Robert J. Duronio - US grants

Controlling the cell division cycle is an essential part of normal animal development. In proliferating tissues, progression through the cell cycle assures that cell division accompanies growth. In contrast, terminal differentiation is typically preceded by cell cycle arrest and the cessation of proliferation. The decision to proliferate or remain quiescent is most often made during the G1 phase of the cell cycle. Somatic cells must correctly manage this decision in order to properly maintain homeostasis. Such management requires regulation of the cell cycle machinery controlling the G1-S transition. This includes gene amplification of positive cell cycle effectors such as cyclin D1, and mutation of negative effectors such as tumor suppressors pRB and p16. These molecules are part of a molecular pathway that controls the cell cycle in response to both positive and negative extracellular effectors of cell growth. A major target of regulation of these molecules is the ES2F/DP family of heterodimeric transcription factors. E2F/DP/1/s control the expression of genes require for growth and DNA replication. Recent results indicate that perturbations of E2F/DP function both in vitro and in vivo can also be oncogenic. E2F/DP and its known upstream regulators are all conserved in Drosophila melanogaster, where we can apply sophisticated genetic approaches to an analysis of E2F/DP function. In Drosophila, dE2F/dDP is required for normal development. Thus, developmental signals that control cell fate may regulate growth and cell cycle progress via altering E2F/DP activity. The goals of this proposal are 1) to genetically identify novel Drosophila genes that regulate the activity of dE2F/dDP and therefore may regulate the G1-S transitions of the cell-cycle, and 2) to understand how dE2F/dDP regulates transcription in vivo and how this affects cell cycle control during development.

[unreadable] DESCRIPTION (provided by applicant): This application requests continued support for predoctoral student training in genetics and molecular biology. Students who receive support are enrolled in the Curriculum in Genetics and Molecular Biology, and interdepartmental predoctoral training program. The goal of this program is to train students to be creative, sophisticated research scientists in the disciplines of genetics and molecular biology. The emphasis of this training is the acquisition of a foundation of knowledge, the accumulation of laboratory skills, and the development of the ability to formulate experimental approaches to solving contemporary problems in the biological sciences. The Curriculum Director, Robert J. Duronio, serves as the Program Director for this NRSA award. The 75 training faculty have appointments in the School of Medicine and the Department of Biology. They participate in student training by acting as dissertation sponsors, serving on dissertation committees, teaching in Curriculum sponsored courses, inviting speakers for the Curriculum's seminar series, and serving on administrative committees such as the Admissions Committee. There are currently 71 students enrolled in the Curriculum and they are training in 35 laboratories in 9 departments on the UNC-CH campus and one laboratory at the NIH Sciences. Student research includes the generation and characterization of mouse models of human diseases, the characterization of molecular mechanisms of replication, recombination and repair, the control of gene expression, and the genetic basis of cancer. The training program requires all students to take courses in genetics and molecular biology, to attend sessions on responsible conduct of research, to attend Curriculum seminars, to act as teaching assistants for one semester, to participate and present in a student seminar series, to present a poster for the Annual Research Day, and to pass a written qualifying exam, an oral preliminary exam, and a final oral exam and written dissertation. Essentially all students publish their dissertation research in peer-reviewed journals. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Determining the mechanisms of normal cell cycle control is critical for our understanding of both development and oncogenesis. During development, cell proliferation occurs by coordinating progress through the cell cycle with growth. Conversely, cell cycle arrest occurs prior to, and is often necessary for, terminal differentiation. Cell proliferation and cell cycle arrest are also highly regulated after the completion of development: stem cells in adult tissues are under tight cell cycle control, as are quiescent cells that only proliferate in response to particular stimuli. Breakdowns in cell cycle control in any of these circumstances can have drastic consequences and contribute to the deregulated growth typical of cancer. The long term objective of this project is to elucidate how developmental programs affect cell cycle progression, a process that remains poorly understood. In this proposal we focus on gene expression mechanisms that control the G1-S transition because this is when most cells decide whether to enter or to exit the cell cycle. PUBLIC HEALTH RELEVANCE: Cell proliferation is a fundamental aspect of the biology of all organisms, and is controlled by a highly orchestrated series of cell biological events termed the cell cycle that directs the accurate duplication and inheritance of the genome. A detailed molecular description of cell cycle events during animal development is critical for our understanding of cell proliferation control, and how such control goes awry in diseases like cancer.

ABSTRACT Proper regulation of histone biosynthesis during the cell cycle is critical for the appropriate coordination of chromatin assembly and DNA replication. Overproduction or underproduction of histone protein results in replication stress and genome instability that contributes to the development of cancer. In this proposal we undertake a comprehensive analysis of replication-dependent (RD) histone mRNA biosynthesis and how this process is coordinated with the cell cycle. Animal RD-histone mRNAs are the only eukaryotic mRNAs that lack a polyA tail, ending instead in a conserved stem loop structure. In contrast, mRNAs for histone variants such as H3.3 and H2A.Z are encoded by polyadenylated mRNAs. Generation of the unique RD-histone mRNA 3' end results from the activity of an evolutionary conserved set of pre-RNA processing factors. The genes encoding all five RD-histone proteins are clustered in metazoan genomes, and transcription and pre-mRNA processing factors required for histone mRNA biosynthesis are organized into a nuclear body (the histone locus body or HLB) that assembles at these gene clusters. We will determine the requirements for the coordinate synthesis of the RD-histone mRNAs using both biochemical and genetic approaches in Drosophila, with a particular focus on the role that the HLB plays in histone transcription and pre- mRNA processing. Histone gene clusters provide a system in which one can readily study the expression of a tightly regulated set of genes at all levels of mRNA biosynthesis, from the organization of genes within the nucleus through activation of transcription, pre-mRNA processing and transcription termination. Our Drosophila experimental paradigm permits the in vivo interrogation of these fundamental processes in gene expression in ways that are unavailable to other systems.

DESCRIPTION (provided by applicant): Chromatin contains information beyond the DNA sequence that is needed to regulate the genome and allow the proper development of multi-cellular organisms. Posttranslational modifications (PTMs) of histones modulate the organization of chromatin for replication, repair and transcription of genetic information, and are hypothesized to be the carriers of information that is inherited 'epigenetically' over multiple cel generations. Importantly, investigations into histone PTM function in multicellular eukaryotes have been limited by pleiotropic effects resulting from pharmacological and genetic manipulation of epigenetic regulatory proteins that create, remove, or respond to histone PTMs. Because of this limitation, there is a gap in knowledge regarding the role of individual histone residues as carriers of information. The objective of this proposal is to generate a comprehensive experimental platform in Drosophila melanogaster for spatial and temporal manipulation of post-translationally modified histone residues during animal development. To study the biological function of a specific histone PTM, the acceptor residue must be changed to an amino acid that cannot be appropriately modified. Then, all wild-type copies of that histone gene must be replaced with the mutant copy. We will use a transgenic strategy for replacement of the entire Drosophila histone multigene family with engineered gene clusters that express specific histone mutants, and create experimental tools for the temporal and spatial restriction of these mutations. These studies will directly test the roles of post- translationally modified histone residues during animal development, and will create a sustainable and expandable resource for the study of epigenetic phenomena in metazoans. Because disruption of evolutionarily conserved histone PTMs is thought to underlie many human diseases, including cancer, this research will directly impact human health.

PROJECT SUMMARY Accurate replication of the genome during cell proliferation is necessary for normal animal development and homeostasis. Disruption of the regulation or fidelity of replication contributes to many human pathologies, particularly cancer. Thus, a complete understanding of the mechanisms governing genome replication is paramount to human health. Replication of large genomes like that found in human cells requires the initiation of bi-directional DNA synthesis at thousands of individual locations on each chromosome. Executing this critical task requires a highly-regulated interaction between DNA and a large set of proteins whose activity must be coordinated with other cellular events such that all regions of the genome are replicated once and only once each cell division. Two decades of research by many laboratories has resulted in the identification of a set of 42 evolutionarily conserved polypeptides that are sufficient for initiation of DNA replication in a cell free setting, as well as how their activity is coordinated with the cell cycle. However, the mechanisms that determine how and where these factors interact with the genome in an intact animal cell, and how they are differentially activated to initiate DNA replication once they bind to the genome, are not well understood. These processes are modulated by the abundance of replication proteins, the chemical composition and relative compaction of chromatin, the folding of large domains of individual chromosomes, and the overall three-dimensional architecture of the genome within the nucleus. A major goal in the field is to determine how each of these levels of organization impact genome replication and stability in different cell types during development and in adult tissues. This project will specifically focus on how chromatin organization influences genome replication and stability during animal development. The basic building block of chromatin is the nucleosome, an octamer of histone proteins encompassed by ~147 base pairs of DNA. Each histone protein has an N-terminal tail that protrudes from the nucleosome core and is subject to a variety of chemical modifications (e.g. methylation, acetylation, and phosphorylation) that modulate chromatin organization and thus influence all aspects of genome function, including DNA replication. We have developed a method in Drosophila for engineering any desired histone tail mutation, providing us a means of manipulating chromatin organization that is not currently available in any other animal system. This genetic method will be combined with cell biological and next generation DNA sequencing methods to determine how chromatin organization modulates DNA replication in different cell types.

ABSTRACT Proper regulation of histone biosynthesis during the cell cycle is critical for the appropriate coordination of chromatin assembly and DNA replication. Overproduction or underproduction of histone protein results in replication stress and genome instability that contributes to the development of cancer. In this proposal we undertake a comprehensive analysis of replication-dependent (RD) histone mRNA biosynthesis and how this process is coordinated with the cell cycle. Animal RD-histone mRNAs are the only eukaryotic mRNAs that lack a polyA tail, ending instead in a conserved stem loop structure. In contrast, mRNAs for histone variants such as H3.3 and H2A.Z are encoded by polyadenylated mRNAs. Generation of the unique RD-histone mRNA 3' end results from the activity of an evolutionary conserved set of pre-RNA processing factors. The genes encoding all five RD-histone proteins are clustered in metazoan genomes, and transcription and pre-mRNA processing factors required for histone mRNA biosynthesis are organized into a nuclear body (the histone locus body or HLB) that assembles at these gene clusters. We will determine the requirements for the coordinate synthesis of the RD-histone mRNAs using both biochemical and genetic approaches in Drosophila, with a particular focus on the role that the HLB plays in histone transcription and pre- mRNA processing. Histone gene clusters provide a system in which one can readily study the expression of a tightly regulated set of genes at all levels of mRNA biosynthesis, from the organization of genes within the nucleus through activation of transcription, pre-mRNA processing and transcription termination. Our Drosophila experimental paradigm permits the in vivo interrogation of these fundamental processes in gene expression in ways that are unavailable to other systems.