Terry Orr-Weaver - US grants

During Drosophila development the genomic DNA content is altered in many tissues as a consequence of changes in the coordination of cell cycle events. Endoreduplication, duplication of the genome without nuclear division and cytokinesis, produces polyploid cells in which the total genomic content has increased. In some tissues specific genomic regions are under or overreplicated as a result of an uncoupling of DNA replication origin usage from the cell cycle. The regulation of these two variations in the cell cycle is investigated in the proposed experiments. The genetic control of the endocycle will be analyzed by obtaining mutants affecting the switch to endoreduplication by the larval precursor cells during embryogenesis. These mutants will be isolated from an embryonic lethal collection generated by single- element transposon tagging. Three reagents that are differential markers for cell and nuclear size will be used to identify mutants causing polyploid cells to remain diploid or diploid cells to become polyploid in the embryo. The mutants in this collection can be readily mapped and cloned, thus permitting their molecular role in regulating developmental changes in the cell cycle to be elucidated. Two gene clusters encoding the chorion genes are overreplicated in the differentiation of the follicle cells. The amplification of these two chromosomal domains will be used as a model for analyzing differential replication. Characterization of the control elements regulating chorion amplification in cis has indicated that a common control element may function in regulating both replication and transcription. This will be tested in a series of experiments testing the linkage of the transcriptional control clement to the amplification control element, the effect of other transcriptional regulatory elements on amplification, and the replicative properties of the control region in cultured cells and embryos.

The regulation of sister-chromatid segregation in Drosophila will be analyzed. While disjunction of sister-chromatids is common to both meiosis and mitosis, in meiosis sister-chromatid separation occurs in meiosis II and is inhibited in meiosis I. Thus there are functions that promote sister-chromatid cohesion as well as those that lead to disjunction. Two Drosophila mutants, mei-S332 and ord, result in precocious sister-chromatid separation in meiosis I of both males and females, providing a key to unraveling this process. We have recently isolated additional alleles of each of these loci; these alleles reveal that the mei-S332 is not required to maintain sister-chromatid cohesion until anaphase I, after the kinetochore has duplicated. The new alleles of mei-S332 will be analyzed for their effects on sister-chromatid segregation in meiosis and mitosis using genetic and cytological approaches. The mei-S332 gene will be cloned and its products identified and characterized. Since an interesting possibility is that the gene encodes a component of the kinetochore which functions to maintain cohesion or the two duplicated halves, the cytological location of the mei-S332 gene product will be analyzed. Sister-chromatid disjunction is central to the ordered segregation of chromosomes is eukaryotic organisms. The information resulting from this work will allow us to understand better fundamental aspect of inheritance.

Abstract 9316168 Orr-Weaver The Drosophila gene mei-S332 has been shown to be critical for maintaining sister-chromatid cohesion in meiosis because mutations in the gene cause the sister chromatids to prematurely disjoin late in the first meiotic division. The defect in mei-S332 mutants is manifest at a time when the sister chromatids are held together only at their centromere regions, suggesting that the product of this gene acts at the centromere or kinetochore to promote cohesion in mitosis. The molecular mechanisms by which mei-S332 controls sister-chromatid cohesion will be elucidated by isolating the gene, identifying the protein it encodes, and analyzing where that protein is localized on the chromosomes. The nature of unusual sex-specific mutations will be determined. Experiments will investigate whether the mei-S332 protein is modified at the metaphase-anaphase transition, the time at which sister-chromatid segregation will be identified by their interaction with the mei- S332 protein, using biochemical and genetic approaches. Two maternal effect mutants have been identified in Drosophila, grauzone and cortex, that have the opposite phenotype as mei-S332 mutants. In these mutants meiosis is arrested at metaphase II, prior to the separation of sister chromatids. These genes may act in a regulatory manner to signal the metaphase-anaphase transition, or they may promote the separation of sister-chromatids, possibly by causing the loss of cohesion. Genetic and cell biology experiments will define the role of these genes in meiosis and mitosis and their relationship to mei-S332. The grauzone gene will be isolated in order to understand the molecular basis of its action in sister-chromatid segregation. *** The proper segregation of chromosomes is essential to ensure that a complete complement of genetic information is transmitted to progeny or daughter cells. Prior to chromosome segregation a copy of each chromosome is replicated. During mitosis th ese sister chromatids segregate to daughter cells. In the first meiotic division the sister chromatids migrate as a unit to the same pole as the homologous copies of each chromosome segregate; the sister chromatids do not segregate until the second meiotic division. One aspect of chromosome segregation that is poorly understood is how the sister chromatids remain attached until their separation in mitosis or meiosis. Sister-chromatid cohesion is likely to be mediated through structural functions that hold the sister chromatids together and regulatory functions that time their dissociation. The control of sister-chromatid segregation will be investigated using Drosophila as a model system because it affords the combined approaches of genetics, cell biology, and molecular biology. %%%

The regulation of DNA replication in multicellular organisms is critical for their development. In addition, the improper regulation of DNA replication can lead to disease states such as malignancy or cell death. The experiments in this proposal address two aspects of the developmental regulation of DNA replication. One of the earliest controls in development is the restart of DNA replication in the embryo in response to fertilization. In most organisms the control of DNA replication in altered in some of the cells such that S phase becomes unlinked from mitosis and cell division, leading to an increase in the DNA content of the cell (polyteny or polyploidy). These two aspects of the developmental regulation of DNA replication will be investigated in the fruit fly, Drosophila melanogaster, because mutations can be isolated in which DNA replication is improperly controlled. Two genes have been identified, plutonium (plu) and pan gu (png), that regulate DNA replication at the onset of development. Unfertilized eggs mutant in either of these genes inappropriately undergo extensive DNA replication; moreover, the DNA in all four meiotic products appears to replicate rather than simply the pronucleus. Analysis of plu and png will be key to understanding how a resting oocyte is converted into a developing embryo in response to maturation and fertilization signals. The mechanisms by which plu and png regulate DNA replication in early development will be elucidated by experiments that will determine whether they control entry into S phase or whether they regulate DNA replication within S phase, possibly by blocking reinitiation. The products encoded by the plu and png genes will be identified by cloning the genes, and their cellular location will be determined. The developmental expression of the genes will be addressed, particularly whether the gene products are altered after fertilization. The interaction between plu, png, and four other genes that regulate the earliest cell cycles of development will be investigated by genetic and molecular approaches. Polytenization in Drosophila results from a modified cell cycle, the endo cell cycle, in which S phase alternates with a gap phase, but no mitosis occurs. The endo cell cycle is under developmental control because the onset of polytenization occurs with precise temporal and spatial regulation during the latter half of embryogenesis. Regulatory genes that trigger the onset of polytenization in development or that control the endo cell cycle will be identified and analyzed. Candidate regulatory genes have been isolated by the criteria that they are transcriptionally activated at the onset of polytenization in several tissues in the embryo. The effect of mutation of these genes on polytenization will be determined, and those that are shown to regulate this process will be cloned. A selection will be done for mutants in which polytenization is affected.

Cell division must be regulated precisely, because when this regulation fails uncontrolled division and cancer ensue. During the development of multicellular organisms cell growth and division have to be coordinated with developmental signals. In addition to exit from the cell cycle to permit differentiation, a variety of cell cycle variants are utilized throughout the plant and animal kingdoms. One of these modified cell cycles, the endo cell cycle, results in DNA replication in the absence of mitosis and produces the polyploid or polytene cells that are found in at least some tissues in almost all organisms. This proposal addresses the three key aspects of how the endo cycle is regulated, using Drosophila as a model. The most significant distinction between the endo cycle and the mitotic cycle is the absence of mitosis. The morula gene is required to inhibit mitosis in the endo cycle. The MORULA protein will be identified and the mechanism by which it blocks mitosis determined. A second difference between the two cell cycles is that DNA replication is differentially regulated in the endo cycle such that some genomic regions are underreplicated while others are overreplicated and the genes amplified. Mutations in the two subunits of the E2F transcription factor, dDP and dE2F, affect differential replication during polyploid S phase. These results reveal a previously unrecognized role for E2F in controlling DNA replication once S phase has initiated. It will be determined whether E2F affects DNA replication via one of its transcriptional targets or whether it has a more direct effect on replication origins. Because the regulatory hierarchy that controls E2F activity is mutated in most human tumors, it is significant for understanding the causes of caner to determine how E2F influences DNA replication. The third level of regulation is common to both the endo cell cycle and the mitotic cycle: the transcripts that are induced at the G1-S transition must be downregulated after DNA replication has initiated in order for a subsequent round of replication to occur. The l(2)52Ec gene is unusual in being necessary both for DNA replication and downregulation of G1-S transcripts, suggesting it is a prerequisite for the cell to recognize that replication has initiated. The hypothesis that the l(2)51Ec protein is a component of the origin complex and necessary for downregulation of previously induced genes will be tested.

The goal of this work is to define the regulatory mechanisms that ensure accurate segregation of the sister chromatids in mitosis and meiosis. Drosophila is a powerful model organism in which to decipher the control of chromosome segregation for three reasons: 1) chromosome dynamics can be visualized directly; 2) genetic tools allow ready detection of mutants defective in segregation; and 3) it is possible to ablate gene function using RNAi in order to evaluate the role of genes identified in other species. This research focuses on proteins that control two crucial aspects of chromosome dynamics: sister-chromatid cohesion and condensation. In order for sister chromatids to move away from each other during anaphase they must first be physically attached to each other. This ensures that each sister chromatid of a pair attaches to microtubules emanating from opposite spindle poles. Cohesion must be precisely regulated to release only as the sisters separate at anaphase. The Drosophila MEI-S332 protein binds to centromeres to maintain cohesion and is essential during meiosis. The cohesin complex of proteins is essential both for the establishment of cohesion during DNA replication and its maintenance until chromatid separation at anaphase. The cohesin complex acts along the length of the chromosomes in mitosis and meiosis I and maintains cohesion at the centromere in meiosis II. This research addresses the mechanism by which MEI-S332 localization to centromeres is regulated and analyzes the relationship between MEI-S332 and the cohesin protein complex. The condensin complex is necessary for chromosome condensation, and mutations have been recovered in the gene encoding one of the subunits. This project will define the interdependency of the cohesin and condensin complexes. The role of the condensin complex in meiosis and gene expression also will be investigated. Three collections of mutants with defects in segregation have been recovered and will be analyzed to determine the activities of the gene products. The phenotypes indicate that these genes play critical roles in chromosome segregation, thus their investigation will provide new insights into chromosome dynamics.

This research investigates the control of chromosome segregation, with the goal of deciphering two fundamental aspects essential for the proper partitioning of chromosomes during cell division. Accurate segregation of chromosomes is essential during the divisions that produce sperm and eggs in order to avoid birth defects such as Down Syndrome. Inaccurate segregation during cell proliferation produces aneuploid cells; these cells with an incorrect number of chromosomes are prone to become transformed cancer cells. The mechanisms that ensure proper segregation remain to be elucidated. This basic research builds on the identification of proteins known to participate in chromosome segregation to unravel the dynamics of chromosome behavior.

DESCRIPTION (provided by applicant): It is essential in all cells to regulate DNA replication precisely, as failures in this lead to cancer or developmental defects. Most, if not all, animals and plants have polyploid or polytene cells, the consequence of altered regulation of DNA replication to produce cells that can function as metabolic factories or serve as large support cells. In addition, some organisms over or underreplicate specific genomic regions to control gene expression. These developmental aspects of the regulation of DNA replication are the focus of this research. The approaches of genomics and cell biology will be used to understand how metazoan DNA replication is controlled during development, innovative methodologies for this biological problem. New examples of amplified and underreplicated genes have been identified in the fruit fly Drosophila melanogaster by a genomic microarray approach. The replicative properties and biological functions of these differentially replicated genes will be deciphered. The microarray methodology will be employed to determine how widely used differential replication is as a developmental strategy for gene expression in Drosophila. Initiation of DNA replication is controlled by a set of proteins conserved from yeast to humans, and a model system has been developed in Drosophila that permits analysis of these proteins and replication origins. Binding of the Origin Recognition Complex (ORC), the Cdtl/DUP initiator protein, and the MCM hexamer can be visualized directly in the ovarian follicle cells. Furthermore after replication initiation, the DUP and MCM) proteins can be seen moving with replication forks during elongation. This cell biological approach, combined with the ability to recover mutants and examine their defects in replication, provides a powerful means to delineate the regulatory circuitry for replication initiation. The role of the DUP protein and its inhibitor Geminin will be defined in cell cycles that produce polyploid or polytene cells. In the final aim, the mechanisms that inhibit mitosis to permit formation of polyploid or polytene chromosomes will be delineated, and it will be determined how proliferating cells are protected from becoming polyploid.

DESCRIPTION (provided by applicant): DNA replication must be regulated precisely to maintain gene copy number. Cancer cells contain many regions in which gene copy number has been amplified and these are associated with tumor progression, but the primary mechanisms causing gene amplification are unknown. Failure of replication may contribute to chromosome fragile sites that lead to chromosomal instability. Mutations in proteins needed for replication initiation are associated with human developmental defects. It is crucial to determine how replication origins are activated or inactivated and how replication fork progression is controlled in metazoan cells. This necessitates the analysis of individual origins, the localization of replication initiation proteins, and ways to examine fork progression. This research will exploit developmental models of genomic regions that become amplified in copy number or under-replicated in response to differentiation in Drosophila. These models provide defined replication origins to which replication initiation proteins, essential for replication in human cells, can be localized and that can be analyzed in differentiated cells as they undergo initiation of DNA replication. They also define specific domains through which replication fork progression is permitted or impeded. These models provide a paradigm for delineating the mechanisms of replication initiation and elongation not present in another metazoan system. The Specific Aims of this proposal are to use these models to answer fundamental questions about replication initiation and fork progression, utilizing interdisciplinary approaches of genomics, genetics and biochemistry. The mechanisms leading to origin activation will be defined for two amplified domains, regions of the genome permissive or restrictive for initiation will be investigated to determine how the Origin Recognition Complex (ORC) is localized and whether there are ORC- independent mechanisms of initiation. In the second aim, genomic regions that block initiation will be used to delineate the effects of chromatin configuration on ORC binding and origin activation. The last aim is to determine how replication fork progression is controlled by chromatin by identifying proteins that promote or block fork movement. Candidate proteins identified by mutants and localization patterns will be investigated. This aim also will analyze th requirement for double-strand break repair at the forks.

? DESCRIPTION (provided by applicant): The size of tissue layers is scaled during the development of organs. In the nervous system, the size of neurons and glia must be coordinated, particularly for glia such as Schwann cells and satellite cells that are in tight association with axons or neuronal cell bodies. Inappropriate growth of Schwann cells and satellite glia cells in response to nerve damage is linked to chronic pain. Despite its importance, little is understood about how neuronal and glial growth are coordinated. In Drosophila, at least two types of glia increase cell size by increasing DNA content (ploidy), a universal strategy to produce large cells throughout the plant and animal kingdoms. The ability to monitor glial cell size by measuring ploidy and to alter glial cell growth by changing ploidy provides additional experimental advantages to the powerful toolkit in Drosophila for analysis of development of the nervous system. We propose to exploit glial growth by ploidy in the Drosophila nervous system to define mechanisms that coordinate growth between glia and neurons in development. We will investigate one type of surface glia, the subperineurial glia (SPG), that provide the blood-brain barrier. SPG grow to accommodate the underlying neuronal mass while retaining an intact envelope for the blood-brain barrier by increasing ploidy rather than dividing. SPG ploidy is controlled by neuronal mass. The wrapping glia (WG) ensheath axons in the peripheral nervous system. They increase up to 50 fold in size without dividing, apparently by increasing ploidy. We will define the role and regulation of polyploidization of WG. Our specific aims test three hypotheses: 1) neuronal mass promotes ploidy and size increases for the SPG by increased neuronal activity and/or mechanical tension; 2) increased ploidy of the WG is necessary for growth to permit ensheathment of elongating axons; and 3) the size of the three glial layers in the peripheral nerves is coordinated. Additionally, we will define the transcriptomes of the SPG and WG under normal conditions and when growth is modified by adjacent tissue layers, as a means to identify genes that specify functions of these glia and alter ploidy in response to cues from other cells. The goals of this proposal will be achieved by using Drosophila genetic tools to label cells, inhibit gene activity, or overexpress genes with exquisite developmental specificity.

? DESCRIPTION (provided by applicant): The goal of this research program is to define how an egg is produced, provisioned, and protected and how the egg is dramatically reorganized for the transition from oocyte to embryo. Analysis of this key window in development is accessible in Drosophila, and due to the conservation of regulators in development, studies in Drosophila will identify regulatory mechanisms and components that will shed light on fundamental features of early development, with implications for human health in understanding developmental defects and infertility. In addition, analysis of how a Drosophila egg is produced has provided models for the control of metazoan DNA replication, generating insights into how gene copy number can become altered in cancer cells. The proposed research will study the crucial processes of DNA replication, mRNA translation, and proteolysis, all of which are subjected to developmental control in the transition from oocyte to embryo. The regulation of replication origins has remained elusive in metazoans because it has not been possible to determine what defines an origin, to localize the essential Origin Recognition Complex (ORC), or to capture origin firing and its control. Drosophila offers the tremendous advantages of developmental control of origin activity and the ability to localize ORC in differentiated tissues to map origins The effect of differentiation and cell cycle modification on ORC localization will be determined, and the relationship between ORC binding, transcription and developmental repression of replication defined. Replication origins that bind ORC across a broad zone and undergo repeated rounds of origin firing in response to differentiation signals will be exploited to deciphr how replication initiation is controlled. The transition from the differentiated oocyte to the totipotent embryo occurs in the absence of transcription, involving rapid changes in osmolarity and extensive remodeling of the proteome by dramatic changes in mRNA translation and proteolysis. Regulators identified by genetic and proteomic screens indicate a crucial role for redox state and a glycine transporter in egg activation that will be delineated. The PNG kinase was shown to be a master regulator of mRNA translation specifically at this developmental transition, providing the link between completion of meiosis and control of mRNA translation in the egg. The mechanism(s) through which PNG activates translation will be explored by testing the hypotheses that it phosphorylates and inactivates translational repressors and affects the localization of mRNAs to RNP complexes in the egg. A unique form of the Anaphase Promoting Complex (APC), APCCort, specifically targets proteins for degradation whose removal is required for the change from meiosis to mitosis. The requirement for APCCort-targeted degradation of recently identified substrates that are critical in the oocyte-to-embryo transition will be evaluated, and the function of other components of the ubiquitin pathway expressed specifically in this developmental period will be defined.