David Arnosti, Ph.D. - US grants

Transcriptional repression plays a key role in regulation of gene expression in development and in disease processes. To understand conserved regulatory processes in animals, we have determined both the molecular workings and physiological relevance of transcriptional represser proteins that play key roles in Drosophila development. Knirps is a key regulator of the even-skipped gene, acting on separate enhancer elements via a short-range mechanism. We have discovered how Knirps and related repressers function via distinct repression mechanisms, involving the evolutionary conserved CtBP corepressor protein and a histone deacetylase Rpd3, and we have developed novel insights into the design of cis regulatory elements that bind these repressers. We have identified key aspects of a "cisregulatory grammar" that predicts how represser proteins can function on defined regulatory elements, laying the groundwork for mathematical and bioinformatic analysis of endogenous enhancers. This work will allow us to apply Drosophila-derived information to general analysis of metazoan cis regulatory element design and evolution. 1. We will quantitatively "map" regulatory surfaces of genes controlled by transcriptional repressors, and use potential function-based mathematical models to predict the regulatory properties of novel enhancers. 2. We will elucidate of mechanisms of short- and long-range repressors using chromatin immunoprecipitation to identify molecular events associated with repression of embryonic reporter genes. 3. We will assess the function of corepressors we discovered associated with the Knirps represser complex, in particular the evolutionary conserved Groucho corepressor, whose role in short-range repression was previously unrecognized. 4. We will identify the physiological significance of conserved residues and splice-variants of the CtBP corepressor, using whole-animal assays to identify the biological significance of the proteins'activities. These aims combine empirical and modeling efforts to obtain a "bottoms up" understanding of transcriptional repression. Our long-term goal is to understand molecular activities of transcriptional control proteins and DNA regulatory sequences that are central to gene expression processes important in development and disease.

DESCRIPTION (provided by applicant): The long-term aim of this project is to elucidate the activity of Drosophila Retinoblastoma family (Rbf) proteins and associated cofactors in a developmental setting. Rbf proteins are key regulators of cell cycle control and play pivotal, although poorly understood, roles in development. Rbf proteins are differentially deployed during development and are thought to have distinct functions and effects on gene expression. The functional properties of Rbf proteins are dictated by the recruiting of distinct cofactors, and we have identified a novel association between the COP9 signalosome complex and Rbf2. Our preliminary data indicates that the COP9 signalosome may function in two important ways to influence Rbf function. First, the COP9 complex regulates Rbf protein stability in the developing embryo. Second, the COP9 complex may serve as a transcriptional co-repressor to enact patterns of transcriptional repression by Rbf proteins. This project will employ powerful biochemical and genetic tools that are available in Drosophila to develop a comprehensive picture of the COP9 signalosome in target gene regulation by Drosophila Rbf proteins. First, we will perform a detailed examination of the mechanism defining the association between the COP9 signalosome and Rbf proteins, as a vital pre-requisite to our subsequent functional assays. Second, we will examine the physiological role of the Rbf-associated COP9 complex using chromatin immunoprecipitation to determine when and where this putative cofactor is associated with Rbf proteins at target gene promoters. Third, the significance of Rbf protein instability and the role for the COP9 complex in this process during developmentally controlled gene repression patterns will be examined. Fourth, we will determine the physiological role of the COP9 signalosome as transcriptional co-regulatory factor for Rbf- mediated gene repression. The function of the COP9 complex as a co-repressor will be examined by means of genetic assays that test Rbf transcriptional regulatory activity during development and by transcription assays to determine the importance of individual COP9 subunits in Rbf regulation of specific genes in the fly. Retinoblastoma proteins have critical roles in development and are key regulators mutated in a high percentage of human tumors. Elucidation of the previously unidentified COP9 function for Rbf gene regulation, its mode of action, and its role in developmental gene regulation will provide important insight into RB action that will facilitate the design of therapeutic interventions in a wide spectrum of human cancers.

Project Summary Transcriptional repressors represent the necessary counterweight to transcriptional activators in metazoan development. The mechanisms of transcriptional repression have been intensively investigated, but we lack crucial insights into how repressors act at a mechanistic level across many targets in the genome. In particular, the coordinated recruiting of transcriptional co-repressors can generate diverse effects on chromatin structure and modification, and we still lack insights on the functional significance of many changes that can be measured in `omics studies. To develop key insights into eukaryotic transcriptional regulatory mechanisms, we use the natural setting of the Drosophila embryo to identify basic biochemical processes in a developmental setting, where differential gene expression is used to drive the developmental fate of particular cells and tissues. In this proposal, 1) we will use genome-wide methods developed in our laboratory to identify direct biochemical, chromatin-based processes that are directed by a set of five endogenous transcriptional regulators that repress through short-range and long-range mechanisms. 2) We will study the in vivo activity of wild-type and mutant repression complexes to identify the contributions of distinct transcriptional co-repressors Groucho and CtBP, testing their contributions to quantitative and/or qualitative effects in chromatin modifications and gene regulation. 3) To identify the cis-regulatory context in which transcriptional repressors act on different enhancers, we will quantitatively measure and mathematically model the output of specific enhancers to uncover the fundamental quantitative properties of specific classes of repressors interacting with activators ? i.e. the common ?rules? by which these proteins interact on many target genes. The three interrelated aims will provide predictive tools for interpretation of genomic cis regulatory content of the metazoan genome, providing essential underpinnings for studies of evolution and disease in higher eukaryotes.