William Theurkauf - US grants

Embryogenesis is initiated by a cleavage stage characterized by rapid mitotic divisions that are under maternal genetic control. During these divisions, there is little or no zygotic gene expression and the simplified cell cycle is composed of alternating S and M phases. Control of embryogenesis switches to the zygotic genome at the midblastula transition (MBT), when the cell cycle slows, zygotic transcription increases dramatically, and G1 and G2 cell cycle phases are introduced. The timing of the MBT is determined by the ratio of nuclei to cytoplasm, suggesting that this transition is triggered as a maternally deposited factor is titrated by DNA or chromatin. Cell cycle checkpoint pathways delay cell cycle progression in response to DNA damage, incomplete replication, or mitotic defects. A conserved DNA replication checkpoint pathway is required for the switch from maternal to zygotic control of development at the Drosophila MBT. This observation supports a model for the MBT in which titration of a maternally deposited DNA replication function triggers the replication checkpoint, which in turn leads to cell cycle delays and the onset of high level gene expression. The long-term goal of this application is to define the molecular mechanism of the transition from maternal to zygotic control of development at the MBT, with particular emphasis on the role of the replication checkpoint pathway at this transition. To address this broad goal, the following specific aims will be pursued. 1) Identify the maternal factors that trigger the replication checkpoint and thus control the development timing of the MBT. 2) Systematically analyze the replication checkpoint pathway that detects titration of these maternal factors and triggers cell cycle delays and activation of the zygotic transcription machinery. 3) Identify the targets for replication checkpoint regulation that mediate zygotic gene activation at the MBT. The Mei-41/ATM tumor suppressor homologue is a component of the checkpoint pathway that controls the MBT. These studies may therefore provide insight into a critical developmental transition, and help define a clinically significant tumor suppressor pathway.

DESCRIPTION (provided by applicant): MRNA localization and local translation are required for processes ranging from mating type switching in yeast to learning and memory in mammals, yet the mechanisms driving mRNA transport and silencing in higher eukaryotic systems remain poorly understood. Axis specification in Drosophila melanogaster provides particularly dramatic examples of coordinated mRNA transport and translational silencing. Bicoid and oskar mRNA are synthesized in a cluster of nurse cells, assemble into transport particles, and move through cytoplasmic bridges (ring canals) to the oocyte. Within the oocyte, microtubules are required for bicoid mRNA localization to the anterior pole and oskar mRNA accumulation at the posterior pole. oskar mRNA is silent until mid-oogenesis, when it is localized to the posterior pole of the oocyte. Bicoid mRNA remains translationally silent until early embryogenesis. We have developed an in vivo assay for bicoid mRNA localization that reveals multiple steps in the anterior mRNA localization pathway, and an in vitro -mRNA transport particle assembly system that recapitulates RNA behavior in vivo. These assays will be used to define RNA sequences and trans-acting proteins that drive anterior localization of bicoid mRNA (Aims 1 and 2). We have also identified a new axis specification gene, armitage, that is essential for oskar mRNA translational silencing and axis specification. The armitage gene is required for RNAi and efficient assembly of the RNA induced silencing complex (RISC), and mutations in 3 additional RNAi components also disrupt oskar mRNA silencing and axis specification. The RNAi machinery thus appears to be essential to embryonic axis specification. The function of armitage in RNAi and RISC assembly, and the role of RNAi in axis specification, are examined in Aims 3 and 4. Aim 5 addresses a possible role for ATR/Chk2 tumor suppressor pathway in regulating RNAi. These studies will thus elucidate molecular mechanisms of mRNA localization and translational silencing that are essential to embryonic axis specification.

Checkpoints delay cellcycle progression when DNA is damaged or S phase is incomplete, allowing time for repair or to complete replication. By contrast, apoptosis eliminates cells carrying irreparable genetic damage. While these interphase-specific DNA damage responses are well characterized, the mitotic response to DNA damage remains poorly understood. When checkpoints fail and genotoxic lesions persist into mitosis, cells often proceed through an aborted division in which chromosome segregation and cytokinesis fail. Following division failure, the resulting cells arrest in GOor die by apoptotic or non-apoptotic mechanisms. This " mitotic catastrophe" response, which eliminates damaged cells, has been observed in systems rangingfrom human cells to fly embryos and may be a major cause of chemotherapy induced cell death in some tumors. Despite the evolutionaryconservation and potential clinical significance of mitotic catastrophe, this process has not been systematically analyzed. We have found that mitotic catastrophe in early Drosophila embryos is linked to centrosome inactivation, chromosome condensation defects, and midbody assembly failures. Further, this mitotic damage response required the Chk2 kinase tumor suppressor homologue, and Chk2 localizes to centrosomes, chromosomes, and the midbody. Our preliminarystudies strongly suggest that Chk2 is also required for mitotic catastrophe is human colorectal cancer cells. Chk2 may therefore function in a conserved mitotic catastrophe pathway that links the division machinery to the integrity of the chromosomal passengers. The goal of this proposal is to define the molecular and cellular mechanism of mitotic catastrophe through systematic cytological, biochemical and genetic studies in Drosophila and human cells.

DESCRIPTION (provided by applicant): The piRNA pathway has a conserved role in transposon silencing and genome maintenance during germline development. Adaptation to new genome pathogens by the Drosophila piRNA genome defense system appears to be driven by transposition into heterochromatic clusters, which produce the primary piRNAs that to initiate silencing. The HP1 homolog Rhino binds to clusters and is evolving rapidly under positive selection, suggesting that it may co-evolve with invading mobile elements. piRNA clusters and Rhino thus function at the adaptive interface between the piRNA pathway and transposons. Aims1 and 2 combine evolutionary divergence and conservation with genetic, genomic, computational and cytological tools to define this critical interface. The ability to differentiate cluster transcripts from mRNAs is critical to the specificity of the piRNA dense system, and our recent data suggest that stalled spicing may mark cluster transcripts for processing into silencing RNAs. Aim 3 focuses on the role of splicing in piRNA precursor sorting into the silencing pathway. These studies address mechanisms that maintain the inherited genome and are likely to directly impact reproductive health.

? DESCRIPTION (provided by applicant): The sole purpose of the germline is to produce gametes-the vehicles of heritable genetic and epigenetic information. In animals, the development and maintenance of a functional germline and gametes requires the activity of PIWI proteins, germline- expressed Argonaut family members that interact with small RNAs known as piRNAs. The best known function for the piRNA pathway is to protect the genome from invading transposable elements, yet most animals encode multiple PIWI genes that interact with distinct classes of piRNAs, suggesting that piRNA pathways have divergent functions even within an organism. Indeed, piRNAs that derive from unique sequences in the genome are abundantly expressed during pachytene in the mouse testis, but whether they promote or simply correlate with sperm development is unclear. These observations raise many important and unresolved questions that are essential to understanding how piRNA pathways promote germline immortality. We propose studies in three model organisms-worms, flies, and mice-to identify the conserved mechanisms and functions of piRNA pathways in animals.

Administrative Core Project Summary The goal or the Program Project is to use powerful bioinformatic approaches and studies in three experimentally tractable and phylogenetically diverse model organisms to define species specific and conserved functions for the piRNA pathway in germline development. This ambitious approach depends on close coordination between three experimental projects, a bioinformatic project, and Mouse and Computational Cores. The Administrative Core is critical to this coordination, and will ensure effective communication between team members, maximize synergy between Projects and Cores, and assure that resources are utilized in the most efficient manner to achieve the overall scientific goals. Relevance The core will facilitate communication between project and cores, aid in dissemination of findings, and provide fiscal oversight. These functions are critical to executing the proposed integrated experimental approach.

Administrative Core Project Summary The goal or the Program Project is to use powerful bioinformatic approaches and studies in three experimentally tractable and phylogenetically diverse model organisms to define species specific and conserved functions for the piRNA pathway in germline development. This ambitious approach depends on close coordination between three experimental projects, a bioinformatic project, and Mouse and Computational Cores. The Administrative Core is critical to this coordination, and will ensure effective communication between team members, maximize synergy between Projects and Cores, and assure that resources are utilized in the most efficient manner to achieve the overall scientific goals. Relevance The core will facilitate communication between project and cores, aid in dissemination of findings, and provide fiscal oversight. These functions are critical to executing the proposed integrated experimental approach.

Project II. piRNA pathway organization and precursor processing William Theurkauf, P. I. Project Summary PIWI interacting RNAs (piRNAs) have a conserved function in transcriptional and post- transcriptional transposon silencing in the germline, which is dedicated to transmitting the inherited genome. Here we focus on identifying conserved organizational principles and molecular mechanisms driving transposon control by the piRNA pathway. Transposon control by piRNAs is best understood in Drosophila, where the primary piRNAs that initiate silencing are derived from large heterochromatic ?clusters? composed of nested transposon fragments. Cluster transcripts appear to be processed into mature piRNAs in nuage, which is an electron dense structure closely associated with the cytoplasmic surface of germline nuclei. In flies, heterochromatic clusters at the nuclear periphery appear to organize the perinuclear nuage, forming a compartment that spans the nuclear envelope. This compartment appears to increase the efficiency and specificity of piRNA production, with is critical to germline development. Studies under Aim1 will define this novel germline specific compartment in flies, mouse and worm. How cluster transcripts are differentiated from mRNAs and processed into mature piRNAs is a critical to the specificity of the system, but remains poorly understood. A complex of Drosophila nuclear proteins suppresses cluster transcript splicing, stalled splicing appears to trigger transposon silencing siRNA production in the pathogenic yeast Cryptocccus, and several putative splicing factors are required for fertility in mouse and humans. These observations suggest that stalled splicing has a conserved role in differentiating piRNA precursors from pre-mRNAs. Studies under Aim 2 will broadly define precursor flow though the piRNA pathway, and test the novel hypothesis that regulated splicing differentiates transposons from protein coding. Relevance Transposons and transposon remnants comprise approximately half of the human genome and represent a potentially explosive source of genome instability. Mobilization of these elements can induce insertions, deletions and rearrangements that cause disease, sterility, and developmental defects. The goals of the proposed studies are to define conserved organizational principles and molecular mechanisms that directly impact human reproductive heath.