Terry L. Orr-Weaver

Identification of functional elements and regulatory circuits by Drosophila modENCODE

From Genome to Regulatory Networks For biologists, having a genome in hand is only the beginning—much more investigation is still needed to characterize how the genome is used to help to produce a functional organism (see the Perspective by Blaxter). In this vein, Gerstein et al. (p. 1775) summarize for the Caenorhabditis elegans genome, and The modENCODE Consortium (p. 1787) summarize for the Drosophila melanogaster genome, full transcriptome analyses over developmental stages, genome-wide identification of transcription factor binding sites, and high-resolution maps of chromatin organization. Both studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized. Overall, the studies provide insights into the organization, structure, and function of the two genomes and provide basic information needed to guide and correlate both focused and genome-wide studies. The Drosophila modENCODE project demonstrates the functional regulatory network of flies. To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Endoreplication cell cycles: more for less.

neatly aligned in parallel arrays (see Urata et al., 1995), but polytene chromosomes greater than 16,000C have been noted in another insect, Chironomus. The distinc-and various intermediate DNA configurations can occur, Seattle, Washington 98109 differing primarily in the degree of association between 2 Whitehead Institute and duplicated chromatids (Figure 2D, Hammond and Laird, Department of Biology 1985). For this reason the term polyploid is often used Massachusetts Institute of Technology to refer to endoreplicated cells with virtually any chro-Cambridge, Massachusetts 02142 mosomal configuration. In endoreplication cell cycles, or endocycles, S Molecular studies from the past decade have revealed phases alternate with distinct gap phases that lack DNA striking conservation in the mechanisms of eukaryotic replication, but there is no cell division (Figure 3). Few, cell cycle control. Yet before the advent of molecular if any, cases of polypoidy resulting from continuous DNA genetics, it was clear that eukaryotes possessed many replication have been reported. Some endocycling cell different cell cycle variations, and thus that there must types retain hallmarks of mitosis, but many examples be diversity in mechanisms of control. One common cell lack all vestiges of mitosis, including chromosome con-cycle variant is the endoreplication cycle, in which cells densation, nuclear envelope breakdown, and the reor-increase their genomic DNA content without dividing. ganization of microtubules that builds the spindle. The Although endocycles are sometimes dismissed as an term endomitosis initially referred to a rare cell cycle in evolutionary peculiarity, they are widespread in protists, which mitosis occurred without nuclear envelope break-plants, and many animals including arthropods, mol-down or cytokinesis (for review see Nagl, 1978). How-lusks, and mammals. Endocycling cells can become ever, now this term is more generally used to describe incredibly polyploid, with chromatin values (C values cycles that proceed through anaphase but lack nuclear denote DNA content as a multiple of the normal haploid division and cytokinesis. In yet another cell cycle variant, nuclear division occurs without cytokinesis, giving rise genome) as high as 24,000 reported in some plant endo-to multinucleate cells. Such cycles are seen in mamma-sperms (Traas et al., 1998). Because cell size for a given lian hepatocytes and osteoclasts, and also in syncytial cell type is generally proportional to the amount of nu-slime molds like Physarum and early insect embryos. clear DNA, endoreplication constitutes an effective The regulation of these cycles is thought to be similar strategy of cell growth, and it is often found …

Mei-S332, a drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions

Mutations in the Drosophila mei-S332 gene cause premature separation of the sister chromatids in late anaphase of meiosis I. Therefore, the mei-S332 protein was postulated to hold the centromere regions of sister chromatids together until anaphase II. The mei-S332 gene encodes a novel 44 kDa protein. Mutations in mei-S332 that differentially affect function in males or females map to distinct domains of the protein. A fusion of mei-S332 to the green fluorescent protein (GFP) is fully functional and localizes specifically to the centromere region of meiotic chromosomes. When sister chromatids separate at anaphase II, mei-S332-GFP disappears from the chromosomes, suggesting that the destruction or release of this protein is required for sister-chromatid separation.

Regulation of APC/C Activators in Mitosis and Meiosis

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase that triggers the degradation of multiple substrates during mitosis. Cdc20/Fizzy and Cdh1/Fizzy-related activate the APC/C and confer substrate specificity through complex interactions with both the core APC/C and substrate proteins. The regulation of Cdc20 and Cdh1 is critical for proper APC/C activity and occurs in multiple ways: targeted protein degradation, phosphorylation, and direct binding of inhibitory proteins. During the specialized divisions of meiosis, the activity of the APC/C must be modified to achieve proper chromosome segregation. Recent studies show that one way in which APC/C activity is modified is through the use of meiosis-specific APC/C activators. Furthermore, regulation of the APC/C during meiosis is carried out by both mitotic regulators of the APC/C as well as meiosis-specific regulators. Here, we review the regulation of APC/C activators during mitosis and the role and regulation of the APC/C during female meiosis.

DNA replication control through interaction of E2F-RB and the origin recognition complex

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro , making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro , but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

A CHECKPOINT ON THE ROAD TO CANCER

Mutations that disrupt a cell-division checkpoint, thereby causing alterations in chromosome number, have been identified in cancer cells. The accompanying increase in mutability helps to explain how tumours acquire large numbers of mutant genes during their development.

8 Chromosome Segregation during Meiosis: Building an Unambivalent Bivalent

Faithful chromosome segregation during anaphase requires that stable microtubule connections are established between chromosomes and both spindle poles by metaphase. Bipolar orientation follows an active period of transient connections between the kinetochores and poles, and tension mediated through attachments between the chromosomes stabilizes those bivalents that have connections to opposite poles. This review focuses on how the chromatids are tied together in the bivalent to ensure proper segregation in the two meiotic divisions. Homologs are partitioned in meiosis I, and reciprocal crossovers, cytologically defined as chiasmata, usually hold the homologs together for this division. The crossovers themselves must be prevented from migrating off the chromatid arms. Binding substances localized to the crossover and sister-chromatid cohesion distal to the crossover have been proposed to prevent loss of chiasmata. Spontaneous nondisjunction events and mutations that disrupt the maintenance of chiasmata are analyzed in the context of these models. Homologs that segregate in meiosis I without chiasmata are briefly discussed. The bivalent must also be constructed so that four chromatids present only two functional kinetochores prior to anaphase I. Cytology and genetic data suggest that the sister kinetochores are duplicated but constrained to act as a single kinetochore. Additionally, centromeric regions of sister chromatids preserve their cohesion until anaphase II, even as cohesion on the sister-chromatid arms is lost at anaphase I. Mutations that specifically disrupt this process are presented.

Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis

N6-methyladenosine is a nonediting RNA modification found in mRNA of all eukaryotes, from yeast to humans. Although the functional significance of N6-methyladenosine is unknown, the Inducer of MEiosis 4 (IME4) gene of Saccharomyces cerevisiae, which encodes the enzyme that catalyzes this modification, is required for gametogenesis. Here we find that the Drosophila IME4 homolog, Dm ime4, is expressed in ovaries and testes, indicating an evolutionarily conserved function for this enzyme in gametogenesis. In contrast to yeast, but as in Arabidopsis, Dm ime4 is essential for viability. Lethality is rescued fully by a wild-type transgenic copy of Dm ime4 but not by introducing mutations shown to abrogate the catalytic activity of yeast Ime4, indicating functional conservation of the catalytic domain. The phenotypes of hypomorphic alleles of Dm ime4 that allow recovery of viable adults reveal critical functions for this gene in oogenesis. Ovarioles from Dm ime4 mutants have fused egg chambers with follicle-cell defects similar to those observed when Notch signaling is defective. Indeed, using a reporter for Notch activation, we find markedly reduced levels of Notch signaling in follicle cells of Dm ime4 mutants. This phenotype of Dm ime4 mutants is rescued by inducing expression of a constitutively activated form of Notch. Our study reveals the function of IME4 in a metazoan. In yeast, this enzyme is responsible for a crucial developmental decision, whereas in Drosophila it appears to target the conserved Notch signaling pathway, which regulates many vital aspects of metazoan development.

Gene Amplification as a Developmental Strategy: Isolation of Two Developmental Amplicons in Drosophila

Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, we identified two additional developmental amplicons in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression.