Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells.

Drosophila contains two members of the E2F transcription factor family (E2f and E2f2), which controls the expression of genes that regulate the G1-S transition of the cell cycle. Previous genetic analyses have indicated that E2f is an essential gene that stimulates DNA replication. We show that loss of E2f2 is viable, but causes partial female sterility associated with changes in the mode of DNA replication in the follicle cells that surround the developing oocyte. Late in wild-type oogenesis, polyploid follicle cells terminate a program of asynchronous endocycles in which the euchromatin is entirely replicated, and then confine DNA synthesis to the synchronous amplification of specific loci, including two clusters of chorion genes that encode eggshell proteins. E2f2 mutant follicle cells terminate endocycles on schedule, but then fail to confine DNA synthesis to sites of gene amplification and inappropriately begin genomic DNA replication. This ectopic DNA synthesis does not represent a continuation of the endocycle program, as the cells do not complete an entire additional S phase. E2f2 mutant females display a 50% reduction in chorion gene amplification, and lay poorly viable eggs with a defective chorion. The replication proteins ORC2, CDC45L and ORC5, which in wild-type follicle cell nuclei localize to sites of gene amplification, are distributed throughout the entire follicle cell nucleus in E2f2 mutants, consistent with their use at many genomic replication origins rather than only at sites of gene amplification. RT-PCR analyses of RNA purified from E2f2 mutant follicle cells indicate an increase in the level of Orc5 mRNA relative to wild type. These data indicate that E2f2 functions to inhibit widespread genomic DNA synthesis in late stage follicle cells, and may do so by repressing the expression of specific components of the replication machinery.

Cell cycle: Flies teach an old dogma new tricks.

E2F transcription factors are thought to influence the G1-S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis. But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase.

Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression.

Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem-loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3' end. Stem-loop-binding protein (SLBP) binds to the histone mRNA 3' end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3' ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3' end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog.

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.

A role for the DP subunit of the E2F transcription factor in axis determination during Drosophila oogenesis.

The E2F family of transcription factors contributes to cell cycle control by regulating the transcription of DNA replication factors. Functional 'E2F' is a DNA-binding heterodimer composed of E2F and DP proteins. Drosophila contains two E2F genes (dE2F, dE2F2) and one DP gene (dDP). Mutation of either dE2F or dDP eliminates G(1)-S transcription of known replication factors during embryogenesis and compromises DNA replication. However, the analysis of these mutant phenotypes is complicated by the perdurance of maternally supplied gene function. To address this and to further analyze the role of E2F transcription factors in development we have phenotypically characterized mitotic clones of dDP mutant cells in the female germline. Our analysis indicates that dDP is required for several essential processes during oogenesis. In a fraction of the mutant egg chambers the germ cells execute one extra round of mitosis, suggesting that in this tissue dDP is uniquely utilized for cell cycle arrest rather than cell cycle progression. Mutation of dDP in the germline also prevents nurse cell cytoplasm transfer to the oocyte, resulting in a 'dumpless' phenotype that blocks oocyte development. This phenotype likely results from both disruption of the actin cytoskeleton and a failure of nurse cell apoptosis, each of which are required for normal cytoplasmic transfer. Lastly, we found that dDP is required for the establishment of the dorsal-ventral axis, as loss of dDP function prevents the localized expression of the EGFR ligand Gurken in the oocyte, which initiates dorsal-ventral polarity in the egg chamber. Thus we have uncovered new functions for E2F transcription factors during development, including an unexpected role in pattern formation.

To differentiate or not to differentiate?

Developmental regulation of the cell cycle is an important determinant of tissue size and shape. Equally important is regulated withdrawal from the cell cycle to allow cells to differentiate. Recent evidence supports a direct link between transcriptional regulation of the cell cycle machinery and cell differentiation.

Establishing links between developmental signaling pathways and cell-cycle regulation in Drosophila.

During development, cell signaling often mediates the choice of cell fate and the accompanying cell biological events that dictate morphogenesis - such as progress through the cell division cycle. Recent genetic analyses in Drosophila are beginning to reveal the molecular connections between developmental signaling pathways and key regulators of the cell cycle.

Fluctuations in cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila.

The precise cell-cycle alternation of S phase and mitosis is controlled by alternating competence of nuclei to respond to S-phase-inducing factors [1]. Nuclei acquire competence to replicate at the low point in cyclin-dependent kinase (Cdk) activities that follows mitotic destruction of cyclins. The elevation of Cdk activity late in G1 is thought to drive cells into S phase and to block replicated DNA from re-acquiring replication competence [2]. Whereas mitosis is normally required to eliminate the cyclins prior to another cycle of replication, experimental elimination of Cdk activity in G2 can restore competence to replicate [3-6]. Here, we examine the roles of Cdks in the endocycies of Drosophila [7]. In these cycles, rounds of discrete S phases without intervening mitoses result in polyteny. Cyclins A and B are lost in cells as they enter endocycles [8,9], and pulses of Cyclin E expression drive endocycle S phases [10-12]. To address whether oscillations of Cyclin E expression are required for endocycles, we expressed Cyclin E continuously in Drosophila salivary glands. Growth of the cells was severely inhibited, and a period of DNA replication was induced but further replication was inhibited. This replication inhibition could be overcome by the kinase inhibitor 6-dimethylaminopurine (6-DMAP), but not by expression of subunits of the transcription factor E2F. These results indicate that endocycle S phases require oscillations in Cdk activity, but, in contrast to oscillations in mitotic cells, these occur independently of mitosis.

Mutations of the Drosophila dDP, dE2F, and cyclin E genes reveal distinct roles for the E2F-DP transcription factor and cyclin E during the G1-S transition.

Activation of heterodimeric E2F-DP transcription factors can drive the G1-S transition. Mutation of the Drosophila melanogaster dE2F gene eliminates transcriptional activation of several replication factors at the G1-S transition and compromises DNA replication. Here we describe a mutation in the Drosophila dDP gene. As expected for a defect in the dE2F partner, this mutation blocks G1-S transcription of DmRNR2 and cyclin E as previously described for mutations of dE2F. Mutation of dDP also causes an incomplete block of DNA replication. When S phase is compromised by reducing the activity of dE2F-dDP by either a dE2F or dDP mutation, the first phenotype detected is a reduction in the intensity of BrdU incorporation and a prolongation of the labeling. Notably, in many cells, there was no detected delay in entry into this compromised S phase. In contrast, when cyclin E function was reduced by a hypomorphic allele combination, BrdU incorporation was robust but the timing of S-phase entry was delayed. We suggest that dE2F-dDP contributes to the expression of two classes of gene products: replication factors, whose abundance has a graded effect on replication, and cyclin E, which triggers an all-or-nothing transition from G1 to S phase.

E2F-induced S phase requires cyclin E.

Both the heterodimeric transcription factor, E2F, and the G1 cyclin, cyclin E, are required for the G1-S transition at the start of the metazoan cell cycle. It has been established that cyclin E can act as an upstream activator of E2F. In addition to this action, we show here that cyclin E has an essential role in DNA replication distinct from activating E2F. We have created transgenic Drosophila capable of inducible, ectopic production of E2F activity. Simultaneous overexpression of both Drosophila E2F subunits, dE2F and dDP, in embryos stimulated the expression of multiple E2F-target genes including cyclin E, and also caused the initiation of S phase. Mutation of cyclin E prevented the initiation of S phase after overexpression of dE2F/dDP without affecting induction of target gene expression. Thus, E2F-directed transcription cannot bypass loss of cyclin E in Drosophila embryos.

The transcription factor E2F is required for S phase during Drosophila embryogenesis.

Overexpression of the E2F-1 cDNA in mammalian cells disrupts normal control of the cell cycle and drives cells into S phase. Whereas eliminating E2F activity would test its inferred involvement in the G1-S transition, elimination is complicated by the existence of gene families encoding mammalian E2F. Here we identify mutations in a single essential Drosophila gene, dE2F, that encodes a homolog of the mammalian E2F gene family. Embryos homozygous for null mutations of dE2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. We conclude that in most cells dE2F is essential for a G1-S transcriptional program and for G1-S progression.

Developmental control of the G1 to S transition in Drosophila: cyclin Eis a limiting downstream target of E2F.

The E2F transcription factor is required for S phase in Drosophila. While it also triggers expression of replication genes at the G1-S transition, the relevance of this transcription is not clear because many of the induced gene products are sufficiently stable that new expression is not required for S phase. However, one unstable product could couple S phase to E2F activation. Here we show that cyclin E expression at G1-S requires E2F, that activation of E2F without cyclin E is not sufficient for S phase, and that early in G1 ectopic expression of cyclin E alone can bypass E2F and induce S phase. We conclude that cyclin E is the downstream gene that couples E2F activity to G1 control. Not all embryonic cycles are similarly coupled to E2F activation, however. The rapidly proliferating CNS cells, which exhibit no obvious G1, express cyclin E constitutively and independently to E2F. Instead, cyclin E expression activates E2F in the CNS. Thus, this tissue-specific E2F-independent transcription of cyclin E reverses the hierarchical relationship between cyclin E and E2F. Both hierarchies activate expression of the full complement of replication functions controlled by E2F; however, whereas inactivation of E2F can produce a G1 when cyclin E is downstream of E2F, we propose that an E2F-independent source of E eliminates G1.

Developmental control of a G1-S transcriptional program in Drosophila.

We have defined a coordinate program of transcription of S-phase genes (DNA polymerase alpha, PCNA and the two ribonucleotide reductase subunits) that can be induced by the G1 cyclin, cyclin E. In Drosophila embryos, this program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. This dynamic pattern of expression is not disrupted by a mutation, string, that blocks the cell cycle. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. We suggest that developmental signals control this transcriptional program and that its activation either directly or indirectly drives transition from G1 to S phase in the stereotyped embryonic pattern.

Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis.

The conserved regulators of cell cycle progression--Cyclins, Cdc2 kinase, and String phosphatase (Cdc25)--accommodate multiple modes of regulation during Drosophila embryogenesis. During cell cycles 2-7, Cdc2/Cyclin complexes are continuously present and show little fluctuation in abundance, phosphomodification, or activity. This suggests that cycling of the mitotic apparatus does not require cytoplasmic oscillations of known regulatory activities. During cycles 8-13 a progressive increase in the degradation of Cyclins at mitosis leads to increasing oscillations of Cdc2 kinase activity. Mutants deficient in cyclin mRNAs suffer cell cycle delays during this period, suggesting that Cyclin accumulation times these cycles. During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of Cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription.

Genetic and biochemical studies of a mutant Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase, nmt72pLeu99-->Pro, that produces temperature-sensitive myristic acid auxotrophy.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that transfers myristate from CoA to the amino-terminal glycine residue of at least 12 cellular proteins. Its reaction mechanism is Ordered Bi Bi with myristoyl-CoA binding occurring before binding of nascent polypeptides and release of CoA preceding release of the myristoylprotein product. nmt1-72 is a temperature-sensitive allele, identified by Stone et al. (Stone, D. E., Cole, G. M., Lopes, M. B., Goebl, M., and Reed, S. I. (1991) Genes & Dev. 5, 1969-1981) that causes arrest in the G1 phase of the cell cycle due to reduced acylation of Gpa1p. We have recovered this mutant allele and determined that it contains a single point mutation resulting in a Leu99 (CTA) to Pro (CCA) substitution. Addition of > or = 500 microM myristate but not palmitate to synthetic or rich media rescues the growth arrest caused by nmt1-72 at 37-39 degrees C, consistent with the observation that purified nmt72p has reduced affinity for myristoyl-CoA and that exogenous myristate but not palmitate increases cellular myristoyl-CoA pools. Metabolic labeling studies in S. cerevisiae and co-expression of nmt72p with several protein substrates of Nmt1p in Escherichia coli indicate that the Leu99-->Pro substitution causes a reduction in the acylation of some but not all protein substrates. Since formation of a myristoyl-CoA.Nmt1p complex appears to be required for synthesis/formation of a peptide binding site, these defects in acylation appear to arise either because Leu99 is a component of the enzyme's functionally distinguishable myristoyl-CoA and peptide recognition sites or because Pro99 alters the interaction between myristoyl-CoA and enzyme in a way that precludes formation of a normal peptide binding site. The reduction in affinity for myristoyl-CoA produced by Leu99-->Pro in nmt72p is less than that produced by the Gly451-->Asp mutation in nmt181p, which also produces temperature-sensitive myristic acid auxotrophy. Isogenic, haploid strains containing NMT1, nmt1-72, and nmt1-181 do not manifest any obvious differences in steady state levels of the acyltransferases during growth at permissive temperatures or in the biosynthesis of long chain saturated acyl-CoAs. The spectrum of cellular N-myristoylproteins whose level of acylation is affected by nmt1-72 and nmt1-181 is distinct.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations of human myristoyl-CoA:protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae.

We have isolated cDNAs encoding human myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) by complementing the nmtl-181 mutation of Saccharomyces cerevisiae, which causes temperature-sensitive myristic acid auxotrophy. Human NMT is derived from a single-copy gene, contains 416 amino acids, is 44% identical to S. cerevisiae NMT (yeast NMT), and can complement the lethal phenotype of an nmtl null mutation. Human and yeast NMTs have overlapping yet distinct protein substrate specificities as judged by a coexpression system that reconstitutes protein N-myristoylation in Escherichia coli. Both enzymes contain a glycine five residues from the C terminus. Gly----Asp or Lys mutagenesis in these orthologous NMTs produces marked reductions in their activities in E. coli as well as temperature-sensitive myristic acid auxotrophy in S. cerevisiae. These results indicate highly conserved structure-function relationships in vivo and underscore the usefulness of these functional assays for identifying factors that regulate protein N-myristoylation in mammalian systems.

Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation.

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1.

While investigating the expression of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT: E.C. 2.3.1.97) by Northern blot analysis, we observed another RNA transcript whose expression resembled that of NMT1 during meiosis and was derived from a gene located less than 1 kb immediately upstream of NMT1. This new gene, designated PWP1 (for periodic tryptophan protein), is divergently transcribed from NMT1 and encodes a 576-residue protein. Null mutants of PWP1 are viable, but their growth is severely retarded and steady-state levels of several cellular proteins (including at least two proteins that label with exogenous [3H]myristic acid) are drastically reduced. New methods for database searching and assessing the statistical significance of sequence similarities identify PWP1 as a member of the beta-transducin protein superfamily. Two other previously unrecognized beta-transducin-like proteins (S. cerevisiae MAK11 and D. discoideum AAC3) were also identified, and an unexpectedly high degree of sequence homology was found between a Chlamydomonas beta-like polypeptide and the C12.3 gene of chickens. A systematic and quantitative comparative analysis resulted in classifying all beta-transducin-like sequences into 11 nonorthologous families. Based on specific sequence attributes, however, not all beta-transducin-like sequences are expected to be functionally similar, and quantitative criteria for inferring functional analogies are discussed. Possible roles of repetitive tryptophan residues in proteins are also considered.

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.