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.

We present here research proposals designed to creatively conceptualize, produce and evaluate vaccines to protect against AIDS. The theme of our National Cooperative Vaccine Development Group is "Immunological and Biological Strategies for an AIDS Vaccine." We intend to thoroughly assess the precise targets of antibodies and T lymphoctyes among SIV1 and HIV1 proteins, to elucidate molecular mechanisms that regulate the expression of SIV1 and HIV1 genes, to use the immunological and biological information to construct the best SIV subunit vaccines using recombinant DNA techniques, to test the immunoprophylactive efficiency of the SIV subunit vaccines in primates and finally, if an SIV vaccine works, to fashion a similar HIV1 subunit vaccine for human AIDS. The Principal Investigator, Dr. Richard Young (Whitehead Institute), will use newly developed recombinant DNA technology to efficiently survey all possible SIV and HIV polypeptides to determine the precise targets of the antibody and helper T lymphocyte (CD4) response during infection. His program will also involve the development of a novel vehicle from M. bovis BCG that can be constructed to produce and deliver HIV protein antigens for immunoprophylaxis. The recombinant DNA tools and products will be provided to Dr. Robert Schooley (Massachusetts General Hospital) to investigate what protein antigens are targets of the cytotoxic T cell (CD8) response during infection. Dr. Herman Eisen (MIT) will collaborate with Drs. Young and Schooley to help define the cellular targets of killing and the mechanisms involved in killing. Dr. David Baltimore (Whitehead Institute) will investigate molecular mechanisms by which viral replication could be attenuated in the cell and will provide information about the regulation of SIV and HIV gene expression. Based on this immunological and molecular biological information, subunit vaccine candidates will be formulates for simian AIDS by the group as a whole and such candidates will be constructed and produced by Dr. Rosenburg (Smith Kline). Dr. Ronald Desrosiers (New England Regional Primate Research Center) will investigate SIV neutralization by antibodies and will test the vaccine candidates produced by Smith Kline in primates for protective efficacy. The immune response to vaccination and subsequent challenge by live virus will be studied by the immunological programs (Young and Schooley, in collaboration with Eisen). The entire group will use information derived from the primate experiments to formulate and produce a vaccine for human AIDS.

The transcription initiation apparatus includes a set of general factors that associate with promoter DNA and with RNA polymerase II during initiation. The general transcription factor TFIID is the first to associate with promoter DNA during assembly of the initiation complex. The native form of the general transcription factor TFIID consists of the TATA binding protein (TBP) and multiple TBP-associated factors (TAFs) in higher eukaryotes, and we have recently found that this is also the case for yeast. This proposal is designed to identify and clone the genes for the complete set of Saccharomyces cerevisiae TBP-associated factors, and to use these genes to pursue molecular genetic and biochemical experiments designed to elucidate the structure of yeast TFIID and the roles of TAFs in transcription initiation. To accomplish these goals, the three specific aims of this proposal are: i) to isolate genes encoding TBP-associated factors in S. cerevisiae using genetic and biochemical approaches; 2) to investigate the effects of mutations on individual TAF functions in vivo; and 3) to study the function of TAFs and the TBP/TAF complex in vitro. To isolate genes encoding TAFs, we will continue to exploit a genetic selection that has already led to the isolation of four TAF genes; the four extragenic suppressors of RNA polymerase II CTD truncation mutants studied to date encode TAFs. As a biochemical approach to isolating TAF genes, we will identify the protein components of a high molecular weight complex containing TBP, subject these proteins to microsequence analysis, and use this information to design oligonucleotides for gene isolation. To investigate the effects of mutations on individual TAF functions in vivo, we will construct null and conditional mutations in the TAF genes and analyze their effects on transcription in vivo. To investigate the functions of the TAFs in the TFIID complex, wild-type and mutant TFIID preparations will be examined in a variety of transcription assays designed to assess the role of TAFs in template assembly and commitment and in basal and activated transcription. The health relatedness of this research derives from its contribution to the understanding of the basic molecular mechanisms that 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): Gene expression programs are fundamental to all living processes. It is now possible to envision mapping the transcriptional regulatory networks that control gene expression programs in living cells. Such maps would be of fundamental value to biologists, and when extended to mammalian cells, might lead to new therapeutic strategies for human diseases. Recently developed technologies allow systematic identification of the genome-wide location of transcriptional regulatory proteins in living cells, and this information can be combined with gene expression and other information to discover transcriptional regulatory networks. We propose to further refine and automate the experimental and computational technologies that enable mapping of transcriptional regulatory networks in living cells. We will use these refined technologies to map the transcriptional regulatory networks that control major biological processes in living yeast cells, and to identify the issues that are key to large scale mapping of mammalian regulatory networks in future studies. The specific aims of this proposal are: 1) further refine and automate the experimental and analytical technologies that enable efficient mapping of transcriptional regulatory networks in living cells; 2) discover the transcriptional regulatory networks that control major biological processes in yeast, including cell cycle, nutrition, environmental response, genome maintenance, and development; 3) refine technologies necessary for mapping mammalian transcriptional regulatory networks and test them by initiating the mapping of regulatory networks involved in control of human cell cycle.

DESCRIPTION (provided by applicant): We propose to use genome-scale chromatin immunoprecipitation analysis (ChIP-on-chips) to map the transcriptional regulatory networks that control normal human pancreatic islet function. In particular, we propose to identify the promoters bound by transcription factors that play a key role in the development and function of human and murine pancreatic islets, including HNF3beta, IPF-1, NKX6.1, NKX2.2, PAX4, ISL1, and NEUROD1. By combining this information with gene expression data (EPConDB), we plan on generating maps of active transcriptional regulatory circuitry in these cells. This information will provide insight into how the functional properties of the beta-cell are acquired and maintained, and will be valuable knowledge in developing cell-based approaches for treating type 1 and type 2 diabetes. Dr. Young's laboratory will provide genomics technologies and will construct the regulatory maps. Dr. Bell's laboratory will provide knowledge in beta-cell biology and expertise in isolating mouse islets and culturing beta-cell lines.

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.

DESCRIPTION (provided by applicant): Gene expression programs are fundamental to all living processes. It is now possible to envision mapping the transcriptional regulatory networks that control gene expression programs in living cells. Such maps would be of fundamental value to biologists, and when extended to mammalian cells, might lead to new therapeutic strategies for human diseases. Recently developed technologies allow systematic identification of the genome-wide location of transcriptional regulatory proteins in living cells, and this information can be combined with gene expression and other information to discover transcriptional regulatory networks. We propose to further refine and automate the experimental and computational technologies that enable mapping of transcriptional regulatory networks in living cells. We will use these refined technologies to map the transcriptional regulatory networks that control major biological processes in living yeast cells, and to identify the issues that are key to large scale mapping of mammalian regulatory networks in future studies. The specific aims of this proposal are: 1) further refine and automate the experimental and analytical technologies that enable efficient mapping of transcriptional regulatory networks in living cells; 2) discover the transcriptional regulatory networks that control major biological processes in yeast, including cell cycle, nutrition, environmental response, genome maintenance, and development; 3) refine technologies necessary for mapping mammalian transcriptional regulatory networks and test them by initiating the mapping of regulatory networks involved in control of human cell cycle.

[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): The concept of a tumor stem cell, a self-renewing cell capable of regenerating the tumor, has shed light on the persistence of human cancers and offers a new focus for translational research. Our goal is to understand the epigenetic mechanisms that distinguish the tumor stem cell from other cells of the tumor and from other normal, self-renewing cells. Epigenetic mechanisms, such as chromatin modifications, DNA methylation and small noncoding RNAs are stable, long-term (typically heritable) changes in the transcriptional potential of a cell that are independent of changes in the underlying genomic sequence. The epigenetic state of a cell serves to define cell identity and the limits of that cell's potential fates. Thus, knowledge of the epigenetic state of cells may identify signature for both tumor stem cell identity and potential. The epigenetic states of histone modifications, DNA methylation and small noncoding RNAs will be mapped using chromatin immunoprecipitation, bisulfite conversion and generation of RNA libraries coupled with genome-wide sequencing. For insight on tumor stem cells, we will take advantage of a previously developed model system that provides a consistent source of cells with tumor stem cell ability. Proposed experiments will examine the differences between a panel of cells, each sorted to enrich for the subpopulation with tumor stem cell ability. For comparisons, non-tumor stem cells will be isolated from the same source and the differences between tumor stem cells and pluripotent and multipotent stem cells will be examined. Additional experiments will be designed to perturb the epigenome of cells and directly test how the epigenetic state of the cell-of-origin affects subsequent tumor phenotype. The knowledge generated could lead to substantial new insights, including the identification of putative markers for early diagnosis, prognosis or monitoring of tumor therapies. Understanding the differences between tumor stem cells and other self-renewing cells could lead to more specific therapies that were also less toxic to normal cells. Manipulating the epigenomic state and examining the results on tumorigenicity would provide direct insight on how these signatures translate into clinically relevant phenotypes.

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.

DESCRIPTION (provided by applicant): MYC is the most frequently amplified oncogene in human cancers occurring in a wide range of tissue types including breast, lung, and prostate. MYC overexpression occurs in at least 30% of all human cancers and frequently correlates with poor clinical outcome and increased chance of relapse. An estimated 450,000 Americans are diagnosed with a MYC-dependent cancer each year. These patients are in need of novel and effective treatment strategies. c-Myc is a helix-loop-helix transcription factor that drives a proliferative cell state by forming a heterodimer with Max, binding sequence-specific DNA elements and stimulating transcription of proliferation-associated genes. Transcription factors are key regulators of cell state as they control the gene expression programs that drive cell type specification and commonly are terminal components of a signaling cascade. These gene expression programs are often deregulated in disease states making transcription factors an ideal class of proteins for therapeutic targeting. However, most transcription factors lack clear pockets for small molecule binding and therefore have been largely considered undruggable with current technologies. A major challenge in the chemical biology field has been to develop potent small molecule inhibitors of transcription factors. While previously published work has identified small molecule inhibitors of c-Myc/Max heterodimerization using truncated proteins in FRET and yeast two-hybrid assays, only a few thousand compounds were screened in each case and the in vitro potency of these inhibitors is limited. Indeed, the potency fails to translate to in vivo activity in animal models. In general, these compounds fail to meet the generally agreed upon criteria for acceptable chemical probes. New chemotypes, which can be successfully developed into chemical probes, are desperately needed.)The proposed research aims to identify inhibitors of c-Myc/Max dimerization and DNA binding using novel technology. A robust high-throughput in vitro assay has been developed to screen for inhibitors of c-Myc/Max dimerization and subsequent binding to its DNA binding site. Secondary biochemical and cellular assays have been developed to validate hits from the primary screen and study them in greater detail. A large high-throughput screen as could be provided through the MLPCN using these robust assays should provide tractable hits for development and validation of biological effect. Medicinal chemistry optimizing these lead molecules through iterative use of downstream assays outlined herein then provides the opportunity to generate useful chemical probes to study c-Myc function. Such probes will hopefully lead the way to new therapeutics against this quintessential cancer target and offer insights into mechanisms for directly inhibiting transcription factors. PUBLIC HEALTH RELEVANCE: Deregulated c-Myc function drives at least 30% of all human cancers and frequently correlates with poor clinical outcome and increased chance of relapse. However, clinically useful inhibitors of c-Myc function have not been developed to treat these patients. The proposed research aims to develop direct inhibitors of c-Myc function, which could lead the way to new therapeutics against this quintessential oncogenic protein. ) )

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.