David Housman - US grants

The focus of this research is the application of recombinant DNA techniques to the study of familial melanoma. Previous studies have demonstrated weak genetic linkage between the Rh locus on the short arm of chromosome 1 and an autosomal dominant gene responsible for familial cutaneous malignant melanoma (CMM) and its associated precursor lesion, the dysplastic nevus syndrome (DNS). We will use recombinant DNA techniques to increase the number of genetic markers on chromosome 1. We will isolate and characterize DNA sequences from chromosome 1 and use these sequences as DNA hybridization probes to detect restriction fragment length polymorphisms (RFLPs) segregating in families carrying the CMM/DNS gene. The analysis of the inheritance of these DNA markers in relation to the CMM/DNS gene will permit a definitive test of the localization of this gene to chromosome 1. If the gene is located on chromosome 1, the use of recombinant DNA techniques will allow its location to be established with a high degree of precision. The precise localization of the gene will permit a number of significant diagnostic questions to be addressed.It will be possible to determine whether all families exhibiting familial melanoma are segregating a gene at the same chromosomal location; it should permit diagnostic evaluation of family members within such families via genetic linkage techniques. The localization of the gene to a specific chromosomal segment will permit the initiation of studies to identify the specific gene responsible for this syndrome. (6)

During the next granting period I plan to continue our research along three major lines. First, we will complete the mapping of the WAGR region of 11p13. Our initial efforts will be directed toward completing a long range restriction map of this region followed by the isolation of the genomic DNA of this region by molecular cloning techniques. From these cloned DNA sequences we will develop a transcriptional map of this region. We will then identify transcripts responsible for the specific functional phenotypes identified with deletions of this region, aniridia, Wilms tumor, urogenital malformations and mental retardation by correlating the location of deletion and translocation breakpoints with the occurrence of these conditions in patients. A second goal of this proposal will be to extend our studies of homology between human chromosome 11 and other mammalian species. One main focus will be to characterize the region of mouse chromosome 2 which is likely to be homologous to the WAGR region of human chromosome 11. We will undertake a detailed effort to study the genetics and functional basis of mutations at the Sey locus on mouse chromosome 2 which we believe is likely to be a homologue to the human aniridia (AN-2) locus on chromosome 11. We will attempt to develop mice which are functionally hemozygous for the mouse homologue to the Wilms tumor gene and determine whether such animals exhibit a high frequency of malignancies derived from embryonic kidney tissue. A further goal of our mouse genetics program will be to continue to characterize homology relationships between other portions of human chromosome II and segments of mouse chromosomes 7 (llp15), 19 (llq13) and 9 (llq23). The third major objective of this proposal will be to continue develop and utilize molecular genetic and somatic genetic techniques to map chromosome 11 in its entirety. Continued effort will be directed towards the extension of studies on the short arm of the chromosome. Increased effort and emphasis will be placed on the long arm of the chromosome during the next granting period. In particular, we will utilize techniques and reagents developed during the previous granting period to isolate somatic cell hybrids for the long arm of the chromosome analogous to those which have been so useful in mapping IIp. Additional cloned genomic DNA segments for IIq will be isolated using these hybrids, and RFLPs will be identified for a subset of these genomic DNA clones, A detailed map of IIq will then be constructed using the combined power of somatic cell genetics, genetic linkage techniques and long range restriction mapping methods. We will utilize map information derived from our studies to test hypotheses regarding the location of genes for specific genetic disorders or predispositions on chromosome 11.

Three basic lines of investigation pertaining to the control of erythroid differentiation of murine erythroleukemia cells will be investigated. The molecular processes that are responsible for the commitment of these cells to terminal erythroid differentiation will be studied. The relationship between globin mRNA synthesis and commitment will be analyzed. The control of genes expressed during the terminal phase of the program will also be studied. (M)

technology /technique development; hypertrophic myocardiopathy; molecular pathology; cardiovascular disorder; atherosclerosis; genetic mapping; nucleic acid probes; ion transport; biological polymorphism; hypertension; diabetes mellitus; blood lipoprotein metabolism; hypertriglyceridemia; kidney disorder; laboratory rat;

The overall objective of this proposal is to understand the processes which control hematopoietic differentiation at the molecular level. The following specific aims will be addressed: 1. To develop a more complete understanding of the factors which control globin gene expression by: a.) Evaluating the impact on the expression of the mouse beta globin gene of mutation at sites in the IVS2 region which exhibit specific binding and footprinting for tissue specific binding proteins B1 and B2 b.) Further characterizing and purifying DNA binding proteins B1 and B2 and c.) Extending DNA binding studies to additional sites in the mouse beta globin gene. 2. To extend our initial studies identifying mRNA regions which contribute to mRNA stability in beta globin and c-fos mRNAs by: a.) Developing additional chimeric constructs between human c- fos and beta globin mRNAs to discriminate with greater precision mRNA sequences or structures which contribute to the differences in stability between these mRNA species. b.) Extending the chimeric mRNA approach to additional mRNA species including alpha globin, c-myc and delta globin mRNAs. C.) Identifying an inducible expression system which will allow us to extend the evaluation of mRNA stability to differentiating hematopoietic cells d.) Developing biochemical and genetic methods for identifying cellular mechanisms which respond to signals within mRNA molecules which lead to differences in half life. 3.) To develop somatic cell genetic approaches to hematopoietic differentiation by: a. Applying gene transfer procedures to determine the significance of changes in expression levels of specific polypeptides in the control of the differentiation program. b. Developing the use of retroviral vectors for insertional mutagenesis in the MEL cell system c. Developing multidrug resistance gene system for use as a selection system for bone marrow progenitor cells and pluripotent stem cells.

neoplasm /cancer pharmacology; neoplasm /cancer genetics; gene expression; antineoplastics; drug resistance; pharmacogenetics; cell bank /registry; complementary DNA; neoplastic cell; tissue /cell culture; molecular cloning;

The basic objectives of this component will continue to utilize genetic strategies to identify and characterize genes of major significance in the development of human tumors as well as the response of tumors to treatment. The main focus of this program has been and continues to be the process of tumorigenesis in nephroblastoma (Wilms tumor). We will continue to study the molecular basis of the action of the Wilms tumor suppressor gene, WT1, discovered and characterized during the previous granting period. We will extend our studies involving gene targeting of mouse ES cells in order to understand the role of the WT1 gene in tumorigenesis and normal development as well as to identify the functional roles of alternatively spliced forms of the WT1 polypeptide. We will continue the biochemical and functional characterization of WT1 by extending our work on interaction partners for the WT1 polypeptide using the yeast two hybrid system, to characterize WT1 interactions with other polypeptides using immunochemistry, to develop cell based systems to directly demonstrate the effects of WT1 on specific target genes and to utilize in vitro transcription and splicing systems for the alternate forms of the WT1 polypeptide. To gain deeper insight into WT1 function, we will explore the role of WT1 in systems other than kidney such as hematopoiesis. We will complete identification and characterization of the WT2 tumor suppressor gene using positional cloning strategies and analysis of mutations and deletions in human tumors. We will further analyze the role of apoptosis in tumor treatment response using genetically based approaches such as further study of the tumor cells deficient for p53. These research strategies will provide the framework for gene identification and characterization extending beyond the immediate province of Wilms tumor to other tumor types which share common mechanisms of tumorigenesis with Wilms tumor as well as to further investigate the underlying basis of response to chemotherapeutic agents and radiation using extensions of the genetic approaches developed during the previous grant period.

The central goal of this proposal is to develop and implement a genotyping strategy for the human genome which permits high throughput genotyping to be achieved at low cost and high efficiency. We have developed an approach based on interspersed repeat sequence per (IRS PCR) which successfully addresses these objectives in the mouse. In this proposal, I propose to develop and implement a related strategy for human genotyping based on the experience we have gained in the development of low cost, high throughput genotyping in the mouse. The adaptation of an IRS PCR based approach to human genotyping will require significant development work. The goals of this proposal are therefore: 1. To carry out the development work necessary to establish an optimal path or paths for implementation of an IRS PCR based strategy for human genotyping. Alternative approaches and strategies will be critically tested and evaluated during this analytical phase of the project. 2. Once optimal strategies are established for identifying and scoring polymorphic markers we propose to implement a genome wide development of genetic markers appropriate for genotyping by the IRS PCR based strategy to support rapid, low cost genotyping of human DNA. 3. The long term goal of the proposal will be to develop a very high marker density which will effectively support complex trait analysis as well as linkage studies for traits which segregate in a simple Mendelian pattern.

The central goal of this proposal is to develop reagents and strategies to support low cost, high volume genotyping efforts for both mouse and human genetics. The principle which underlies our approach is the use of PCR based methods which reproducibly yield a set of hundreds to thousands of single copy genomic fragments distributed across the genetic map. Genetic markers based on single nucleotide polymorphisms (SNPs) identified in such sets of genomic sequences can be genotyped in a simple hybridization based format at low cost with high efficiency and accuracy. Interspersed repetitive sequence polymerase chain reaction (IRS PCR) is a methodology we have explored and developed during the previous granting period to achieve this objective. In the mouse, we have utilized this methodology in pilot studies to identify SNP markers among common inbred strains which can be genotyped using a simple high throughput allele specific oligonucleotide (ASO) procedure. We have also carried out parallel studies which support the feasibility of this approach for human genotyping. In the current application we propose to scale up the current marker generation program to build a collection of genetic markers which can support a low cost, high throughput genotyping effort for both mouse and human genetics. One set of specific aims is directly focused on the process of SNP marker generation and characterization based on processes which have already been developed at pilot scale. A second group of specific aims is focused on technical development efforts to support the central goal of the program.

DESCRIPTION (Adapted from Applicant's Abstract) The hemostatic balance is regulated by vascular bed-specific endothelial cell signaling pathways. The applicants propose that coronary artery thrombosis arises through local alterations in one or more of these pathways. The overall goals of the Collaborative Program are to elucidate the molecular basis of endothelial cell subtype-specific gene expression in the heart and to identify the critical components of cardiac hemostasis. In this project , Dr. Rosenberg will study the role of a platelet-derived growth factor signaling pathway in mediating expression of a gene program within cardiac microvascular endothelial cells that includes tissue factor (TF). He will also optimize a recently developed mouse model of coronary artery thrombosis. In this project, Dr. Aird will examine the role of the Egr-l transcription factor in mediating cardiac-specific hemostasis. He will ask how a single gene can serve to "fine tune" hemostasis according to the local needs of the tissue. In this project, Dr. Mackman will evaluate the role of a thrombin-PAR-1 signaling pathway in governing local levels of procoagulant (TF) and fibrinolytic (tissue-type plasminogen activator) molecules within the heart. In addition, he will address the contribution of monocytederived TF to cardiac hemostasis. In this project, Dr. Housman will use genetic approaches in large populations to identify genotypes which significantly contribute to coronary thrombosis. The three basic science projects are interrelated by several common themes. Each component involves: (1) the study of a cardiac endothelial cell type-specific signaling pathway, (2) the determination of the effects of cell type-specific signaling pathways on global hemostasis (fibrin deposition) (3) the study of TF gene regulation and its role as the initiator of coagulation in the cardiac circulation, and (4) the use of transgenic mouse technology for studying vascular-bed specific hemostasis in the heart. The clinical project will serve as a vital link to validate the role of local hemostatic components in human populations.

The overall goal of Project IV (Housman) is to utilize the DMPK deficient mouse to analyze the genetic interactions that lead to cardiac conduction defects and heart block in this model system. The DMPK knockout mouse was developed to model characteristics of myotonic dystrophy, an inherited disorder in which cardiac conduction defects in the AV node and infra-Hisian and supra-Hisian tissue are a primary cause of premature death. The DMPK knockout mouse model strikingly resembles this pattern of cardiac conduct defect. Mice with absence of function for DMPK show normal cardiac conduction during the first two months of life. Between two and five months these animals develop lengthening of PR interval, lengthening of HIV interval and secondary and tertiary heart block. To define the molecular pathways that lead to this pathological outcome, we will carry out focused efforts to 1) determine the substrates for the protein kinase activity of DMPK 2) resolve the relative roles of two alternative splices splice forms of DMPK that show differential subcellular localization and 3) investigate the role of novel genes identified by transcriptional profiling, proteomic analysis and genetic modifier screens. We will develop a baseline for proteomic analysis of mouse cardiac development and pathology that will be applicable to other Projects in this program. We will also develop for executing mouse modifier gene mapping that will support the efforts of other Projects in this program in identifying gene interactions through genetic modifier mapping.

Shared Research Resources differs from the other core facilities in the MIT CCR in that it covers a range of distributed equipment and services that broadly support the research of the CCR. This facility oversees the maintenance of infrastructure and large shared equipment essential for the research of all research groups within E17/18 as well as the CCR Facilities that benefit all CCR members. It also supports necessary safety operations and related activities. A major role of the facility is the implementation and continuance of a comprehensive Environment, Health &Safety management system being developed by MIT that encompasses safety inspections, laboratory safety and staff training. In addition, this facility provides technical support and service for both shared and laboratory equipment within the CCR.

The CCNE will develop novel nanomaterials and devices for the improved detection and treatment of cancer. A critical aspect of this process is to test promising materials in the context of tumors in a preclinical setting, preferably in vivo. The Mouse Models Core (MMC) will take advantage of the extensive infrastructure available the MIT Center for Cancer Research for the development and characterization of mouse models of cancer to support the science of the CCNE. The overall function of MMC is to provide technical, methodological and analytical support to each of the Projects in the area of in vivo testing. The Core will function as a centralized resource to establish, maintain and effectively utilize a series of animal models of cancer for the evaluation and preclinical testing of the nanomaterials generated by the individual groups. The MMC will provide a combinatior xenograft tumor models and genetically-engineered mouse models (GEMMs). The Core will import established GEMMs for lung, prostate and brain tumors developed at MIT and elsewhere. It will also use cutting-edge methods in gene targeting to construct additional models tailored to fit the needs of the Projects. It will take advantage the gene targeting and transgenic technologies available from the MIT CCR Transgenic Animal Core Facility The Core will utilize in-house bioluminescence imaging capabilities as well as micro-computed tomography (micro-CT) to follow tumor development and response to therapy for various models. Other imaging technologies will be accessed through the MGH Molecular Imaging Center, including for biodistribution analysis. In addition the provision of mouse cancer models, the MMC staff will carry out efficacy studies for nanomaterial-based therapeutics (Projects 1 and 2). It will also perform dosing and other necessary animal manipulations for imaging based studies, for which imaging will be performed at the MGH Molecular Imaging Center (Projects 3-5). The MMC will be directed by Dr. David Housman (MIT). Dr. Alain Charest (MIT) will directly supervise the activities of the Core and participate in strain construction. Dr. Tyler Jacks will serve as a consultant for the Core.

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy, with an incidence of about 1 in 8,000 adults. The most common form of the disease, DM1, is caused by an expanded CTG repeat in the 3'UTR of the DMPK gene, and CUG repeat RNAs from this gene fold into hairpins that accumulate in nuclear foci, resulting in effective depletion of the alternative splicing factor Muscleblind (MBNL1) and hyperactivation of the splicing factor CUG Binding Protein 1 (CUGBP1). Misregulation of splicing by these factors is central in the disease. Thus, characterization of the spectrum of changes in the transcriptomes of DM patients is central to understanding disease pathogenesis. This project seeks to understand the molecular basis of DM and to identify genes and mRNA isoforms suitable for therapeutic intervention using an approach based on next-generation sequencing of mRNAs. The project has the following specific aims: 1) To generate a comprehensive catalog of genes, exons and mRNA isoforms whose expression is altered in DM, and to assess the variability of these changes between individuals. 2) To characterize gene and mRNA isoform expression changes in mouse models of DM. 3) To associate gene and isoform changes with clinical and pathological features in DM. Achieving these aims will lay the foundation for a deeper understanding of DM and will generate leads for future molecular genetics and screening studies and is likely to identify candidate therapeutic targets. PUBLIC HEALTH RELEVANCE: This research project will comprehensively determine the changes in RNA and protein molecules that occur in the muscles of patients affected by myotonic dystrophy, which is the most common adult onset form of muscular dystrophy, affecting 1 in 8,000 adults. Knowledge of these molecular changes will help to identify which molecules and genes underlie specific symptoms of the disease and will aid in identifying targets for therapy.

The mission of the Shared Research Resources (SRR) Core Facility is to maintain the infrastructural underpinnings of all aspects of the Koch Institute's research program. This Core differs from all other Core Facilities in that it does not have a specific locafion but instead functions building-wide. The Core has several key responsibilities. First, it oversees the smooth running of the Kl building, interfacing with MIT's physical plant to address any emerging issues. This includes overseeing maintenance of cold rooms, warm rooms, fissue culture rooms and darkrooms. Second, the Core provides, supports and maintains a wide range of distributed equipment that is used as a shared resource by the Core Facilities and/or the researchers in the Kl building. Third, it supports the comprehensive Environment, Health & Safety management system (EHS-MS) developed by MIT in compliance with federal, state and local regulafions. In this capacity, the Core provides safety inspections, laboratory safety, staff training and hazardous waste management to investigator laboratories and Core Facilifies. Finally, the SRR Core oversees dedicated research spaces in the Kl building that have been developed to provide oversight of, and enable safe working pracfices with, certain high titer VSVg-pseiJdotyped viruses, high-risk isotopes, and the cesium g-cell radiation source essential of our research programs. As oufiined below, much of the cost of the SRR Core's activity is supported direcfiy by MIT. However, CCSG funds provide crifical contributions towards the partial salary support of the SRR Core personnel and central services.

The simple genetic cause of Huntington?s disease contrasts starkly with the vast number of pathways that are affected by the mutation. Some of these pathway-level changes may persist even if the mutated allele of the disease-causing gene (HTT) can be corrected through gene therapy or related methods. During the first granting period, our analysis of HD models identified several potential therapeutic directions, including ones closely tied to epigenetics (the transcriptional regulators NEUROD1, WNTand ELK-1), as well as pathways that interact with epigenomic changes (energy metabolism and lipid biochemistry). Some of these effects were restricted to particular cell types in the brain. We also found evidence that mutant HTT (mHTT) expression causes neurodevelopmental impairments, changing the distribution of cell types in the brain. We and others have also identified a significant number of genetic variants in the human population for which there is significant support for an impact of that variant on HD age of onset (AOO). In the current proposal, we examine the therapeutic potential of interventions based on these findings. We will target these pathways in mice, measuring how interventions alter transcription, the epigenome, signaling and metabolomics. A critical innovation is our use of single-cell and spatially resolved methods to examine how responses to mHTT and therapeutics vary among different types of cells. Equally important, we will differentiate specific cell types from induced-pluripotent stem cells (iPSC) in vitro to examine cell-type specific effects in human cells. Using an approach based in systems biology we will look for common pathways that are affected by the genetic AOO modifiers, the candidates from our prior grant period and leads from the literature. Our approach is highly innovative, as it uses cutting edge experimental methods with single-cell and spatial resolution to reveal aspects of HD that cannot be detected in homogenates. We also computationally integrate multi-omic data (genomics, epigenomics, transcripts, proteins and metabolites) from the individual cells and brain regions to uncover therapeutic pathways. The research is highly significant, as it seeks to guide therapeutic discovery for an invariably fatal neurodegenerative disease. We expect that the impact of our work will extend beyond HD, by providing a model for how to measure and model cell-type specific neurodegeneration to identify therapeutic approaches.