heat shock dDP rescues the dDP mutants, the developmental period during which ectopic dDP expression is capable of rescuing the lethality of the dDP mutants was defined. Ectopic expression of dDP results in 100% rescue of dDP mutant animals. Thus, the late lethality of dDP mutants is not a manifestation of a defect in the early development of the organism, but rather it stems from defects in larval/pupal life (Royzman, 1997).

The striking observation from the Drosophila dDP and dE2F mutants is that although cyclic transcription of cyclin E, PCNA, and ribonucleotide reductase 2 (RNR2) is not detectable, S phase still occurs. Although the possibility that cyclic transcription of these genes occurs at a low level driven by maternal pools of dDP and dE2F cannot be excluded, the bursts of transcription that normally precede S phase are not essential for the G1-S transition. In these mutants the cell cycle may be driven by basal levels of transcripts and post-transcriptional regulation. The maternal pools of components of the replication machinery can persist until late in development, as evidenced by the fact that mutations in PCNA and MCM2 cause late larval lethality (Royzman, 1997 and references).

E2F positively regulates many of the genes required for initiation of S phase (the DNA synthetic phase). In mammals, the tumor suppressor RB interacts with, and negatively regulates, E2F, but it is not clear whether the function of pRB is solely mediated by E2F. In addition, E2F has been shown to mediate both transcription activation and repression; it remains to be tested which function of E2F is critical for normal development. Drosophila homologs of the RB and E2F family of proteins Rbf and E2f have been identified. The genetic interactions between Rbf and E2f were analyzed during Drosophila development, and the results show that Rbf is required at multiple stages of development. Unexpectedly, Rbf null mutants can develop until late pupae stage when the activity of E2f is experimentally reduced, and can develop into viable adults with normal adult appendages in the presence of an E2f mutation that retains the DNA binding domain but lacks the transactivation domain. These results indicate that most, if not all, of the function of Rbf during development is mediated through E2f. In turn, the genetic interactions shown here also suggest that E2f functions primarily as a transcription activator rather than a co-repressor of Rbf during Drosophila development. Analysis of the expression of an E2F target gene Pcna in eye discs shows that the expression of PCNA is activated by E2f in the second mitotic wave and repressed in the morphogenetic furrow and posterior to the second mitotic wave by Rbf. Interestingly, reducing the level of Rbf restores the normal pattern of cell proliferation in E2f mutant eye discs but not the expression of E2f target genes, suggesting that the coordinated transcription of E2f target genes does not significantly affect the pattern of cell proliferation (Du, 2000).

Given that E2f is just one of many potential targets of Rbf, the dramatic suppression of the rbf mutant phenotypes by E2f mutants is very unexpected. (1) Lowering the activity of E2f can suppress the early larval lethality of the rbf mutants as well as the developmental phenotypes observed in the adult eyes and bristles. (2) An allele of E2f with an intact DNA binding domain but with no transactivation domain or Rbf binding domain can suppress the lethality of rbf null mutants, allowing the double mutant flies to develop into viable adults. Furthermore, these suppressed rbf null adults show normal adult structures. Thus the uninhibited E2f in rbf mutants mediates both the lethality as well as the observed eye and bristle phenotypes in adults. These observations provide strong evidence that E2F mediates most, if not all, of the phenotypes of rbf during development (Du, 2000).

Interestingly, lowering the activity of Rbf can also partially suppress the E2f null phenotypes. There are at least two possible explanations for this observation. One possibility is that Rbf has a function downstream of E2f; the other possibility is that Rbf can affect the E2f mutant phenotypes through a parallel pathway (for example Rbf may be able to regulate the expression of E2f target genes through another target such as dE2F2). The second explanation seems to be more likely for the following reasons: (1) in the suppressed E2f null mutants, the expression of E2f target genes is not restored, and the larvae growth is still greatly retarded, suggesting that reducing the level of Rbf bypasses rather than restores the function lost by E2f mutation; (2) the rbf null mutant phenotype is fully suppressed by an E2f mutant that lacks transcription activation and an Rbf binding domain, demonstrating that Rbf functions upstream of E2f. In summary, these results do not point to a role for Rb downstream of E2f. In contrast, loss of Rbf indeed causes deregulation of the expression of PCNA even in the absence of transcription activation by E2f, supporting the notion that Rbf can regulate the expression of PCNA by targets other than E2f (Du, 2000).

E2F transcription factors can function both to activate transcription and to repress transcription by recruiting RB family proteins to specific promoters. Although analysis of E2f mutants shows that E2f is required for the coordinated expression of replication functions such as PCNA and Ribonucleoside diphosphate reductase small subunit (RnrS), it is not clear whether the lack of transcription of these set of genes is the cause of the larval lethality. It is formally possible that the lethality of E2f mutant is caused by the failure to repress certain critical E2f target genes. Depending on the function of E2f as a transcription activator or a co-repressor of Rbf, completely different predications are expected regarding the genetic interaction between Rbf and E2f. The observation that lowering the level of Rbf can suppress the larval lethality of E2f mutants and allow the E2f mutants to develop into pharate adults, suggests that during Drosophila development, the function of E2f is mainly to activate transcription and not to recruit Rbf to repress transcription (Du, 2000).

In addition to the first E2f to be identified in Drosophila, a second, termed E2f2, has been identified (Sawado, 1998). 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. Taken together, these data suggest a model for the function of Rbf, E2f and E2f2. In this model, E2f functions mainly to activate transcription of the E2f target genes. Rbf negatively regulates the activity of E2f to inhibit the expression of E2f target genes. In addition, Rbf can also repress the expression of E2F targets genes through other targets of Rbf such as E2f2. Thus the expression of E2F target genes will have three different states: activated, when there is free E2f/Dp to activate transcription; repressed, when there is E2f2/Dp/Rbf (and possibly E2f/Dp/Rbf) to repress transcription; and basal, when there is neither activation nor repression. At present, it is not clear whether E2f also has a function to repress transcription during development, nor is it is clear about the function of free E2f2/Dp (Du, 2000).

S phase-coupled E2f1 destruction ensures homeostasis in proliferating tissues

Precise control of cell cycle regulators is critical for normal development and tissue homeostasis. E2F transcription factors are activated during G1 to drive the G1-S transition and are then inhibited during S phase by a variety of mechanisms. The single Drosophila activator E2F (E2f1) was genetically manipulate to explore the developmental requirement for S phase-coupled E2F down-regulation. Expression of an E2f1 mutant that is not destroyed during S phase drives cell cycle progression and causes apoptosis. Interestingly, this apoptosis is not exclusively the result of inappropriate cell cycle progression, because a stable E2f1 mutant that cannot function as a transcription factor or drive cell cycle progression also triggers apoptosis. This observation suggests that the inappropriate presence of E2f1 protein during S phase can trigger apoptosis by mechanisms that are independent of E2F acting directly at target genes. The ability of S phase-stabilized E2f1 to trigger apoptosis requires an interaction between E2f1 and the Drosophila pRb homolog, Rbf1, and involves induction of the pro-apoptotic gene, hid. Simultaneously blocking E2f1 destruction during S phase and inhibiting the induction of apoptosis results in tissue overgrowth and lethality. It is proposed that inappropriate accumulation of E2f1 protein during S phase triggers the elimination of potentially hyperplastic cells via apoptosis in order to ensure normal development of rapidly proliferating tissues (Davidson, 2012).

Thus stabilizing the single Drosophila activator E2f1 in S phase results in apoptosis is necessary to prevent hypertrophy of wing imaginal discs. It is concluded from these data that hyper-accumulation of E2f1 during S phase represents a form of proliferative stress during development that is sensed by the apoptotic machinery and results in the elimination of cells with excess E2f1 activity to maintain homeostasis during tissue growth (Davidson, 2012).

What might be the function of a Drosophila cell's ability to detect abnormal accumulation of E2f1 protein during S phase and subsequently trigger apoptosis? One possibility is that accumulation of E2f1 during S phase resembles instances of abnormally high E2f1 activity that might occur sporadically during rapid growth of a population of precursor cells such as those in the wing imaginal disc. These events could be caused by stochastic or even genetic changes that affect either E2f1 gene transcription or the ability of the CRL4^Cdt2/PCNA pathway to destroy E2f1 after replication factor genes are activated in late G1. The cell's ability to detect E2f1 accumulation in S phase clears these potentially hyperplastic cells from developing tissues via apoptosis, consequently contributing to the balance between cell proliferation and cell death that is necessary for normal tissue growth (Davidson, 2012).

Growing Drosophila imaginal discs possess another mechanism of homeostasis in which a process of compensatory proliferation is activated in order to achieve normal tissue development when as many as 50% of cells are killed by external stimuli like radiation-induced DNA damage. Indeed, in spite of high levels of apoptosis (15% of the cells), 50% of en-Gal4>E2f1^Stable progeny survive until adulthood with about 2/3 of these surviving flies containing wings with somewhat mild morphological defects. Blocking apoptosis with baculovirus p35 when E2f1^Stable is expressed shifts the cell proliferation/apoptosis balance too strongly in favor of cell proliferation, resulting in massive hypertrophy and 100% lethality (Davidson, 2012).

p35 is a broad caspase inhibitor that blocks effector caspase activity at a step downstream of their proteolytic activation. Therefore, cells expressing p35 can initiate apoptosis, but lack the capacity to complete it and are referred to as 'undead cells.' These undead cells produce signals that stimulate unaffected neighboring cells to proliferate. Thus, the dramatic hypertrophy seen in E2f1^Stable/p35 wing discs might be the result of two synergizing growth signals: hyper-active E2f1 and compensatory proliferation from undead cells. The current experiments cannot precisely discern the relative contribution of these two inputs, but E2f1 activity appears to make a larger contribution because E2f1^{Stable/DBD Mut} expression does not cause dramatic overgrowth (Davidson, 2012).

What might explain the 32% of en-Gal4>E2f1^Stable discs that displayed a reduced posterior compartment rather than an overgrown one? The DNA damage observed in eye discs experiments provides a possible answer. Perhaps early in development the 'arrest' class of wing discs sustained enough genomic damage to prevent proliferation, resulting in too small a pool of cells that could respond to the hyper-active E2f1 and undead cell signals to support disc overgrowth. Thus, the wide range of phenotypes that were observed in E2f1^Stable/p35 wing discs may result from multiple influences that act stochastically within the population (Davidson, 2012).

Because endogenous E2f1 is quantitatively destroyed only in S phase, the relative amount of hyper-accumulation of E2f1^Stable is greater during S phase than during any other stage of the cell cycle. Therefore, one possibility is that E2f1^Stable-induced phenotypes result from the stability of E2f1 protein in S phase, and not from general over-expression throughout the cell cycle. Failure to detect E2f1^Stable induced apoptosis in G1-arrested embryonic cells is consistent with this possibility. However, another difference between these embryonic cells and wing discs cells is that the former are cell cycle arrested and the latter are continuingly proliferating during larval development. Thus, another possibility is that S phase-destruction of E2f1 modulates the levels of E2f1 in proliferating cells, and cells that fail to destroy E2f1 during S phase have an increased chance of activating apoptosis at any point in the cell cycle. In either model, S phase E2f1 destruction is not essential for proliferation per se. In marked contrast, E2f1^Stable expression blocks endocycle progression, suggesting that knocking in E2f1^Stable to the endogenous locus would be lethal during development, perhaps even dominant lethal (Davidson, 2012).

E2f1^Stable induces apoptosis at least in part through expression of the pro-apoptotic gene hid. Surprisingly, these events still occur after expression of an E2f1^Stable variant that cannot bind DNA and therefore lacks the ability to stimulate transcription of replication factor genes or cell cycle progression. Instead, E2f1^Stable requires the ability to bind Rbf1 to induce hid gene expression and apoptosis. Genetic disruption of Rbf1 is well known to result in increased hid expression. It is therefore proposed that the inappropriate accumulation of E2f1 in S phase disrupts some aspect of Rbf1 function leading to hid expression and apoptosis (Davidson, 2012).

The data do not discern either the function of Rbf1 that is disrupted by E2f1^Stable or the mechanism of hid induction. While the mechanism connecting Rbf1/E2f1 function and hid may be indirect, some studies suggest that Rbf1 and/or E2f1 could regulate hid directly. It has been demonstrated that Drosophila wing disc cells undergo apoptosis in response to ionizing radiation independently of p53 and that this response requires E2f1 and is triggered by hid expression. In eye discs, loss of Rbf1 function in the MF results in apoptosis that requires E2f1 transactivation function and is accompanied by hid expression. However, whether these effects represent a direct induction of hid by E2f1 is not clear. E2f1 binding at the hid locus has been observed, but the binding site is located ~1.4 kb upstream of the of the start of hid transcription, which is more distal than in well characterized E2F-regulated promoters. When located this far upstream the hid E2f1 binding site fails to activate gene expression in S2 cell reporter assays. hid is also a target of p53, and so any DNA damage resulting from stabilizing E2f1 during S phase, as was observed in eye discs, may also contribute to the activation of hid expression via p53-mediated DNA damage response pathways (Davidson, 2012).

Another possibility is that E2f1, in combination with Rbf1, plays primarily a repressive role at the hid locus. In this model, the result that E2f1^Stable or E2f1^{Stable/DBD Mut} both induce apoptosis would be explained by disruption of Rbf1/E2f1 repressive complexes at the hid locus causing de-repression of hid expression. This model has interesting caveats: what protects the Rbf1/E2f1 complex at the hid locus from being disrupted by Cyclin E/Cdk2, which is active during S phase and inactivates Rbf1-mediated repression of E2f1, or by CRL4^Cdt2-mediate destruction of E2f1? Recent data indicate that the dREAM/MMB complex is required for the stability of E2F/Rbf1 repressive complexes in S phase, and acts to protect these complexes from CDK-mediated phosphorylation at non-cell cycle-regulated genes. While there is yet no evidence that dREAM/MMB regulates hid , this work provides precedent for gene specific Rbf1 regulation during S phase (Davidson, 2012).

Finally, while hid might be a critical player in the response to E2f1^Stable, there are likely other mechanisms responsible for sensing and modulating the apoptotic response to E2f1 levels. For instance, it has been demonstrated that a micro-RNA, mir-11, which is located within the last intron of the Drosophila E2f1 gene, acts to dampen expression of pro-apoptotic E2f1 target genes following DNA damage. In this way, the normal controls of E2f1 gene expression modulate apoptosis. In addition, transgenic constructs lack the normal E2f1 3' UTR, which serves as a site for suppression of E2f1 expression by pumilio translational repressor complexes. Thus, several modes of E2f1 regulation have been bypassed via transgenic expression of E2f1^Stable (Davidson, 2012).

The finding that stabilized Drosophila E2f1 can induce apoptosis independently of transcription and cell cycle progression parallels previous observations made in mammalian cells, albeit with important differences. In mammalian cells, E2F1 can induce apoptosis independently of transcription and cell cycle progression, but apoptosis required E2F1 DNA binding activity, unlike in the current experiments. These studies suggested that DNA binding by E2F1 prevented pro-apoptotic promoters from binding repressor E2F family members (Davidson, 2012).

This comparison of results highlights the way similar phenotypic outcomes in different species can arise from different mechanisms. While mammalian activator E2Fs are also inhibited during S phase, they are not subject to CRL4^Cdt2-mediated, S phase-coupled destruction like Drosophila E2f1. Instead, mammalian activator E2Fs are inhibited by direct Cyclin A/Cdk2 phosphorylation, targeted for destruction by SCF^Skp2, and functionally antagonized by E2F7 and E2F8. The regulation provided by E2F7 and E2F8 is of particular note, as it is essential for mouse development. These atypical E2Fs homo and hetero-dimerize and act redundantly to repress E2F1 target genes independently of pRb family proteins, thus blocking E2F1 from inducing apoptosis. Moreover, the E2F7 and E2F8 genes are E2F1 targets, consequently creating a negative feedback loop that limits E2F1 activity after the G1/S transition. A similar negative feedback loop among factors that regulate G1/S transcription exists in yeast. The analogous Drosophila negative feedback loop involves E2f1 inducing its own destruction by stimulating Cyclin E transcription, which triggers S phase. Therefore, the evolution of eukaryotes has resulted in the use of different molecular mechanism to achieve negative feedback regulation of G1/S-regulated transcription, and in the case of activator E2Fs this regulation is essential for normal development (Davidson, 2012).

Cyclin A-Myb-MuvB-Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease (Rotelli, 2019).

Endoreplication is a common cell cycle variant that entails periodic genome duplication without cell division and results in large polyploid cells. Two variations on endoreplication are the endocycle, a repeated G/S cycle that completely skips mitosis, and endomitosis, wherein cells enter but do not complete mitosis and / or cytokinesis before duplicating their genome again. In a wide array of organisms, specific cell types switch from mitotic cycles to endoreplication cycles as part of normal tissue growth during development. Cells also can switch to endoreplication in response to conditional inputs, for example during wound healing, tissue regeneration, aging, and cancer. It is still not fully understood, however, how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication (Rotelli, 2019).

There are common themes across plants and animals for how cells switch to endoreplication during development. One common theme is that developmental signaling pathways induce endoreplication by inhibiting the mitotic cyclin dependent kinase 1 (CDK1). After CDK1 activity is repressed, repeated G / S cell cycle phases are controlled by alternating activity of the ubiquitin ligase APC/CCDH1 and Cyclin E / CDK2. Work in Drosophila has defined mechanisms by which APC/CCDH1 and CycE / Cdk2 regulate G / S progression, and ensure that the genome is duplicated only once per cycle. Despite these conserved themes, how endoreplication is regulated can vary among organisms, as well as tissues within an organism. These variations include the identity of the signaling pathways that induce endoreplication, the mechanism of CDK1 inhibition, and the downstream effects on cell cycle remodeling into either an endomitotic cycle (partial mitosis) or endocycle (skip mitosis). In many cases, however, the identity of the developmental signals and the molecular mechanisms of cell cycle remodeling are not known (Rotelli, 2019).

To gain insight into the regulation of variant polyploid cell cycles, two-color microarrays have been used to compare the transcriptomes of endocycling and mitotic cycling cells in Drosophila tissues (Maqbool, 2010). Endocycling cells of larval fat body and salivary gland have been shown to have dampened expression of genes that are normally induced by E2F1, a surprising result for these highly polyploid cells given that many of these genes are required for DNA synthesis. Nonetheless, subsequent studies showed that the expression of the E2F-regulated mouse orthologs of these genes is reduced in endoreplicating cells of mouse liver, megakaryocytes, and trophoblast giant cells. Drosophila endocycling cells also had dampened expression of genes regulated by the Myb transcription factor, the ortholog of the human B-Myb oncogene (MYBL2). Myb is part of a larger complex called Myb-MuvB (MMB), whose subunit composition and functions are mostly conserved from flies to humans. One conserved function of the MMB is the induction of periodic transcription of genes that are required for mitosis and cytokinesis. It was these mitotic and cytokinetic genes whose expression was dampened in Drosophila endocycles, suggesting that this repressed MMB transcriptome may promote the switch to endocycles that skip mitosis. It is not known, however, how E2F1 and Myb activity are repressed during endocycles, nor which of the downregulated genes are key for the remodeling of mitotic cycles into endocycles (Rotelli, 2019).

In addition to endoreplication during development, there are a growing number of examples of cells switching to endoreplication cycles in response to conditional stresses and environmental inputs. These cells will be called induced endoreplicating cells (iECs) to distinguish them from developmental endoreplicating cells (devECs). For example, iECs contribute to tissue regeneration after injury in flies, mice, humans, and the zebrafish heart, and evidence suggests that a transient switch to endoreplication contributes to genome instability in cancer. Cardiovascular hypertension stress also promotes an endoreplication that increases the size and ploidy of heart muscle cells, and this hypertrophy contributes to cardiac disease. It remains little understood how similar the cell cycles of iECs are to devECs (Rotelli, 2019).

Similar to the developmental repression of CDK1 activity to promote endocycles, it has been shown that experimental inhibition of CDK1 activity is sufficient to induce endoreplication in flies, mouse, and human cells. These experimental iECs in Drosophila are similar to devECs in that they skip mitosis, have oscillating CycE / Cdk2 activity, periodically duplicate their genome during G / S cycles, and repress the apoptotic response to genotoxic stress. This study uses these experimental iECs to determine how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication cycles, and how similar this remodeling is between iECs and devECs. The findings indicate that the status of a CycA-Myb-AurB network determines the choice between mitotic cycles and endoreplication cycles in both iECs and devECs (Rotelli, 2019).

This study has investigated how the cell cycle is remodeled when mitotic cycling cells switch into endoreplication cycles, and how similar this remodeling is between devECs and experimental iECs. Repression of a CycA-Myb-AurB mitotic network promotes a switch to endoreplication in both devECs and iECs. Although a dampened E2F1 transcriptome of S phase genes is a common property of devECs in flies and mice, this study found that repression of the Myb transcriptome is sufficient to induce endoreplication in the absence of reduced expression of the E2F1 transcriptome. Knockdown of different components of the CycA-Myb-AurB network resulted in endoreplication cycles that repressed mitosis to different extents, which suggests that regulation of different steps of this pathway may explain the known diversity of endoreplication cycles in vivo. Overall, these findings define how cells either commit to mitosis or switch to different types of endoreplication cycles, with broader relevance to understanding the regulation of these variant cell cycles and their contribution to development, tissue regeneration, and cancer (Rotelli, 2019).

The findings indicate that the status of the CycA-Myb-AurB network determines the choice between mitotic or endoreplication cycles (The CycA-Myb-AurB network regulates the choice between cell cycle programs). These proteins are essential for the function of their respective protein complexes: CycA activates CDK1 to regulate mitotic entry, Myb is required for transcriptional activation of mitotic genes by the MMB transcription factor complex, and AurB is the kinase subunit of the four-subunit CPC. While each of these complexes were previously known to have important mitotic functions, the data indicate that they are key nodes of a network whose activity level determines whether cells switch to the alternative growth program of endoreplication. The results are consistent with previous evidence in several organisms that lower activity of the Myb transcription factor results in polyploidization, and further shows that repressing the function of the CPC and cytokinetic proteins downstream of Myb also promotes endoreplication. Importantly, genetic evidence indicates that not all types of mitotic inhibition result in a switch to endoreplication. For example, knockdown of the Spc25 and Spc105R kinetochore proteins or the Polo kinase resulted in a mitotic arrest, not a switch to repeated endoreplication cycles. These observations are consistent with CycA / CDK, MMB, and the CPC playing principal roles in the mitotic network hierarchy and the decision to either commit to mitosis or switch to endoreplication cycles (Rotelli, 2019).

While knockdown of different proteins in the CycA-Myb-AurB network were each sufficient to induce endoreplication cycles, these iEC populations had different fractions of cells with multiple nuclei diagnostic of an endomitotic cycle. Knockdown of cytokinetic genes pav and tum resulted in the highest fraction of endomitotic cells, followed by the CPC subunits, then Myb, and finally CycA, with knockdown of this cyclin resulting in the fewest endomitotic cells. These results suggest that knocking down genes higher in this branching mitotic network (e.g. CycA) inhibits more mitotic functions and preferentially promotes G / S endocycles that skip mitosis, whereas inhibition of functions further downstream in the network promote endomitosis. Moreover, different levels of CPC function also resulted in different subtypes of endoreplication. Strong knockdown of AurB inhibited chromosome segregation and cytokinesis resulting in cells with a single polyploid nucleus, whereas a mild knockdown resulted in successful chromosome segregation but failed cytokinesis, suggesting that cytokinesis requires more CPC function than chromosome segregation. It thus appears that different thresholds of mitotic function result in different types of endoreplication cycles. This idea that endomitosis and endocycles are points on an endoreplication continuum is consistent with evidence that treatment of human cells with low concentrations of CDK1 or AurB inhibitors induces endomitosis, whereas higher concentrations induce endocycles. The results raise the possibility that in tissues of flies and mammals both conditional and developmental inputs may repress different steps of the CycA-Myb-AurB network to induce slightly different types of endoreplication cycles that partially or completely skip mitosis. Together, these findings show that there are different paths to polyploidy depending on both the types and degree to which different mitotic functions are repressed (Rotelli, 2019).

The findings are relevant to the regulation of periodic MMB transcription factor activity during the canonical mitotic cycle. Knockdown of CycA compromised MMB transcriptional activation of mitotic gene expression, and their physical association suggests that the activation of the MMB by CycA may be direct. The MMB-regulated mitotic genes were expressed at lower levels in CycA iECs, even though Myb protein levels were not reduced. This result is consistent with the hypothesis that CycA / CDK phosphorylation of the MMB is required for its induction of mitotic gene expression. Moreover, misexpression of Myb in CycA knockdown follicle cells did not prevent the switch to endoreplication, further evidence that CycA / CDK is required for MMB activity and mitotic cycles. While the dependency of the MMB on CycA was not previously known in Drosophila, it was previously reported that in human cells CycA / CDK2 phosphorylates and activates human B-Myb in late S phase, and also triggers its degradation. While further experiments are needed to prove that CycA / CDK regulation of the MMB is direct, interrogation of the results of multiple phosphoproteome studies using iProteinDB indicated that Drosophila Myb protein is phosphorylated at three CDK consensus sites including one, S381 that is of a similar sequence and position to a CDK phosphorylated site on human B-Myb (T447). The hypothesis is favored that it is CycA complexed to CDK1 that regulates the MMB because, unlike human cells, in Drosophila CycA / CDK2 is not required for S phase, and Myb is degraded later in the cell cycle during mitosis. Moreover, it is known that mutations in CDK1, but not CDK2, induce endocycles in Drosophila, mouse, and other organisms. A cogent hypothesis is that CycA / CDK1 phosphorylates Myb, and perhaps other MMB subunits, to stimulate MMB activity as a transcriptional activator of mitotic genes, explaining how pulses of mitotic gene expression are integrated with the master cell cycle control machinery. It remains formally possible, however, that both CycA / CDK2 and CycA / CDK1 activate the MMB in Drosophila. The early reports that CycA / CDK2 activates B-Myb in human cells were before the discovery that it functions as part of the MMB and the identification of many MMB target genes, and further experiments are needed to fully define how MMB activity is coordinated with the central cell cycle oscillator in fly and human cells (Rotelli, 2019).

Endocycles were experimentally induced by knockdown of CycA to mimic the repression of CDK1 that occurs in devECs. The data revealed both similarities and differences between these experimental iECs and devECs. Both iECs and SG devECs had a repressed CycA-Myb-AurB network of mitotic genes. In contrast, only devECs had reduced expression of large numbers of E2F1-dependent S phase genes, a conserved property of devECs in fly and mouse. In CycA iECs, many of these key S phase genes were not downregulated, including Cyclin E, PCNA, and subunits of the pre-Replicative complex, among others. This difference between CycA dsRNA iECs and SG devECs indicates that repression of these S phase genes is not essential for endoreplication. In fact, none of the E2F1 -dependent S phase genes were downregulated in Myb dsRNA iEC. Instead, the 12 E2F1-dependent genes that were commonly downregulated in Myb dsRNA iEC, CycA dsRNA iEC, and SG devEC all have functions in mitosis. These 12 mitotic genes are, therefore, dependent on both Myb and E2F1 for their expression, including the cytokinetic gene tum whose knockdown induced endomitotic cycles. This observation leads to the hypothesis that downregulation of the E2F transcriptome in fly and mouse devECs may serve to repress the expression of these mitotic genes, and that the repression of S phase genes is a secondary consequence of this regulation. These genomic data, together with the genetic evidence in S2 cells and tissues, indicates that in Drosophila the repression of the Myb transcriptome is sufficient to induce endoreplication without repression of the E2F1 transcriptome. The observation that both CycAdsRNA iECs and devECs both have lower CycA / CDK activity, but differ in expression of E2F1 regulated S phase genes, also implies that there are CDK-independent mechanisms by which developmental signals repress the E2F1 transcriptome in devECs (Rotelli, 2019).

The results have broader relevance to the growing number of biological contexts that induce endoreplication. Endoreplicating cells are induced and contribute to wound healing and regeneration in a number of tissues in fly and mouse, and, depending on cell type, can either inhibit or promote regeneration of the zebrafish heart. An important remaining question is whether these iECs, like experimental iECs and devECs, have a repressed CycA-Myb-AurB network. If so, manipulation of this network may improve regenerative therapies. In the cancer cell, evidence suggests that DNA damage and mitotic stress, including that induced by cancer therapies, can switch cells into an endoreplication cycle. These therapies include CDK and AurB inhibitors, which induce human cells to polyploidize, consistent with the fly data that CycA / CDK and the CPC are key network nodes whose repression promotes the switch to endoreplication. Upon withdrawal of these inhibitors, transient cancer iECs return to an error-prone mitosis that generates aneuploid cells, which have the potential to contribute to therapy resistance and more aggressive cancer progression. The finding that the Myb transcriptome is repressed in iECs opens the possibility that these mitotic errors may be due in part to a failure to properly orchestrate a return of mitotic gene expression. Understanding how this and other networks are remodeled in polyploid cancer cells will empower development of new approaches to prevent cancer progression (Rotelli, 2019).

Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication

DNA endoreplication has been implicated as a cell strategy to grow in size and in tissue injury. This study demonstrates that barrier to autointegration factor (BAF), represses endoreplication in Drosophila myofibers. This study shows that BAF localization at the nuclear envelope was eliminated either in mutants of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, in which the LEM-domain protein Otefin was similarly excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope required the activity of the BAF kinase VRK1/Ball, and consistently non-phosphorytable BAF-GFP was excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlated with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication was found to overlap BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope resulted with increased E2F1 levels in the nucleoplasm, and subsequent elevated DNA content. It is suggested that LINC-dependent, and phospho-sensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation at the nucleoplasm (Unnikannan, 2020).

Endoreplication emerges as an important strategy of differentiated cells, enabling them to grow in size or rescue tissue integrity following injury, in a wide range of non-dividing cell types. Recent experimental studies have proposed a functional link between mechanical inputs and endoreplication events in various cell types. Moreover, mechanical signals transmitted across the nuclear membrane have been implicated in the regulation of cell cycle, epigenetic events and gene transcription. As part of the mechanism linking cell cycle events with mechanical inputs, the translocation of specific essential factors into the nucleus has been proposed. However, the molecular link between nuclear translocation of such factors, mechanical inputs on the nuclear envelope and endoreplication is still elusive (Unnikannan, 2020).

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been suggested to mediate mechanically induced nuclear entry of essential factors (Driscoll, 2015; Horn, 2014; Osmanagic-Myers, 2015). It physically connects the cytoskeleton and the nucleoskeleton at the interface of the nuclear envelope and has been associated with various human myopathies. The LINC complex is composed of Nesprin protein family members, which associate at their cytoplasmic N-terminal end with distinct cytoskeletal components, and on their nuclear C-terminal end with SUN domain proteins at the perinuclear space. SUN domain proteins bind to various nuclear lamina components, resulting in a physical link between the cytoskeleton and the nucleoskeleton. Recent results indicate that, in Drosophila larval muscles, the LINC complex is essential for arresting endoreplication in the muscle nuclei (myonuclei) and that LINC mutants exhibit additional rounds of DNA replication, resulting in elevated polyploidy. The molecular nature of this process is currently elusive (Unnikannan, 2020).

In an attempt to reveal the components downstream of the LINC-dependent arrest of DNA endoreplication, a screen was performed for genes whose transcription changes in Drosophila Nesprin/klar mutant muscles. One of the identified genes was barrier-to-autointegration factor (baf), shown to be significantly reduced at the transcription level. BAF is a small protein of 89 amino acids that binds dsDNA as well as the nuclear envelope, and in addition forms homodimers. Furthermore, BAF binds to the inner components of the nuclear membrane, including the Lap-2, Emerin, MAN1 (LEM) domain proteins, as well as to lamins A/C and B. Thus, BAF dimers might bridge between dsDNA and the nuclear envelope. Proteomic analysis of BAF partners indicate its potential association with additional proteins, including transcription factors, damage-specific DNA binding proteins and histones. Furthermore, the binding of BAF to its potential partners might be regulated by its phosphorylation state. For example, phosphorylated BAF associates with LEM-domain proteins, whereas de-phosphorylated BAF favors binding to dsDNA. One kinase that has been implicated in BAF phosphorylation is the threonine-serine VRK1 kinase, whose homolog in Drosophila is Ballchen (Ball, also known as NHK-1) (Unnikannan, 2020).

BAF has a crucial role in the condensation and assembly of post-mitotic DNA. Its interaction with both dsDNA and the nuclear lamina enables DNA compaction through cross-bridges between chromosomes and the nuclear envelope, a process essential for the assembly of DNA within a single nucleus following mitosis. Likewise, BAF is recruited to the sites of ruptured nuclear membrane, where it is essential for resealing the ruptured nuclear membrane. Interestingly, in humans a single amino acid substitution of BAF causes Nestor-Guillermo progeria syndrome (NGPS); however, the molecular basis for the disease awaits further investigation (Unnikannan, 2020).

Previous studies demonstrated that in Drosophila, muscle-specific knockdown of BAF increases the levels of DNA endoreplication, phenocopying the LINC mutant outcome. This led to the hypothesis that BAF acts downstream of the LINC complex-dependent mechanotransduction in promoting the arrest of DNA endoreplication in muscle. This study demonstrates that BAF localization at the nuclear envelope is crucial for that process, and that it is downstream of the LINC complex, depends on nucleus-sarcomere connections, and is phosphosensitive. Importantly, elimination of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei and a concomitant increase in E2F1 levels in the nucleoplasm. Taken together, these findings suggest a model in which a LINC-dependent localization of BAF at the nuclear envelope promotes E2F1 tethering to the nuclear envelope to inhibit its accumulation in the nucleoplasm (Unnikannan, 2020).

This study demonstrates the contribution of a novel mechanosensitive component, BAF, in controlling the nuclear accumulation of E2F1, a crucial transcription factor required for the regulation of endoreplication. Whereas previous reports implicated BAF in promoting the condensation and assembly of post-mitotic dsDNA into single nuclei, this study demonstrates that BAF is also essential for the arrest of DNA endoreplication in fully differentiated muscle fibers. Importantly, only BAF that localizes to the nuclear envelope appears to be relevant for this function in post-mitotic differentiated cells. The contribution of BAF to larval muscle functionality is unclear, as baf mutants did not survive up to third instar stage and BAF knockdown in muscles by using RNAi did not eliminate BAF very efficiently (Unnikannan, 2020).

In Drosophila muscle fibers, it was found that BAF was detected in various subcellular sites, including the cytoplasm, nuclear envelope, nucleoplasm and at the nucleolus borders. Yet, only the portion of BAF localized at the nuclear envelope was found to change following elimination of a functional LINC complex. It is well accepted that the LINC complex transmits cytoplasmic mechanical inputs from the cytoskeleton to the nucleoskeleton in various cell types. Moreover, nuclear deformations (from oval into spheroid shape) observed both in larval muscles of LINC complex mutants and in conditions where nuclei detach from the sarcomeres or following Sls knockdown are indicative of changes in the mechanical inputs applied on the nuclear envelope. Because BAF localization at the nuclear envelope was specifically impaired in both conditions, it is proposed that maintenance of BAF at the nuclear envelope is mechanically sensitive (Unnikannan, 2020).

In control myofibers, BAF exhibited a relatively broad distribution along the outlines of the nuclear envelope, often extending beyond the Lamin C expression domain towards the cytoplasm, overlapping with the nucleus-associated microtubules. This suggested that, in addition to its association with the inner aspects of the nuclear membrane through binding to LEM-domain proteins and Lamin A/C, BAF associates with the outer aspects of the nuclear membrane. Previous experiments indicate that despite its small size BAF does not diffuse passively from the cytoplasm to the nucleus. Furthermore, photobleaching experiments with GFP-BAF indicate that BAF-dependent repair of nuclear ruptures occurs when cytoplasmic BAF, but not nuclear BAF, rapidly associates with the ruptured sites and further recruits LEM-domain proteins to establish membrane sealing. The authors suggest that their findings are consistent with a dynamic exchange of BAF between cytoplasmic and nuclear pools, where BAF in the cytoplasm primarily responds to mechanical signals. Because the current experiments indicate that BAF phosphorylation is crucial for its maintenance at the nuclear membrane, it is possible that the exchange of BAF localization between the cytoplasm and the nucleus is stabilized by its phosphorylation. The contribution of the LINC complex to BAF association with the nuclear envelope could be either direct (e.g. by binding to components of the LINC complex) or indirect (e.g. through an effect of the LINC complex on the distribution of LEM proteins at the nuclear envelope). The results support the latter model, in which the LINC complex maintains the localization of the LEM protein Otefin at the nuclear envelope to mediate BAF association with the nuclear envelope. Hence, a model is suggested in which the contribution of the LINC complex to BAF localization at the nuclear envelope is through an effect on Otefin localization at the nuclear envelope (Unnikannan, 2020).

Endoreplication has been implicated in a wide variety of differentiated cells in a broad range of species, including human tissues. A link between mechanical tension and endoreplication has been recently suggested. However, the molecular mechanism coupling mechanical tension with the endoreplication process is still elusive. This study found that a key regulator of endoreplication, E2F1, exhibits a specific distribution at the nuclear envelope in fully differentiated myofibers, where it probably resides non-actively. Changes in the mechanical environment of the nuclear envelope correlate with the localization of E2F1 and promote its accumulation within the nucleoplasm, where it is expected to promote DNA synthesis. It will be of interest to find which proteins associate directly with E2F1 at the nuclear envelope. Attempts to co-immunoprecipitate BAF with Msp300 or E2F1 failed to show a specific protein interaction between these proteins. From a physiological point of view, no detectable changes in muscle size or movement were observed in the baf knockdown muscles, and the larvae developed up to adult stage. The baf homozygous mutant did not develop up to the third instar larval stage, so the full physiological contribution of BAF to muscle growth awaits experiments in which a more efficient reduction in BAF levels is induced in muscle tissue (Unnikannan, 2020).

In summary, these results reveal a novel insight into the role of the LINC complex in coupling endoreplication with changes in the nuclear envelope composition in mature muscle fibers. In particular, the mechanosensitive component, BAF, whose localization at the nuclear envelope is tightly regulated by the LINC complex, is shown to negatively control the nuclear accumulation of the cell cycle regulator E2F1 at the level of the nuclear envelope. The localization of Otefin in the nuclear envelope and BAF phosphorylation by Ball kinase are both crucial in this context. This process might be part of a mechanosensitive pathway that regulates polyploidy in a wide variety of differentiated cells (Unnikannan, 2020).

The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. From an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), this study identified a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. It was demonstrated that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, this study found that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, this study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation (Zho, 2022).

Prohibitins (PHBs) are members of the conserved SPFH superfamily. PHB1 was first identified by its antiproliferative activity upon ectopic expression, which was later attributed to its 3' untranslated region instead of the PHB protein itself. The PHB complex contains two homologous members: PHB1 and PHB2. PHB1 and PHB2 are ubiquitously expressed and are present in the mitochondria, the nucleus, cytosol, and the lipid rafts of the plasma membrane. A number of studies have described a role of the PHB complex within the mitochondria, where it forms a supramacromolecular structure at the inner membrane of the mitochondria acting as a scaffold (or a chaperone) for proteins and lipids regulating mitochondrial metabolism. The PHB complex has been implicated in various cellular and developmental processes and diseases, such as mitochondrial respiration, signaling, and mitophagy depending on its cellular localization. Disruption of the PHB genes has effects ranging from decreased replicative lifespan in yeast, to larval arrest in Drosophila, and to embryonic lethality in mice. However, it remains unexplored whether they play a role in intestinal stem cell regulation in Drosophila (Zho, 2022).

Proliferation and differentiation of adult stem cells must be tightly controlled to maintain tissue homeostasis and prevent tumorigenesis. However, how stem cell proliferation is properly controlled and in particular how the cell cycle is regulated in stem cells is not fully understood. This study identified Yun as an ISC proliferation regulator from a large-scale RNAi screen. Loss of yun function in progenitors restricts them from proliferating, such that they remain in a quiescent state under normal conditions and during tissue regeneration following acute tissue damage. Yun was shown to act as a scaffold for the PHB complex, and the Yun/PHB complex is regulated by EGFR signaling and functions through E2F1 to sustain proliferation of normal stem cells for tissue homeostasis/regeneration and transformed stem cells in tumorigenesis (Zho, 2022).

In addition to EGFR signaling, the levels of the Yun/PHB complex are also elevated upon activation of JAK/STAT signaling or in the absence of Notch, indicating that the Yun/PHB complex may also be regulated by JAK/STAT and Notch signaling directly or indirectly. Previous studies and the current data show that EGFR signaling acts downstream of JAK/STAT, Notch, and Wnt signaling in ISC proliferation and that ectopic expression of yun/Phb could rescue proliferation defects in the absence of EGFR signaling. Therefore, it is proposed that EGFR signaling is the major upstream of signal of the Yun/PHB complex, although the possibilities cannot be fully excluded that the complex may also be regulated by the other signaling pathways directly or indirectly. The EGFR/MAPK pathway and E2F1 are differentially required for stem cell proliferation (mitosis) and differentiation (endoreplication). How E2F1 is differentially regulated during these two processes is not clear. The regulation of E2F1 levels by EGFR/MAPK signaling has been proposed to be due to increased translation or/and increased protein stability, possibly involving some unknown cytoplasmic factors. The identification of the Yun/PHB complex may account for the differential regulation of E2F1 by EGFR/MAPK signaling. The Yun/PHB complex is expressed in progenitors and mediates EGFR/MAPK signaling for ISC proliferation but not progeny differentiation, indicating that the Yun/PHB complex is more specifically required for ISC proliferation. Interestingly, a previous study identified another target of EGFR signaling, the transcription factor Sox100B/dSox9B, which has a critical role in progeny differentiation, indicating that the control of the ISC proliferation and progeny differentiation by EGFR/MAKP signaling is likely differentially mediated by different effectors (Zho, 2022).

The levels of E2F1 protein, along with the expression of PCNA-GFP, were significantly diminished in yun/Phb-defective progenitors or imaginal wing discs, suggesting that Yun affects E2F1 levels. Biochemical analysis shows that the Yun/PHB complex associates with E2F1 in vivo, indicating that the Yun/PHB complex interacts and stabilizes E2F1 protein to regulate ISC proliferation. Furthermore, ectopic expression of single components of the Yun/PHB complex increased E2F1 protein levels, which were further increased when two or three components of the Yun/PHB complex were coexpressed, supporting the notion that the Yun/PHB complex regulates E2F1 protein levels. Moreover, ectopic expression of E2F1/Dp significantly restored ISC proliferation in yun/Phb-defective intestines. Consistently, knockdown of the negative regulator of E2F1, Rb, also restored ISC proliferation in these yun/Phb-defective intestines, albeit at a weaker level than that of E2F1/Dp overexpression. Together, these data demonstrate that E2F1 acts downstream of the Yun/PHB complex for ISC proliferation. Interestingly, overexpressing PCNA alone is not sufficient to restore ISC proliferation in yun-depleted intestines, indicating that collective activation of multiple downstream targets of the E2F1/Dp complex are required to restore ISC proliferation. It has been previously proposed that PHB1 binds to Rb and functions as a negative regulator of E2F1-mediated transcription. These studies contrast with previous work suggesting that the PHB complex is required for cell proliferation. This in vivo study uncovered how E2F1 is differential regulated by EGFR/MAPK signaling and acts downstream of the Yun/PHB complex in ISC proliferation to maintain tissue homeostasis under normal and stress conditions and during tumorigenesis in Drosophila, which is in striking contrast to the proposed antiproliferation role of PHB1 (Zho, 2022).

Unlike Yun, Phb1 and Phb2 are conserved and have been reported to form a complex that localizes to the nucleus, plasma membrane, and mitochondria in mammalian cells. In mitochondria, the PHB complex functions as chaperones in mitochondria of some cell types. Although in flies, the Yun/PHB complex is partially localized in mitochondria in progenitors, the results indicate that the ISC proliferation defects observed in yun/Phb-defective progenitors are unlikely due to their roles in mitochondria. Human Phb1 and Phb2 could significantly restore ISC proliferation defects in Phb1- and Phb2-depleted intestines, respectively, and were required for the proliferation of different human cancer cell lines, indicating that the function of the PHB complex in proliferation is conserved. Finally, the observations that 1) Yun acts as a scaffold of PHBs for their proper function; 2) the Yun/PHB complex acts downstream of EGFR/MAPK signaling; and 3) PHBs and EGFR/MAPK signaling are evolutionarily conserved, suggest that a functional counterpart of Yun exists in mammals, which is different in primary sequence but possibly similar in structure (Zho, 2022).

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. This study used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. The impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk(ΔE2F) mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk(ΔE2F) mutants. Moreover, Pgk(ΔE2F) animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, these results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the Pgk(ΔE2F) animals underscore the importance of E2F regulation on a single E2F target, Pgk (Zappia, 2023).

E2F regulates thousands of genes and is involved in many cellular functions. One of the central unresolved issues is whether E2F function is the net result of regulating all E2F targets or only a few key genes. However, the importance of individual E2F targets cannot simply be deduced by either examining global transcriptional changes in E2F-deficient cells or analyzing ChIP-validated lists of E2F targets. The most straightforward approach is to mutate E2F sites in the endogenous E2F targets, yet this was done in very few studies. Genome editing tools provided an opportunity to begin addressing this question by systematically introducing point mutations in E2F sites of genes involved in the same biological process. Despite using the same stringent criteria in mining proteomic, transcriptomic, and ChIP-seq to select E2F targets, this knowledge was found to be largely insufficient to predict the functionality of E2F binding sites, as well as the contribution of E2F in controlling the expression of individual targets. Notably, that among five, similarly high-confidence metabolic targets, the loss of E2F regulation is particularly important only for the Pgk gene. These findings underscore the value of this approach in dissecting the function of the transcription factor E2F (Zappia, 2023).

The availability of orthogonal datasets for proteomic changes in Dp-deficient muscles combined with genome-wide binding for Dp and Rbf in the same tissue allowed selection for target genes that should be highly dependent on E2F regulation. Surprisingly, significant variation was found in the recruitment of E2F/Dp/Rbf and the expression of the target gene upon mutating E2F sites among five metabolic genes (Table 1). Neither the number of E2F sites, their positions, nor the response of the luciferase reporter to E2F1 is sufficient to predict E2F regulation of the endogenous targets in vivo. Strikingly, although Cyt-c-p contained multiple E2F binding sites, the introduction of mutations in all these sites had no effect on the recruitment of E2F/Dp/Rbf and the expression of Cyt-c-p. For the remaining genes, mutations in the core element of the E2F binding sites reduced the recruitment of E2F/Dp/Rbf and, consequently, the expression of the target gene albeit to various degrees. This work illustrates that the contribution of E2F in gene expression regulation is highly context- and target-dependent and that one could not simply predict the functionality of E2F binding sites or the importance of a target by relying on existing transcriptomic, proteomic, and ChIP-seq datasets. One explanation for the partial loss of binding for Dp and Rbf in the GpdhΔE2F and kdnΔE2F lines is that since the whole animal was used for ChIP-qPCR, whether the reduction in the recruitment is due to the loss of E2F binding in either a subset of cells or across all cells could not be distinguished, thus consistent with a tissue-specific role of E2F. Another possibility is that E2F still retains a weak affinity to the mutant sequence. It is also possible that E2F sites are not the only DNA elements that recruit E2F complexes to chromatin. Indeed, other DNA sequences, such as cell cycle homology region sites, were shown to help in tethering the E2F complex to DNA as a part of the MuvB complex (see Myb). Additionally, it has been suggested that other DNA binding factors may facilitate E2F binding. Overall, these results suggest that unlike in vitro assays with recombinant proteins, mutation of E2F sites does not always fully prevent the recruitment of E2F in vivo (Zappia, 2023).

In Drosophila, the E2F activity is a net effect of the activator E2F1/Dp and the repressor E2F2/Dp complexes that work antagonistically at cell cycle promoters during development. One limitation of the approach described in this study is that it does not distinguish the relative contribution of activator and repressor E2Fs. In earlier studies, in which the roles of E2F1, E2F2, and Rbf were examined during flight muscles development, this study found that unlike E2F1, the loss of E2F2 is largely inconsequential, while Rbf acts as an activator. Given that the mutations in E2F sites did not lead to an increase in gene expression, the data are consistent with the idea that the repressive E2F2/Dp complex does not have a major contribution in this particular context for these genes (Zappia, 2023).

Among five genes that were analyzed this study, mutating E2F sites upstream Pgk gene completely prevents the recruitment of E2F/Dp/Rbf and results in several-fold reduction in the expression of the Pgk gene. The neuron-specific Pgk knockdown markedly decreases the levels of ATP and results in locomotive defects. In another study, flies carrying a temperature-sensitive allele of Pgk, nubian, exhibit reduced lifespan and several-fold reduction in the levels of ATP. Thus, altered generation of ATP and shorten lifespan appear to be the hallmarks of diminished Pgk function in flies. Notably, the PgkΔE2F mutant animals show low glycolytic and TCA cycle intermediates and low ATP levels and die earlier, thus suggesting that in the absence of E2F regulation, the normal function of Pgk is compromised (Zappia, 2023).

The loss of E2F function on the Pgk gene leads to defects in high energy-consuming organs, such as flight muscles and ovaries. The morphology of mitochondria is abnormal in PgkΔE2F and muscles are dysfunctional, as revealed by the flight test. The ovaries in PgkΔE2F females were smaller and contained a high number of degenerated egg chambers at the onset of vitellogenesis. Intriguingly, just like in the PgkΔE2F mutant animals, occasional degenerating early egg chambers (stage 8 and earlier) were previously described in the Dp and E2f1 mutants (51) raising the possibility that this aspect of the Dp mutant phenotype may be due to loss of E2F regulation on the Pgk gene. Interestingly, degenerating egg chambers have previously been associated with metabolic defects, including alteration in the TCA cycle intermediates (52), nutritional status (39, 40), and lipid transport (53). Egg chambers can degenerate before investing energy into egg production, in particular vitellogenesis. Given that multiple signals are integrated during the midoogenesis checkpoint, it reasoned that low levels of glycogen in PgkΔE2F mutants may contribute, at least to some extent, to egg chamber degeneration in PgkΔE2F mutants. Collectively, these data strongly argue that E2F is important for regulating the function of Pgk (Zappia, 2023).

Recent studies revealed that metabolic intermediates can function as cofactors for histone-modifying enzymes by regulating their activities and therefore change chromatin dynamics. For example, alpha-KG is a cofactor for Jumonji C domain-containing histone lysine demethylases, and low alpha-KG levels can lead to histone hypermethylation. In contrast, fumarate and succinate can serve as alpha-KG antagonists and inhibit lysine demethylases (31). Metabolic analysis of PgkΔE2F animals revealed a severe reduction in glycolytic and TCA cycle intermediates, including alpha-KG, succinate, and fumarate, thus, raising the possibility that activities for the histone demethylase are altered in PgkΔE2F mutants. Another metabolite affected in PgkΔE2F is lactate that serves as a precursor for a new histone modification known as lactylation that was shown to directly promote gene transcription. The findings that the loss of E2F regulation on the Pgk gene is accompanied by changes in several metabolites, which can regulate the activity of multiple histone-modifying enzymes, raises the possibility that the epigenetic landscape may change. This idea is supported by altered chromatin accessibility at multiple loci in PgkΔE2F mutants, as revealed by ATAC-seq. Notably, it is accompanied by a reduction in the expression of several genes, including metabolic genes. The effect was specific to the loss of E2F regulation on the Pgk gene because it has been validated in two independent lines carrying the mutation on the E2F sites, while no changes in gene expression were observed in Pgk loss of function in heterozygous animals (hypomorph PgkKG06443). Interestingly, published microarray analysis for Pgknubian animals revealed changes in the expression of genes involved in glucose and lipid metabolism (37). In sum, the data suggest that abnormal flux through Pgk leads to changes in chromatin accessibility and, subsequently, transcriptional changes (Zappia, 2023).

Comparison of transcriptomes, proteomes, and metabolomes for Rbf-deficient animals led to unexpected conclusion that very few of these metabolic changes actually corresponded to matching transcriptional changes at direct Rbf targets (55). This result raised the question of the relative importance of transcriptional regulation mediated by E2F/Rb function. The observation that the loss of E2F regulation of a single glycolytic gene can affect chromatin accessibility and elicit broad transcriptional changes at numerous metabolic genes may provide an explanation for this discordance. Ironically, many glycolytic and mitochondrial genes that are misexpressed in PgkΔE2F mutants are E2F target genes based on ChIP-seq data. However, the changes in their expression occur without changes in E2F recruitment. Thus, the transcriptional changes observed in the E2F-deficient animals are indeed a complex combination of both direct effects of E2F at the target genes and indirect, yet E2F-dependent, effects at other targets as exemplified by Pgk. These indirect effects were missed when an entire E2F/Rbf module was inactivated. This finding further highlights the value of mutating E2F binding sites in individual E2F target genes to dissect different tiers of E2F regulation (Zappia, 2023).

GENE STRUCTURE

Bases 5' UTR -701

Bases in 3' UTR - 1261

PROTEIN STRUCTURE

Amino Acids - 805

Structural Domains

Although Drosophila and human proteins share three regions with a high degree of homology, the fly protein is much larger, in part due to the insertion of a 300-aa block with a unique sequence between the two homologous blocks closest to the C-terminal. The Drosophila gene is equally related to each of the human family members. Amino acids 249-318 of the fly protein share a striking homology with DNA-binding domains of the human E2F genes. In addition, a region termed the "marked box" is highly conserved. This domain may function in protein dimerization. The C-terminal Rb-binding domain has also been largely conserved in the fly protein, sharing 56% similarity to the human proteins (Dynlacht, 1994 and Ohtani, 1994).

date revised: 10 June 2024  

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