Nathaniel Heintz - US grants

The major objective of the proposed research is to elucidate the molecular mechanisms governing periodic expression of specific mRNAs during the cell cycle. The primary focus of the studies described herein is to understand the transcriptional and post-transcriptional regulatory events controlling the accumulation of human histone mRNA during the S phase of the HeLa cell cycle. Additional studies, employing the methodologies developed for the analysis of histone gene expression, will be pursued discover those processes controlling the cell cycle specific expression of other cellular genes. The proposed research will be conducted according to the following course. First, the expression of several individual genes encoding each of the histone subtypes will be analysed in vivo to identify those that code for the most abundant cell cycle regulated mRNAs. Second, in vitro studies of both the transcriptional and post-transcriptional mechanisms which control the accumulation of each histone mRNA will be conducted in soluble extracts from synchronized HeLa cells. Current studies of the pHu4A histone H4 gene (see below) provide a useful paradigm for the analysis of other cell cycle regulated histone genes. Thus, these studies will focus on the identification of both nucleotide sequences and protein factors which are involved in the expression of histone genes in vitro. Third, in vivo studies employing either transiently or stably transfected histone genes will be pursued to demonstrate that the nucleotide sequences which are important for histone gene transcription in vitro are also utilized in vivo. This methodology will also be employed to determine whether specific sequences in histone mRNA provide a signal for its rapid and specific degradation after the inhibition of DNA synthesis. Fourth, similar experiments will be conducted to identify those events controlling the cell cycle specific expression of other cellular genes. The initial emphasis in this area will be to gain insight into the dramatic transcriptionsl regulation of the human HSP 70 gene we have observed during the HeLa cell cycle. It seems apparent that studies of this type can generatecrucial information concerning the regulation of specific genes, as well as provide an avenue toward an increased understanding of general processes required for progression through the cell cycle.

The development and mature function of the mammalian brain must require the precise regulation and concerted action of thousands genes whose products are restricted to the nervous system. The purpose of this grant is to further develop and utilize bacterial artificial chromosomes (BACs) as tools for the discovery and analysis of genes predominantly expressed in the mammalian CNS. The proposal is organized into three specific aims: 1) To utilize BAC transgenic analysis for the characterization of CNS specific gene expression patterns and for the localization of their encoded protein products [BACexpress]. 2) To utilize CNS specific BAC/EGFP or beta-lactamase expressing mice and fluorescence activated cell sorting (FACS) to prepare cell specific probes for gene expression analysis. To use these probes to interrogate either DNA chips or microarrays to profile gene expression for specific CNS cell types (BAC array). 3) To develop and utilize gene trapping in BAC clones to accelerate discovery of CNS specific genes [BACtrap]. The further development of these techniques and their adoption for high throughput analysis can yield: accurate temporal and cell type specific expression profiles for nearly all CNS specific genes; the size, abundance and subcellular distribution of proteins encoded by each of these genes; a library of defined BAC vectors for genetic manipulation of the great variety of CNS cell types; phenotypes resulting from increased dosage of any of the assayed genes; and a "molecular histology" of the CNS that includes the definition of molecularly marked subtypes of morphologically identified neurons.

neurogenetics; artificial chromosomes; gene expression; biomedical resource; central nervous system; macromolecule; serial analysis of gene expression; mass spectrometry; genetically modified animals; clinical research;

DESCRIPTION (provided by applicant): The purpose of this grant is to generate a novel series of agents for genetic manipulation of receptors, ion channel, and signaling molecules in vivo. These agents (tethered toxins) are chimeric molecules derived from tethering of naturally occurring peptide neurotoxins to the cell surface via GPI anchors or transmembrane. These studies derive from the discovery of mammalian prototoxin genes (e.g. Lynx 1) which are the evolutionary antecedents of snake venom toxins, and which can function as modulators of nAChRs in their native GPI-anchored form. Preliminary results are that tethered bungarotoxins retain their activity on nAChRs, and that they are not cleaved from the cell surface to inhibit adjacent cells. The existence of many thousands of naturally occurring peptide neurotoxins (e.g. bungarotoxins, conotoxins, conantokins, etc.), their exquisite target specificities, and the ability to target expression of the agents in vivo using BAC transgenic mice, suggests that the development of a generic strategy for harnessing their potency for in vivo use will permit genetic control over a wide variety of neuronal functions. For example, cell specific genetic control of neural activity, neurotransmitter receptor function (e.g. ACh, NMDA, 5-HT3 receptors), and specific GPCR signal transduction cascades would become possible. The specific aims are to: 1) Construct additional tethered toxins, particularly tethered conotoxins, and test their activity in Xenopus oocytes; 2) Produce BAC transgenic mice expressing tethered toxins in specific CNS cell types in vivo. Assess the efficacy of tethered toxin action by evaluating phenotypes that would be expected based on results obtained in Specific Aim 1 and current knowledge of the roles of the targeted receptors and ion channels in vivo; 3) Develop inducible tethered toxins to improve the temporal resolution of this strategy for genetic manipulation of specific cells and signaling pathways. These studies will allow unprecedented precision in the genetic dissection of functions required for CNS development, function and dysfunction in vivo.

mass spectrometry

The overall objective of this contract is to 1) continue the high-throughput analysis of gene expression patterns in the mouse nervous system during development;2) generate new mouse genetic research tools for the scientific community. Using two complementary and coordinated technologies (standard radiometric in situ hybridization and BAC transgenic reporter mice), the contract screens probes for a large number (e.g., thousands) of gene products on a relatively limited number (10 to 20) of sections of the nervous system. Planes of section are chosen by the NINDS to include the structures of most general interest to the neuroscience community (e.g., neocortex, basal ganglia, hippocampus, cerebellum, spinal cord, etc.). The spatial locations/patterns of gene expression are analyzed at 3 or 4 stages of development (early and late embryonic, and early postnatal) and in adult mice. The resulting images of gene expression are digitized and posted in Web-accessible databases, and all BAC transgenic mice generated by the contract are deposited in an NIH-sponsored mouse repository.

DESCRIPTION (provided by applicant): Project Summary/Abstract: Recent studies of addiction have highlighted several regions of the brain that are thought to be involved in goal directed and drug seeking behaviors. The specific neuronal classes involved in the regulation of these behaviors are beginning to be identified, and attempts to profile the molecular changes in these cell types occurring as a consequence of addiction have met with some success. While these studies demonstrate that it is possible to discover changes in cell types that are correlated with, and in some cases contribute to, addiction, our knowledge in this area is fragmentary and incomplete. This is due to technical obstacles that have faced this field for decades, and that have recently been overcome by novel methodologies developed in our laboratories. The objective of this program of research is to identify all of the changes in gene expression and accompanying alterations in epigenetic regulation that contribute to the addictive state in cell types known to be important in the neural circuitry controlling addiction. The approach we will take in this program is to: 1) employ TRAP methodology and bacTRAP transgenic mouse lines to comprehensively profile the translated mRNAs from fifteen mouse CNS cell types that are components of the neural circuitry controlling addiction;2) to collect translational profiles from each of these cell types in mice exposed to cocaine, methylphenidate, and methamphetamine 3) perform in depth comparative analysis of these resulting microarray datasets to identify changes in gene expression that accompany the addictive state in each cell type;4) to concurrently isolate nuclei from each cell type and map cell specific sites of mC and hmC modification to the neuronal genomes;5) to conduct follow up studies on genomic loci identified in the preceding aims to map additional epigenetic regulatory events by ChIP assays using H3K9m2 and H3K27m3 specific antibodies to identify genomic loci associated with suppression of gene expression in euchromatin. This project will provide information and experimental animals that will stimulate addiction research in a broad spectrum of laboratories, and provide materials and a paradigm for comprehensive studies of these same neural circuits under other experimental conditions. As such, this program will have an enormous impact on modern molecular neurobiology, and on the development of novel targets for pharmacological interventions in CNS disorders. PUBLIC HEALTH RELEVANCE: According to the National Institute on Drug Abuse, "Estimates of the total overall costs of substance abuse in the United States ..... exceed half a trillion dollars annually". The goals of the project are to comprehensively profile molecular changes occurring in specific cell types in regions associated with addiction, and to concurrently map epigenetic changes occurring in these cell types. It will stimulate in depth research into cell specific molecular events that are responsible for establishment of the addictive state, and identify novel targets for the development of new therapies for drug addiction.

The importance of new technological approaches for the advance of science cannot be overestimated. Although an impressive array of new technologies for investigation of complex tissues has been developed over the past decade, the nervous system is composed of hundreds (at least!) of distinct cell types and many interiocking circuits that control behavior in normal or pathophysiological situations. The purpose of the Pilot Project Program is to stimulate new and more efficient approaches for the discovery of novel molecular mechanisms associated with drug abuse, and to aide in breaching the experimental barriers that restrict current discovery methodologies to genetically tractable organisms. This program is intended to support less established investigators because more established laboratories normally have sufficient resources to pursue creative ideas as they arise.

SUMMARY: The Molecular and Epigenetic Profiling Core (MEPC) will provide specialized behavioral and experimental services for the conduct of TRAP translational profiling and epigenetic mapping studies in the context of chronic drug treatment, drug self-administration, and drug withdrawal. In particular, the MEPC will perform TRAP gene expression analysis and epigenetic mapping of 5hmC and 5mC on cell types labeled by bacTRAP transgenic mice generated in the BAC Recombineering and Transgenic Targeting Core. The MEPC will be a critical component for the success of the NIDA P30 Center in that it will serve as a centralized facility providing the expertise and resources for performing these sensitive techniques, thus increasing the quality and reproducibility of data and streamlining the productivity of collaborating investigators. MECP personnel will characterize all new bacTRAP lines generated by the Center and disseminate these data to other participating laboratories. In addition, the MEPC will perform on-site, comprehensive pilot experiments using the self administration behavioral paradigm to analyze cell type specific molecular and epigenetic changes that occur in animal models of addiction. The MEPC will also train personnel from all participating laboratories on using TRAP translational profiling and epigenetic methods used in this core. In addition, the MEPC will host investigators to carry out more in-depth profiling projects that reach beyond the scope of the pilot experiments. This obviates the need to ship mice to laboratories with only short-term experimental goals or that do not have access to resources needed to carry out these experiments. The data and facilities provided by this research support core will offer immediate high-impact application of the transgenic tools generated by the NIDA PSO Center.

SUMMARY: The Administrative Core for the Center for Molecular and Epigenetic Research of Cell Types Mediating Addictive Behaviors will be house in the Collaborative Research Center within the Laboratory of Molecular Biology at The Rockefeller University. It will maintain all financial records and interactions within the Center, coordinate budgets between the Research Support Cores and Pilot Research Project Support Cores, and assist with preparation of financial documents for Progress Reports. It will also oversee data quality control, and data presentation to the public through the Center Website. A critical second role of the Administrative Core will be to foster and maintain interactions between the Center, Center Investigators and NIDA Program Staff. Accordingly, it will administer the Center Advisory Committee and it will host quarterly videoconferences with the committee. It will also manage interactions with the administration of The Rockefeller University, and provide feedback to queries that come to the Center from interested external investigators.

This INSPIRE award brings together research areas traditionally supported in the Division of Integrative Organismal Systems in the Directorate for Biological Sciences, in the Division of Chemical, Bioengineering,Environmental, and Transport Systems in the Directorate for Engineering, and in the Division of Behavior and Cognitive Sciences in the Directorate for Social, Behavioral and Economic Sciences. Cognition arises from the activity within complex brain circuits. These brain circuits are laid out according to a species' genetic blueprint. Past and current successes in uncovering the biological basis of cognition include the discovery of areas in the human brain supporting specific cognitive functions, the determination of the roles that specific cell types play in the behavior of animals like the mouse, and the increasingly detailed understanding of the impact specific genes and their alterations can have on cognitive functions in health and disease. The goal of this interdisciplinary project, conducted at The Rockefeller University, is to directly determine the genetic specificity of brain circuit elements that are critically important for high-level cognitive function. This project will be significant by 1) elucidating the complexity of biological organization from the level of genes, through cell types, brain areas, and neural circuits to behavior, 2) developing new technology that will allow researchers to dissect brain circuits underlying cognition with the precision and specificity of model organisms, and 3) improving the understanding of how genetic alterations impact cognition. The interdisciplinary project at the interface of cognitive neuroscience, neural systems, and neurotechnology, is expected to have broader impacts on society by providing insights into some of the deepest questions about the human mind and by offering unique educational and outreach opportunities to improve public understanding of the organization and function of the brain.

The project will investigate the genetic specificity of a multi-node brain circuit that supports cognitive function. The circuit will be localized with functional and structural magnetic brain imaging. Genetic expression patterns of projection neurons within multiple circuit nodes will then be determined using cutting edge molecular techniques. The functions of the projection neurons linking the nodes will then be determined through advanced and custom-designed optogenetic and electrophysiological techniques. The same optogenetic approach will then be used for causal interrogation of projection neurons in cognitive and emotional behaviors. Combining the gene expression and functional data, predictions for how specific polymorphisms in human genes may alter cognitive-emotional abilities will be generated. These predictions will be tested through functional brain imaging and behavioral testing of genotyped subjects. Together, these investigations will provide deep insights into the brain circuits and genetic underpinnings that make possible the cognitive functions of the human mind.