Monica J. Justice - US grants

The primary goal of the proposed research is to understand the molecular genetic basis of the development of B-cell lymphomas in mice. The identification of new loci that are implicated in causing murine B

Genes on the distal portion of mouse Chr 11 are also found on human Chr 17. This extreme linkage conservation allows us to utilize the physiologic, pathologic, and molecular similarities of mouse and human in studies that exploit the experimental genetic advantages of the mouse. The primary goal of the proposed research is to obtain new mutations that reflect single gene function on the distal region of mouse Chr 11. Establishing genotype/phenotype in a specific region by mutagenesis can provide a cost effective approach to determining gene function, particularly in gene rich regions. The conserved synteny between mouse and human will provide a direct link to predications of gene function in humans. We will enhance the genetic analysis of mouse Chromosome 11 by inducing mutations with the chemical mutagen N-ethyl-N-nitrosourea. Appropriate genetic markers will be used to isolate recessive embryo and juvenile lethal and visible phenotypes in the second generation (G/2) animals. We will extend the mutational analysis by screening for multiple medically relevant recessive phenotypes that include: a) neurological, behavioral, and sensory organ defects, b) hematopoietic defects, c) autoimmune disease, d) obesity, e) infertility, and f) morphological abnormalities. Homozygous recessive embryos lethal phenotypes will be classified to a particular stage of embryonic death to understand unique aspects of mammalian development. Selected new mutations will be mapped to a higher resolution using deletion sub-intervals and complementation analysis. Some pairwise complementation tests will be performed on mutations like phenotypic classes. We will identify the genes involve din selected mutations, and correlate the molecular lesion with the biological phenotype. Mutations will be provided to the community and availability will be posted on the World Wide Web. Mutant mouse stocks will be frozen as embryos and/or sperm for archival purposes. Our work will generate many new models of human diseases, to help treat and understand diverse disorders such as behavioral maladies, birth defects, infertility, aging, and obesity that will be useful to members of the biomedical community.

DESCRIPTION (adapted from investigator's abstract): The investigators propose to conduct a large scale mutagenesis screen using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to isolate recessive mutations that affect mammalian development. They will use a strategy that incorporates balancer chromosomes that cover approximately 25 percent of the mouse genome. These Cre-loxP engineered balancer chromosomes will be used as genetic tools to isolate region-specific mutations that result in all stages of embryonic or postpartum death. Mutations that result in embryonic death will be characterized by determining the stage of embryonic arrest, developmental histopathology, and a molecular profile through in situ hybridization. They will isolate viable mutations genome- wide using (1) X-ray analysis for skeletal and limb dysmorphology and bone density, (2) tandem mass spectrometry for biochemical metabolites, (3) urinalysis for glucose, and (4) a complete blood or urogenital abnormalities will be further characterized by endocrine tests. Mutations that result in death during neonatal to juvenile development will be displayed on a web site and distributed to the scientific community on request. Numerous human birth defects will be modeled including axial- limb patterning, neural tube and cardiac defects, and placental failure. Further, models of pediatric metabolic disorders and endocrinopathies will be isolated and characterized.

DESCRIPTION: (Adapted from the investigator's abstract) The primary goal of the proposed research is to understand the molecular genetic basis for the development of B-cell leukemias and lymphomas. Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice by insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Some AKXD recombinant inbred strains of mice have a high incidence of B-cell lymphomas caused by MuLV insertion, making them valuable resources for identifying new proto-oncogenes using the retrovirus as a molecular tag. Viral insertion site amplification (VISA) can quickly identify proviral flanking sequences in the AKXD somatic tumors by obtaining a viral sequence tag (VST). An analysis of four AKXD strains 35 genes well as many unknown VSTs altered by retroviral insertion. We will extend this study to obtain VSTs for the remaining five AKXD strains that develop primarily B-cell lymphomas. Insertions at one locus called lymphoid viral insertion site 1 (Lvisl) account for 23 percent of the proviral insertion mutations in the AKXID B-lineage tumors, suggesting that genes at Lvis1 play a primary role in the development of hematopoietic disease. Two genes proximal to Lvisl are misexpressed: the hematopoietic homeobox gene, Hex, and a kinesin-related spindle protein, Eg5. Either of these genes could play a role in leukemogenesis, and we will examine this hypothesis by 1) overexpressing the genes in transgenic mice, and 2) transducing the genes into hematopoietic cells using retroviral gene transfer. These genes may act singly or together to potentiate leukemogenesis. Further, we propose to examine the role of Hex in hematopoeisis by eliminating function by a tissue-specific targeted gene disruption. The VSTs represent the majority of genes that contribute to disease onset and progression within the hematopoietic lineages of the AKXD strains. To make the data publicly accessible, we will develop a database describing VSTs and tumor phenotypes. Through collaborations, we will examine the potential involvement of some genes in human leukemias and lymphomas. These data can be integrated with both genomic and gene expression profiles from human cancers to uncover the pathways involved in the development of leukemia and lymphoma in both mouse and human.

DESCRIPTION: (provided by applicant) The International Mammalian Genome Society (IMGS) sponsors the annual Mouse Genome Conference with alternating locations in North America, Europe and Asia. Since 1990, attendance has grown from 100 to 450, and is expected to increase. Many leaders in the field are regular participants. The meeting has also attracted young investigators and newcomers to the field, providing an opportunity for contacts with established investigators. This conference provides a forum for discussion of new initiatives for understanding the function of genes identified by the human genome project. Current programs in large scale regional and genome-wide mutagenesis of the mouse, in this country and abroad, had their inception in discussions at the 1994 and 1995 meetings. The Conference has stimulated international collaborations and provided a sense of community to investigators in the field. It hosts meetings of the Editorial Board of the IMGS journal, Mammalian Genome, Mouse Chromosome Committees, and the Mouse Nomenclature Committee, a body that has maintained systematic nomenclature for mouse for more than 40 years. Speakers and session chairs include strong representation of women. This application requests support for the next five meetings in this series, the 2001 meeting to be organized by Ian Jackson, Ph. D. in Edinburgh, Scotland, the year 2002 meeting to be organized by Monica Justice, Ph. D., in San Antonio, Texas, and the years 2003-2005 meetings, which are in various stages of planning. The intellectual focus of these meetings will be directed towards functional mammalian genomics, an area in which the mouse plays an increasingly central role, and towards analysis and annotation of large scale sequence data. Topics will include mutagenesis screens, analysis of quantitative and multigenic traits, functional evaluation of new mutants, modeling human disease, pharmacogenetics, microarray systems, and comparative large scale human/mouse sequence analysis.

[unreadable] DESCRIPTION (provided by applicant): Now that the mouse and human genome sequences are complete, biologists need systematic approaches to determine the function of each gene. Nucleotide sequence alone does not predict gene function, so functional genomic studies are required. One of the most powerful ways to reveal gene function is to generate mutations and determine their consequence in the living organism. Here, our goal is to provide functional information for genes that map to human chromosome 17, a linkage group that is conserved on mouse chromosome 11, through mouse mutagenesis. Among the nearly 700 genes that will be investigated in this study, many will be causally associated with human disease. Genetic resources for functional genetic studies of this targeted region were generated previously. The purpose of this R01 is to use these genetic resources to ask 1) how many genes are essential, 2) what proportion of genes are likely to mutate to a readily detectable phenotype, 3) what phenotypes are most commonly observed after mutation, and 4) what diverse functions can mammalian genes perform? Our underlying hypothesis is that forward genetics using N-ethyl-N-nitrosourea (ENU) mutagenesis is an efficient way of asking questions about gene function in mammals. Our specific aims are to 1) query genetic function on mouse Chromosome 11 using high- efficiency ENU mutagenesis, 2) identify the molecular lesions in mutations, 3) determine the molecular and cellular basis for the defects in mutants with blood cell and/or cardiovascular defects, and 4) extend the depth of an established sperm/DNA archive for future gene-based screens. The mutations we generate have been shared, and will continue to be shared with the scientific community, through a public website www.mouse-genome.bcm.tmc.edu. Our previous work has made a difference in the way we can approach mouse genetics, and our proposed work will influence the way we understand what genes do and how they work in mammals. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The International Mammalian Genome Conference is a popular stanchion of the mouse community, and the annual conference of the International Mammalian Genome Society. This meeting began nineteen years ago in Paris, France, to provide a forum for promoting the understanding of the mouse genome. Initially, the focus was on building physical maps, understanding mouse strains and their variation, and developing new genetic strategies for modeling human disease. Now, understanding the functional relevance of mammalian genes is a major goal. Today, with sequence of the mouse genome available, and multiple genome sequences being completed, focus remains on modeling human diseases, and identifying new genes involved in simple and complex genetic disorders. New technologies are an emphasis, but are moving towards understanding evolutionarily conserved non-coding sequences and developing phenotyping strategies to understand mouse biology and physiology. The aims of the Mammalian Genome Conferences are 1) [unreadable] To communicate important discoveries of the genome project, 2) To coordinate the generation and dissemination of information on new mutant mice, 3) To evaluate the status of informatics resources for comparative vertebrate bioinformatics, 4) To develop and establish priorities for 'functional genomics' strategies, including development of new assays for large scale phenotypic screening, and research resources, 5) To foster international and national collaborative research among investigators using the mouse as a genetic model, and 6) To mentor students and postdoctoral fellows in the area of mouse genetics, genomics and biology. [unreadable] Relevance: The mouse is a primary genetic model for human disease. The International Mammalian [unreadable] Genome Conferences, in particular, promote human disease modeling through genetic strategies and through international collaborations to better utilize mouse resources. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A comprehensive functional annotation of all genes is a key goal for the future investigation of mammalian systems and biomedical sciences. We have established a consortium for the large-scale phenotyping of mouse mutants, which is fundamental to the investigation of gene function. The BaSH consortium, Baylor College of Medicine (BCM), Houston, Texas, the Wellcome Trust Sanger Institute Mouse Genetics Programme, Hinxton, United Kingdom, and the Medical Research Council Harwell, (Mammalian Genetics Unit and Mary Lyon Centre), United Kingdom, will undertake broad-based phenotype analysis of 300 IKMC mouse lines per year with the aim of identifying perturbations on developmental, physiological and biochemical pathways that will guide experimenters to develop hypothesis-driven research into disease systems. Our aims are to 1) complete the broad-based disease phenotyping of over 1500 lines of mutant mice in the C57BL/6N genetic background, 2) validate an optimized and enhanced broad-based phenotyping pipeline that will detect a variety of disease phenotypes and increase throughput, and 3) submit phenotypic data to the designated data coordination center, ensuring an interface with the wider biomedical scientific community that will inform human genetic studies. Our approach is to build on our unique expertise in mouse phenotyping and the successful operation of major pilot projects for mouse phenotyping of EUCOMM and KOMP mutants to deliver a phenotyping pipeline with strategic breadth that serves the needs of the medical community. Our pipeline design aims to deliver mouse models in key therapeutically relevant areas - for example in Cardiovascular, Metabolic, Neurological, Respiratory and Immunological Systems. Assessment of mouse mutants using our phenotyping pipeline will discover novel preclinical models of therapeutic importance, encompassing many of the diseases that account for the highest rates of disease morbidity throughout the developed world. RELEVANCE: Most of the genes in a person are normal, but we also carry several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little of no consequence, or function only under stress. Currently, we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes using model organisms such as the mouse.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DESCRIPTION (provided by applicant): Annotation of the reference human genome has identified approximately 20,000 protein coding genes as well as 3,000 non-coding RNAs. Together these genes orchestrate the development of the organism, supporting all aspects of the function of cells, tissues, organ systems as well physiology and behavior. The l000 genomes project has revealed extraordinary levels of diversity in human genomes, yet for most genes neither the function of normal version nor the disease consequence of loss-of-function variants is known. The mouse provides a route to understand the function of genes and their variants. Mice share developmental, physiological, anatomical and metabolic parallels with humans, which are evident in healthy as well as diseased states. These reflect similarities of the genes in both species. Mutant mice generated using ES cell technology are a sensitive biological assay system from which a deep understanding of function can be gleaned and they also provide a long lasting biological resource for further study. The objective of this proposal is to generate mutant mice from a resource of ES cells with conditionally targeted, lacZ-tagged alleles generated under previous NIH (KOMP) and EU (EUCOMM) funded programs. We and others will use these mice to discover the function of genes. We are proposing to carry out this work at scale and have formed a consortium of three Institutes (Baylor College of Medicine, the Sanger Institute and MRC Harwell) to work together as equal partners to achieve this objective. We will generate mice corresponding to 1500 mutant genes from ES cells distributed by the Sanger Institute. We will characterize the adult and embryonic expression pattern of the each targeted gene and by breeding determine the requirement of each for embryonic development and fertility, if any. All of the alleles will be cryo-preserved and placed in a repository and all of the data will be deposited in a centralized data coordination center to support further studies. RELEVANCE: Most of the 20,000 genes in a typical person are normal but we also have several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little or no consequence, or function only under stress. Currently we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes.

DESCRIPTION (provided by applicant): Annotation of the reference human genome has identified approximately 20,000 protein coding genes as well as 3,000 non-coding RNAs. Together these genes orchestrate the development of the organism, supporting all aspects of the function of cells, tissues, organ systems as well physiology and behavior. The l000 genomes project has revealed extraordinary levels of diversity in human genomes, yet for most genes neither the function of normal version nor the disease consequence of loss-of-function variants is known. The mouse provides a route to understand the function of genes and their variants. Mice share developmental, physiological, anatomical and metabolic parallels with humans, which are evident in healthy as well as diseased states. These reflect similarities of the genes in both species. Mutant mice generated using ES cell technology are a sensitive biological assay system from which a deep understanding of function can be gleaned and they also provide a long lasting biological resource for further study. The objective of this proposal is to generate mutant mice from a resource of ES cells with conditionally targeted, lacZ-tagged alleles generated under previous NIH (KOMP) and EU (EUCOMM) funded programs. We and others will use these mice to discover the function of genes. We are proposing to carry out this work at scale and have formed a consortium of three Institutes (Baylor College of Medicine, the Sanger Institute and MRC Harwell) to work together as equal partners to achieve this objective. We will generate mice corresponding to 1500 mutant genes from ES cells distributed by the Sanger Institute. We will characterize the adult and embryonic expression pattern of the each targeted gene and by breeding determine the requirement of each for embryonic development and fertility, if any. All of the alleles will be cryo-preserved and placed in a repository and all of the data will be deposited in a centralized data coordination center to support further studies. RELEVANCE: Most of the 20,000 genes in a typical person are normal but we also have several hundred broken ones. While some broken genes can cause severe disease such as cystic fibrosis or cancer, others have little or no consequence, or function only under stress. Currently we have some understanding of the function of just one third of human genes. If we are to fully understand human health and disease we must expand knowledge of gene function to all of our genes.

DESCRIPTION (provided by applicant): Drug resistance and cancer relapse are attributed to the persistence of stem cells. The PR/SET domain protein PRDM14 regulates a cell's pluripotent potential to initiate and progress cancer through epigenetic events that are molecular signatures of stem cells. Normally, PRDM14 uses epigenetic mechanisms to establish the pluripotency of primordial germ cells, and its role in cancer is similar. PRDM14's widespread expression in many tumor types, including breast, ovarian, colon, lung, melanoma and lymphoblastic leukemia implies that it is commonly involved in epigenetic events to initiate cancer. This proposal challenges paradigms by suggesting that PRDM14 regulates a somatic cell's pluripotent potential to generate C-ICs as it promotes genomic instability, allowing it to cause tumors in many cell types. PRDM14 is a prime target for therapeutic intervention, because of its restricted expression to only a few cells in the body. Key to this project is that unique inducible mouse models will be exploited to examine events during tumor development, progression and relapse. Experiments that address three basic questions will be performed: what biochemical mechanisms does Prdm14 use to establish self-renewal to initiate cancer, how do the initiating cells maintain self-renewal in the tumor, and how does PRDM14 perturb genome integrity within these cells to progress malignancy? The first aim will use cutting edge genomic approaches to confirm Prdm14's binding targets and identify its protein partners, while it determines how Prdm14 alters chromatin to reset pluripotency in pre- leukemia cells. The second aim will determine the properties of Prdm14 pre-leukemia cells using flow cytometry, cellular and molecular profiling approaches. The third aim will determine how PRDM14 catalyzes chromosomal rearrangements, leading to copy number variation in the tumors that cause specific driver mutations such as activated NOTCH1 through recombination. These studies propose a genetic strategy to inhibit recombination and a drug treatment strategy designed against targets downstream of PRDM14's action to prevent the growth of tumors in the mouse model. The mouse models allow the behavior of cancer cells to be monitored from the beginning, mark cells for isolation, and follow their lineage potential. Data from human tumors will be mined and compared with mouse tumors throughout the study and mouse leukemia will be compared with human leukemia in Aim 3 to demonstrate clinical relevance. Although mouse leukemia models are exploited, the work will have far-reaching applications to all cancers. PRDM14's limited normal expression in adults indicates that PRDM14 or its initial regulatory targets are key candidates for a therapy directed against cells that express it abnormally. Therefore, PRDM14 could prove to be a universal target in C-ICs.