Richard A. Young - US grants

The DNA-dependent RNA polymerases play a central role in gene expression. The aim of the proposed research is to contribute to a more precise genetic and biochemical description of the yeast RNA polymerase II. It is proposed that a combination of genetic and biochemical approaches are used in an effort to determine A) whether the polypeptides that copurify with RNA polymerase activity are all genuine subunits, B) whether other subunits exist which have thus far escaped biochemical detection and C) whether different forms of RNA polymerase II are responsible for the transcription of different kinds of genes. To accomplish these goals, the specific aims of the experiments outlined in this proposal are: 1) To identify and isolate the genes which specify each of the yeast RNA polymerase II subunits by probing a recombinant DNA expression library with antibodies and 2) To construct yeast mutants with lesions at defined RNA polymerase gene loci, resulting in conditional phenotypes, by using isolated subunit DNA sequences mutagenized in vitro to replace the wild type gene. The conditional mutants constructed through this procedure will be used in an effort to: A) identify genuine RNA polymerase II subunits through biochemical analysis, B) elucidate RNA polymerase subunit interactions and reveal previously undetected components of the enzyme through the isolation and analysis of extragenic suppressors, C) identify subunits through which specific regulatory factors act to control gene expression, and examine the possibility that there exist multiple forms of RNA polymerase II, by analyzing the effects of the mutant alleles on the relative ability to express specific genes. The health relatedness of this project derives from its contribution to the understanding of the basic molecular mechanisms which control gene expression.

This proposal is designed to further develop three types of vaccine candidates: recombinant stress protein fusions, HIV and SIV genome-less particles, and live recombinant BCG vehicles that express antigens and lymphokines. We plan to engineer novel recombinant fusion proteins that incorporate mycobacterial stress proteins and retroviral proteins, exploiting the unusual immunological properties of stress proteins. We will continue to develop genetically inactivated HIV/SIV particles as vaccine candidates by constructing stable cell lines that produce the noninfectious retroviral mutants particles. We will also continue to generate and test the immune response to new forms of recombinant BCG vaccines, especially those that secrete interleukin. We are undertaking the simultaneous development of these three vaccine candidates because we recognize that an efficacious HIV vaccine may require multiple components. Our strategy is to investigate the humoral and cellular immune response to each reagent in mice, to optimize conditions required for eliciting CD8+ CTL, and to provide these data and reagents to investigators in other NCVDG programs and at NIAID for further studies in primates. The stress protein fusions, genetically inactivated retroviral particles, and BCG recombinants will be provided to specific NCVDGs and to NIAID for tests of efficacy in animal models. A vaccine study with BCG-SIV recombinants is already underway in collaboration with the NCVDG under Dr. Murray Gardner. The long-term goals of this research program are to improve our understanding of HIV biology and immunology and to develop an HIV vaccine candidate.

The objective of this proposal is to construct novel protein and DNA vaccines that are maximally effective for eliciting potent primary and memory CD8 CTLs and to determine whether such vaccines protect macaques against SIV infection and disease. MHC class I restricted CTLs are thought to play an important role in immunity to retroviruses, and we have discovered that proteins fused to the mycobacterial chaperonin Hsp70 are able to enter into cellular compartments that lead to MHC class I antigen presentation and elicit potent CTLs. Hsp70-SIV fusion proteins and DNA vaccines that express these fusion proteins will be constructed and purified. These constructs will be provided to collaborating investigators to evaluate their ability to elicit potent primary and memory CTLs in mouse models. They will also be provided to collaborating investigators in order to explore the interactions of the heat shock proteins with antigen presenting cells and determine their intracellular fates. Macaques will be vaccinated with those constructs that produce maximal CTL responses in mice. Macaques inoculated with these vaccines will be evaluated for humoral and cellular immune responses in collaboration with Dr. Norman Letvin (Subcontract with Beth Israel Deaconess Medical Center), and for protection against a live challenge with SIV in collaboration with Dr. Michael Wyand (Subcontract with GTC Mason Laboratories).

DESCRIPTION (provided by applicant): It is fundamentally important to understand how human innate immune cells respond to pathogens because these cells are frequently the first line of defense against infection. Recent gene expression profiling studies have shown that pathogen exposure causes changes in the gene expression programs of dendritic cells and macrophages, but have not revealed how these gene expression programs are regulated. With a sequenced human genome and advanced genome-wide analytical technologies, it is possible to begin mapping the transcriptional regulatory networks that control the host cell response to infection by priority pathogens. Such information would allow us to discover precisely how pathogens perturb host cell gene expression and might suggest new strategies for pathogen control. We propose to use newly developed experimental and computational technologies to begin mapping the transcriptional regulatory networks that control the response of innate immune cells to pathogen exposure. To accomplish this, we propose to; 1) identify the genomic targets of key transcriptional regulators in human macrophages and dendritic cells; 2) deduce the transcriptional regulatory networks that regulate macrophage and dendritic cell gene expression programs using a combination of genome-wide location and expression data; 3) discover how the transcriptional regulatory networks of macrophages and dendritic cells are modified when these cells are exposed to priority pathogens.

DESCRIPTION (provided by applicant): We propose to use newly developed experimental and computational technologies to begin mapping the transcriptional regulatory networks that control the cell cycle in human cells. It is fundamentally important to understand these regulatory networks, as many biological processes and diseases are connected in some manner to cell cycle control. Transcriptional regulatory networks can be mapped by identifying the sites bound by regulators throughout the genome of living cells, and by combining this information with gene expression data. To accomplish this, the specific aims of the proposal are: 1) Determine the genomic binding sites of candidate cell cycle transcriptional regulators in human cells; 2) Identify the set of human genes whose expression oscillates during the cell cycle in human cells; 3) Construct a model of the transcriptional regulatory network that controls the human cell cycle and test its key features; and 4) Identify differences between cell cycle regulation in primary fibroblasts and fibroblast tumor cells. The transcriptional regulatory networks that control the human cell cycle will be of fundamental value to biologists because they will reveal pathways by which cellular phenotypes are regulated and may suggest new strategies to diagnose and combat diseases associated with defects in cell cycle regulation.

[unreadable] DESCRIPTION (provided by applicant): We propose to begin mapping the transcriptional regulatory circuitry that controls the gene expression programs of embryonic stem cells and selected differentiated cells. A short-term goal of this study is to learn how the regulatory circuitry of embryonic stem cells contributes to pluripotency, and a long-term goal is to use knowledge of this circuitry to facilitate efforts to manipulate cellular fates. Mapping transcriptional regulatory circuitry can be initiated by identifying the active and silent portions of the genome and determining how master regulators control a core set of genes. These cell-type specific maps will define active and repressed chromatin structure for the entire non-repeat genome and identify the core regulatory circuitry that defines cellular phenotype. To accomplish this, the specific aims of the proposal are: 1) further develop experimental and analytical technologies that identify the genome-wide location of proteins associated with vertebrate genomes in vivo, 2) define the active and repressed chromatin structure for the entire non-repeat genome in human embryonic stem cells and compare it to that of two differentiated cell types, 3) determine how master regulators contribute to the core transcriptional regulatory circuitry of human embryonic stem cells and two differentiated cells, and 4) identify conserved components of the epigenetic state and core transcriptional regulatory circuitry in human and mouse cells to facilitate genetic tests and help identify the key controls of cell state. Improved understanding of vertebrate chromatin structure and transcriptional circuitry from these studies should lead to new insights into embryonic stem cell pluripotency, will generate maps of regulatory circuitry that may facilitate efforts to manipulate cell fates for regenerative medicine, and will provide the foundation for further mapping regulatory circuitry in human and other vertebrate cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Project Summary/Abstract The Whitehead Institute requests $499,968 to purchase a complete Genome Analyzer-IIx sequencer package including cluster station and paired-end read module from Illumina technologies. The system will provide critically needed capacity for an overburdened sequencing pipeline that supports researchers at the Whitehead Institute using the Illumina platform for a wide variety of applications. The new system will be integrated with an established and successful Illumina sequencing platform at the Whitehead Institute's Genome Technology Core (GTC). The GTC is a fee-for-service core facility that serves the entire Whitehead Institute as well as labs at MIT and dozens of other labs throughout the greater Boston area and the world. Founded in 2002 the GTC has supported hundreds of research projects in a wide variety of experimental systems. The rapid adoption of Illumina sequencing for a wide variety of applications has created a demand for sequencing that will soon exceed the capacity of the existing systems. The addition of the Genome Analyzer system requested in this application will ensure that the dozens of labs supported by the GTC, including the 12 NIH funded labs describing projects in this application, will continue to get the access and support that they need.

The main goal of this RAISE Award project is to discover fundamental aspects of gene regulation in mammals. Many diseased cellular states (including cancer and autoimmunity) are associated with aberrant regulation of transcription of genes regulated by super-enhancers (SEs), large clusters of enhancers that regulate the transcription of genes important for cell type specific processes in both healthy and diseased (e.g., cancer) states.. Therefore, the proposed fundamental studies are of significance to the design of therapies for diseases that have a large toll on human health. The immediate potential impact of the studies concerns inhibitors of SEs that are currently being tested in clinical trials to treat cancer and other diseases. This effort also has an important training component. Undergraduate research is an integral part of labs at MIT. Formal mechanisms such as the MIT Undergraduate Opportunities Program (UROP), the MIT Summer Research Program and Amgen Scholars Program for underrepresented minorities, will be used to recruit undergraduates to the PI's laboratories. One PI will also participate in ACCESS, a weekend at MIT for underrepresented minority students designed to make them aware of opportunities for graduate study. The impact of this research on science and technology will be disseminated to the broader scientific community through the production of a video learning module targeted at both the broad community of citizens and specifically the K-12 educational audience. MIT has been at the epicenter of research at the convergence of the physical, life, and engineering sciences. The PIs will teach courses wherein this interdisciplinary work will be highlighted.

Super-enhancers are occupied by an unusually high density of interacting molecules, and are able to drive higher levels of transcription than typical enhancers. Several lines of evidence suggest that they form via cooperative processes, and SEs are far more vulnerable than typical enhancers to perturbation of components that are commonly associated with most enhancers. Recently, the PIs proposed that a phase separated multi-molecular assembly regulates the formation and function of SEs (Cell, 2017). They suggested that some puzzles associated with SE function are consistent with such a model. These results provide just a starting point to explore the role of phase separation in gene control in mammals. By bringing together sophisticated theoretical studies (rooted in statistical physics) and biological experiments, the PIs now aim to study their novel proposal with the goal of developing a conceptual framework for understanding gene regulation in mammals, and why SEs evolved to regulate key genes. The mechanistic insights thus gleaned will also apply to diverse processes in eukaryotic cells that are mediated by phase separated membraneless organelles. By bringing together approaches rooted in physics and biology, the PIs aim to address the following significant questions: 1] What are the fundamental physical and biological principles that determine how SEs form and function to regulate gene transcription in mammals? 2] Why have genes with the most prominent roles in cell identity evolved to be regulated by SEs? 3] Why do cancer cells have SE-regulated oncogenes, and why are these so vulnerable to drugs that inhibit certain transcriptional regulators? In order to take steps toward answering these questions, the PIs propose to study two major topics: 1] Understanding the nature of the phase transition and its implications for gene regulation. 2] Understanding how super-enhancers nucleate and form.

This RAISE project is being jointly funded by the Physics of Living Systems program in the Division of Physics in the Mathematical and Physical Sciences Directorate, by the Cellular Cluster in the Division of Molecular and Cellular Biosciences in the Biosciences Directorate, and by the Office of Integrative Activities.