Christopher Alan Bradfield, PhD - US grants

The Ahr-locus encodes the structural gene for the AH-receptor (AHR), a ligand activated transcription factor that mediates many of the biological responses of 2,3,7,8-trachlorodibenzo-p-dioxin (TCDD) and related polychlorinated biphenyls (PCBs). One of the directions of our research is to develop new models of the AHR signalling pathway and to use these models to identify the molecular basis of species dependent responses to TCDD (NIEHS R29-ES05703). In this proposal, we describe experiments that represent a new direction for our laboratory that should provide information valuable in characterizing the risks that compounds like TCDD and PCBs pose to the environment. Over the last two years, we have cloned the AHR cDNA and found that it contains a basic-region/helix-loop-helix domain similar to that found in its dimerization partner ARNT. More recently, our mutagenesis studies have provided a functional map of the AHR and have allowed us to localize those domains required for DNA binding, agonist binding, dimerization and transcriptional activation. In addition, we have cloned and characterized the AHR's structural gene, identified many of the 5' regulatory elements that control its expression and mapped its chromosomal location in mice and humans. In this application, we propose to use this new information and the related molecular probes to identify those developmental stages most sensitive to TCDD-toxicity and to construct transgenic mouse lines with mutant and controllable Ahr- and Arnt-loci. One of our highest priorities will be directed towards development of a targeting vector for use in "knocking- out" the AHR and ARNT in embryonic stem cells (Bs cells). By injecting these recombinant Es cells into blastocysts, we propose to generate transgenic mice which are heterozygous and homozygous for a null mutation at the Ahr- and Amt-loci. We propose that the mouse strains gene rated under this project will prove valuable in the following ways, 1) to determine the phenotype of mice lacking an AHR or ARNT, 2) to identify the effects of TCDD and PCB mixtures that are not mediated by the AHR or ARNT, 3) as prototype strains for subsequent in vivo mutation studies of the Ahr- and Arnt-loci (e.g. humanizing the murine model), and 4) determining the role of altered gene expression in TCDD toxicity.

DESCRIPTION (provided by applicant): Understanding the molecular details of dioxin/Ah receptor (AHR) signal transduction will yield a number of benefits. First, identifying modifiers of AHR signaling will help explain the tissue and species specificity of dioxin toxicity and thus will aid attempts to predict the human and environmental risk that results from exposure to these pollutants. Second, the AHR is a prototype of a large super family of environmental sensors. What we learn here will shed significant light on a variety of environment-gene interactions. Third, it is likely that the AHR will be involved in a variety of human disorders. Detailed information about signaling can guide the pharmacology necessary to develop related therapeutics. Finally, identifying endogenous activators of the AHR will provide one of the most significant clues as to how and why this protein signals in development and begin to explain the conservation of this protein throughout evolution. In an effort to answer these questions, we propose the following specific aims: Aim 1: Identify/understand modifiers of AHR signaling through the use of genetic screens in the yeast, S cerevisiae. We will employ two screening approaches in yeast. First we will use a library of deletions to determine the role of each yeast gene product on AHR signal transduction. Second, we will expand upon these screens by using mammalian cDNAs to perform high copy modifier screens in yeast. Identified modifiers will then be characterized using both statistical clustering of their pharmacological action, as well as biochemical approaches to elucidate mechanism. Aim 2: Understand the mechanism of AHR modifiers through the use of signaling kinetics in mammalian cell culture. We propose to use fluorescently tagged molecules and reporters to elucidate the cellular mechanisms of AHR modifiers using a mammalian culture system. Once developed, this system will also serve as the basis for an activator screen described in Aim 3. Aim 3: Determine how the AHR signals during development. The phenotype of Ah null mice suggests the existence of a developmental activator of the AHR. To identify this factor, we propose to: 1) identify those developmental sites where the activator is most likely to be expressed; 2) screen for cDNAs encoding protein factors that activate the AHR using an activator trap protocol in cell culture; and 3) characterize the soluble factor derived from murine heart that activates the AHR in cell culture. Aim 4: Complete the high-resolution domain mapping studies of the AHR.

The recently established Automated DNA Sequencing Facility in the McArdle Laboratory for Cancer Research provides scientists access to state-of-the- art machines for rapid DNA sequencing. All five projects of this program- project grant currently make heavy use of this common facility. The projected needs for this facility will increase in future years. Uses include sequence verification of recombinant DNAs, and screening of candidate cDNA from screening projects such as yeast two-hybrid and protein sequencing. Additionally, all faculty of this PPG are using rodent liver as a model of carcinogenesis. Therefore, an expressed sequence tag data base will be established on this important tissue. Funds are requested to support the costs for running the facility proportional to its use by the projects supported by this program-project grant. Only costs incurred for running samples on the common machines and the maintenance of those machines are requested under this core. All other costs for the preparation of sequencing samples are borne by individual projects. This core will be administered by Dr. Bradfielde.

Our objective is to understand the AHR's role in liver development, as well as the mechanism by which 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor interactions are related to tumor promotion organ toxicity and birth defects. We propose to use the power of murine genetics, embryonic stem (ES) cell technology and gene targeting to understand the molecular details of this signaling pathways. Our experiments are derived from recent gene targeting of the murine Ahr and Arnt loci. The surprising phenotypes of these animals have left us with a number of important questions, such as: What is the AHR's role in normal liver growth and development and is the related to the mechanism of TCDD carcinogenicity/toxicity? What receptor domains are involved in toxic endpoints? Given that homozygous Arnt null alleles are embryonic lethal, how can we characterize ARNT's role in TCDD toxicity and carcinogenesis? To address these questions, we have proposed to generate informative allelic series at both the Ahr and Arnt loci and define the molecular basis for their phenotypes. In addition, we propose that these mutant strains will provide insights into the roles of the corresponding proteins in liver development, toxicity and hepatocarcinogenesis. Our specific aims are as follows: AIM #1: Characterize the Ahr null allele and understand the variable that affect expression of phenotype. AIM#2: Generate an allelic series at the Ahr locus to delineate the roles of the AHR and its subdomains on liver development, TCDD-induced hepatotoxicity and carcinogenesis. AIM#3: Generate an allelic series at the Arnt locus to delineate the role of the ARNT protein in TCDD-induced toxicity, teratogenesis and carcinogenesis. AIM#4: Determine the AHR' role in modulating liver size and in hepatotoxicity and carcinogenicity of compounds like TCDD.

DESCRIPTION (Applicant's Abstract): This proposal is directed at understanding the toxicology of halogenated dibenzo-p-dioxins ("dioxins"), as well as the biology of a basic-helix-loop-helix-PAS (bHLH-PAS) protein known as the Ah receptor (AHR). The goal is to better understand the biology of a number of modifiers of AHR signal transduction. The specific questions that will be answered are whether any of these modifiers play a role in dioxin toxicity or if their activity is limited to the induction of xenobiotic metabolizing enzymes (XMEs). The factors to be examined include the small immunophilin-like molecule we refer to as ARA9, a novel homologue of the Drosophila kelch protein, referred to as ARA3, and members of the nonreceptor tyrosine kinase superfamily, cYes and cSrc. The other modifiers to be included in these screens are members of the bHLH-PAS superfamily. These include the dominant negative "Ah receptor related" protein (AHRR), the steroid receptor coactivator (SRC-1), and the AHR partners, ARNT and ARNT2. The final putative modifier we will examine is a cis-acting intragenic element we have recently identified within the AHR's structural gene. We propose that this element regulates AHR expression and thus may be a modulator of cellular sensitivity to receptor agonists. By monitoring toxic endpoints, in parallel with XME induction and liver development, we will be able to identify factors that are shared by these three processes and those which are specific to only a subset of pathways. Identification of pathway-specific factors will provide insight into the molecular mechanisms that underlie each of these biological processes. Information from these studies will have a significant impact on how we estimate risk from chemicals like dioxin, either by providing support for the use of XME induction as a surrogate for toxicity or an argument against such a paradigm.

DESCRIPTION (provided by applicant): This is a proposal to understand Ah receptor (AHR) biology and also an attempt to develop new tools for the toxicologist. In this proposal, we will couple recombinant mouse models, liver cell isolation techniques and modern genomic technology to identify those transcriptional outputs that define the AHR's role in the hepatotoxic effects of potent agonists like 2,3,7,8-tetrachlorodibenzo-pdioxin ("dioxin"). In parallel, we will use the AHR as a model system to develop a new generation of tools, reagents and resources that will aid biologists in their efforts to understand toxicant action. Our specific aims are: Aim 1: Develop cDNA clone sets and microarray technologies (MAT) that are optimal for the study of AHR signaling in parenchymal and nonparenchymal cell types. Aim 2: Develop statistical tools for the optimal design of toxicogenomics studies, as well as to interpret these studies by incorporating data from mutant animal models, pathology and the biomedical literature. Aim 3: We will test the hypothesis that various cell types are interacting and that this interaction can be detected through the presentation of waves of temporally linked transcriptional change. Aim 4: Elucidate the roles of the candidate genes, AHR, Cyplal, Cypla2, Cyplbl, TNF-a and IL6. Using recombinant mouse models, each of these genes will be tested for roles in aspects of hepatotoxicity or for roles in dioxin-induced signaling across hepatic cell types. Aim 5: Test the idea that global aberrations in gene expression can serve as a new definition of toxicity. We will also test the idea that patterns of gene expression will define various pathologies. Aim 6: The MAT we develop will have significant application across toxicant classes. Therefore, we will develop a resource where toxicologists can view the transcriptional responses to dioxin and classic hepatotoxicants.

[unreadable] DESCRIPTION (provided by applicant): Our laboratory has recently identified a spontaneous mouse mutant with an ocular phenotype that we will refer to as Big Eyes (BE). Histological analysis of the BE mice indicated an enlarged anterior chamber that correlated with the appearance of an epithelialization of the corneal endothelium and proliferation of these epithelial cells into the iridocorneal angle. It is our hypothesis that blockage of the iridocorneal angle in BE mice produces an increase in intraocular pressure and that this mouse is a model for human diseases such as posterior polymorphous dystrophy (PPD) or iridocorneal endothelial syndrome (ICE) and associated conditions including glaucoma. The preliminary genetics of the BE phenotype suggest a single gene displaying autosomal dominance and numerous genetic modifiers within the mouse genome. The objectives of this proposal are to characterize the clinical and pathological features of the BE phenotype and to identify its underlying genetic causes using a gene mapping approach. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to understand the mechanisms by which environmental chemicals influence liver tumor promotion. To accomplish this task, we propose to use the prototype carcinogen, 2,3,7,8-tetrachlorodibenzo- [unreadable] p-dioxin (dioxin), as a promoter of hepatocellular carcinoma in the two-stage mouse model. The choice of dioxin is related to its biological potency and the large volume of genetic and pharmacological evidence that suggests its effects are mediated through a single ligand-activated transcription factor known as the Ah receptor (AHR). Given the central nature of this signaling protein, efforts will be made to elucidate the molecular details of the dioxin-AHR pathway as it relates to liver tumor promotion. To this end, we will first optimize the murine model of liver tumor promotion, with particular emphasis on genetics and statistical power. We will then use gene-targeting approaches to manipulate various functional domains and expression patterns of the AHR, as well as its heterodimeric partner ARNT. In particular, focus will be on determining if dioxin's tumor promoting activity is a cell autonomous process and if it is the direct result of the AHR's or ARNT's activity as transcription factors. This understanding will guide the later experiments that will be directed at identifying those particular transcriptional outputs that lie in the pathway to tumor promotion. In this last respect, emphasis will be placed on direct test for the involvement of candidate genes such as Cyp1a1, Cyp1a2 and Cyp1b1, as well as the IL1-like inflammatory cytokines. As a backup approach, we will begin to develop novel screening approaches to identify candidate genes using high throughput transcriptional profiling of preneoplastic lesions and SiRNA technologies to elucidate functional roles in promotion. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this Program-Project Grant (PPG) is to use the tools of modern genetics to elucidate the basic mechanisms that underlie the early stages of hepatocellular carcinogenesis. The focus of the research will be to understand those steps that influence the growth of initiated Cells and to define those events that lead to expression of the malignant phenotype. In classical terms, we are attempting to understand the steps of promotion and progression. Each of these projects will employ complementary approaches to understand these processes, with an emphasis on the use of murine models, chemical modifiers of carcinogenesis, and the tools of molecular genetics. The projects include: (1) Genetic modifiers of murine hepatocarcinogenesis by N. Drinkwater; and (2) Quantifying gene effects on hepatic cancer in vivo by E. Sandgren. Three modest core services (Administration, Animal Technology and Histotechnology) will support this effort. The program maintains a clear focus on hepatocellular cancers, yet, the experiments are designed so that the resultant understanding of liver cancer can be directly applicable to all types of solid tumors. Because of the unique focus and expertise of each of the co-investigators, and due to the exceptional collaborative environment generated by this PPG, the overall contributions that result from the whole program will greatly exceed the sum of the contributions that could be obtained if each project was performed in isolation. Upon[unreadable] completion of this proposal, it is anticipated that we will have furthered our understanding of how chemicals[unreadable] influence the growth controls of initiated cells within the liver. Furthermore, we will have gained molecular[unreadable] insights into the murine model of liver cancer that will allow us to more effectively extrapolate cancer data[unreadable] from animal models to the human condition. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The PPCD1 mouse arose from a spontaneous mutation in our mouse colony and exhibits an enlarged anterior chamber secondary to metaplasia of the corneal endothelium and blockage of the iridocorneal angle by the epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human corneal endothelial dystrophies, notably posterior polymorphous dystrophy (PPD), and the sporadic condition, iridocorneal endothelial syndrome (ICE). Secondary phenotypes observed in PPCD1 mice include corneal neovascularization, retinal ganglion cell loss, and photoreceptor loss, all significant causes of blindness in humans. The mouse PPCD1 phenotype exhibits an autosomal dominant pattern of inheritance, with complete penetrance on the sensitive DBA/2J background and significantly decreased penetrance on the C57BL/6J background. The objective of this proposal is to provide a molecular explanation for the murine PPCD1 phenotypes with hopes of shedding light on the human disease. We have mapped the mouse PPCD1 gene, designated "Ppcd1", to a 6.2 Mbp interval on Chromosome 2, and identified a hemizygous 87,000 bp duplication in this interval. The endpoints of the duplication are located in positions which disrupt the genes Csrp2bp and 6330439K17Rik. LOC100043552, a pseudogene located in the center of the sequence, is also presumably duplicated. Our primary candidate for the Ppcd1 gene is Csrp2bp. This prediction is based on decreased Csrp2bp expression levels in PPCD1 animals, preliminary evidence for abnormal eye size and corneal opacities in induced null alleles of Csrp2bp and the known physical interaction of this gene product with the product of the Zeb1 locius (the other known hereditary cause of PPCD in humans). We propose to confirm our hypothesis that mutation of Csrp2bp is responsible for the PPCD1 phenotype by replicating corneal endothelial cell metaplasia in independent recombinant mouse models. We propose to identify mechanisms by which Csrp2bp interactions with Zeb1 cause the PPCD1 phenotype. We will determine the temporal and spatial localization of normal and abnormal Csrp2bp gene expression, identify Csrp2bp-regulated gene promoters and determine if they are also regulated by Zeb1. We will develop methods to test the function and regulation of Csrp2bp and demonstrate how disruption of its function produces the PPCD1 phenotype. We will extend our findings to human PPCD through screening for mutations in CSRP2BP and the orthologs of 6330439K17Rik and LOC100043552, respectively, C20ORF12, and ZNF 133 in PPCD1 patients in whom ZEB1 has been excluded as a causative gene. Putative causative mutations will be tested using in vitro functional assays. Finally, through linkage analysis, we will look for C57BL/6J modifier loci that influence the penetrance of PPCD1. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to determine the gene responsible for "PPCD1", a mouse model of the human ocular disorder posterior polymorphous corneal dystrophy. Studies will also be carried out to determine the genes and mechanisms responsible for other corneal and retinal abnormalities associated with PPCD1 that are a significant cause of vision loss.

DESCRIPTION (provided by applicant): The Molecular and Environmental Toxicology Summer Research Program (MET-SRP) will provide an intense learning and research training experience in cellular and molecular mechanisms of toxicity for undergraduates who are members of underrepresented ethnic/racial minorities. This proposal seeks support of six undergraduate trainees each summer. The proposed program will have 21 faculty trainers, most of whom also are trainers on the investigators'NIEHS pre- and postdoctoral T32 Training Grant. The trainers have recognized expertise in molecular and cellular approaches to carcinogenesis, developmental biology and immunology, and are particularly interested in the mechanistic effects of toxicants on these biological processes. At the beginning of the program, the trainees will meet with the principal investigator, Christopher Bradfield, for a general orientation to the program. Each week, Dr. Bradfield will conduct a tutorial session featuring an interactive overview of environmental health sciences and a discussion of weekly reading assignments. In addition, the investigators will provide an advisory session on graduate study and careers in environmental health sciences. Each trainee will have a Molecular and Environmental Toxicology graduate student mentor, and trainees will meet with other graduate students at various scheduled events. At the end of the program, trainees will give a 20-minute scientific research presentation at a symposium open to participants in the various campus summer programs in the biomedical sciences. The MET-SRP will partner with the Integrated Biological Sciences Summer Research Program (IBS-SRP), which coordinates seven summer research programs serving 40-46 students in various biological sciences disciplines at the University of Wisconsin- Madison. Overall, the IBS-SRP is one of twelve Summer Research Opportunity Programs (SROP) at the University of Wisconsin-Madison, which served a total of 110 students in the summer of 2010. The trainees will participate in a variety of joint events as part of the IBS-SRP and SROP communities. These will include a Welcome Dinner hosted by the Graduate School, a movie night and ethics discussions. These activities will provide a chance for the trainees to interact with many other trainees from underrepresented groups with an interest in a scientific career. Public Health Relevance: This application is for support of a summer research training experience for undergraduate students who are members of underrepresented racial or ethnic minorities. One goal of this program is to increase the exposure of the targeted undergraduate populations to the possibility of careers in the Environmental Health Sciences. Along with encouraging the trainees'interest in scientific research, the summer program will also provide them with valuable experience and improve their chances of acceptance to advanced degree programs in the biomedical sciences. Additional, the program aims to increase the diversity of the scientific workforce.

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the biological effects of polycyclic aromatic hydrocarbons (PAHs), halogenated-dioxins (dioxins), - dibenzofurans, -biphenyls (PCBs), and -diphenylethers. Because of its central role in acute toxicity, teratogenesis and carcinogenesis of a large number of environmental contaminants, mechanistic information about AHR signaling is used by regulatory agencies to establish acceptable exposure levels for these pollutants. The overarching hypothesis of this proposal is that at least three distinct AHR signaling pathways exist that are of toxicological significance to human populations and vertebrate species in general. We propose that these three pathways can be distinguished by their unique impacts on transcriptional profiles within target cell populations and that these pathways can be further defined based upon their dependence on fractional activation of the AHR, by their cellular context, and their influence on gene expression through enhancer elements known as AHREs. The goal of this proposal is to identify biomarkers that reflect exposure to agonists and their induced toxic responses. We will accomplish this through the identification of low and high affinity AHREs in the hepatocyte and endothelial cell compartments of novel recombinant mouse models. To these ends, we propose the following specific aims: Specific Aim 1: Use gene targeting in ES cells to generate recombinant alleles derived from a ligand responsive version of Ahr. Specific Aim 2: Demonstrate the importance of the cellular expression and concentration of AHR. Specific Aim 3: Classify the high and low affinity AHRE batteries using genomic approaches. Specific Aim 4: Provide evidence that environmental AHR agonists can stimulate the endogenous AHR pathway. We propose that the results of this effort will provide new models that will inform the mechanism of dioxin toxicity in humans, characterize DNA signatures of toxicity, and test a novel hypothesis that developmental toxicity arises from interference with the endogenous AHR pathway. Collectively these studies will set the stage for more accurate and informative risk assessment in man.

Project Summary/Abstract The Molecular and Environmental Toxicology Training Program (MET-TP) involves 32 trainers that have outstanding training records and well-funded research programs. These trainers cover a variety of research areas such as developmental toxicology, predictive toxicology, structural biology, high throughput toxicology, stem cell toxicology, mammalian genetics, molecular biology and biochemistry. Predoctoral trainees are typically supported for a period of two years and can perform molecular toxicology research in over a dozen unique departments housed in multiple colleges on the UW campus. Postdoctoral trainees are also supported for two years and have the same access to the trainer pool as predocs, but are required to submit an Individual NRSA application in their first six months. The selection of trainees and oversight of the grant will be carried out by the Training Grant Leadership (TGL) which consists of the director and two deputy directors. In addition, four subcommittees monitor student progress, curriculum, recruitment and trainee input. The MET- TP will be reviewed by an Internal review process from eth School of Medicine and Public Health (SMPH) and External Advisory Board composed of nationally recognized toxicologists. This proposal seeks continued support for eight predoctoral and three postdoctoral positions and requests and additional position for a predoctoral trainee from an underrepresented minority. All predoctoral trainees take a MET-TP core curriculum comprised of courses in basic mechanistic toxicology, the environment and human disease, research ethics and career development. They are also required to attend a weekly toxicology research seminar, and complete additional courses required by their Trainer. Pre- and postdoctoral trainees will also be mentored in grant writing, ethics, managing techniques and teaching and will complete two courses in Responsible Conduct of Research. Finally each trainee will be trained as lifelong learners through the participation in multiple continuing education courses and in their role developing a scientific symposia related to environmental health. Predoctoral trainee progress is monitored by Research Advisory Committees for each trainee and by the TGL. Postdoctoral training is facilitated by appointment of a Postdoctoral Career Advisory Committee (two faculty mentors in addition to a trainer) that will recommend selected didactic continuing education courses, collaborations and professional development credits. The recruitment of minorities to the Program is given high priority and is facilitated by a NIEHS-funded Summer Minority Research Program for undergraduates. Upon completion of the training program, graduates usually undertake a period of postdoctoral training, or assume responsible career positions in toxicology in academic, governmental, or other public or private research institutions, or industrial laboratories.

Abstract (Summary) The Molecular and Environmental Toxicology Summer Research Opportunities Program (MET-SROP) will provide an intense learning and research training experience in cellular and molecular mechanisms of toxicity for undergraduates who are underrepresented ethnic/racial minorities, economically disadvantaged, or disabled. This proposal seeks support for eight undergraduate trainees each summer for five years. The proposed program will provide access to over 30 faculty trainers, most of whom also are trainers on our NIEHS Pre- and Postdoctoral T32 Training Grant. The trainers have recognized expertise in molecular and cellular approaches to chemical toxicity, molecular epidemiology, teratology and immunotoxicology. Trainers are particularly interested in the mechanistic effects of toxicants and opportunities for translation of this information into human risk assessment. At the beginning of the program, the trainees will meet with the Directors, Christopher Bradfield and Kristen Malecki for a general orientation to the program and the week's objectives. Each week, Directors or Trainers will conduct a tutorial session featuring an interactive overview of environmental health sciences and a discussion of weekly reading assignments. In addition, we will provide advisory sessions on graduate study, professional development, Lab Safety, animal care, ethics and human subjects training, as well as on careers in environmental health sciences. Each trainee will have both a faculty and a graduate student mentor, and trainees will meet with other graduate students at various scheduled events. At the beginning of the program, each trainee will present a poster on his/her scientific proposal and at the end of the program, trainees will give a 20-minute oral scientific research presentation at a symposium open to participants in the various campus summer programs in the biomedical sciences. The MET-SROP will partner with the Integrated Biological Sciences Summer Research Program (IBS-SRP), which oversees eight additional summer research programs in various biological science disciplines at UW- Madison. The IBS-SRP will provide safety and responsible conduct of research training. In addition, through a partnership with the IBS-SRP, our trainees will participate in a variety of joint events as part of the IBS-SRP community, including seminars, career and GRE counseling, and social events. These activities will provide a chance for the trainees to interact with many other trainees from diverse backgrounds with an interest in a scientific career.

PROJECT SUMMARY This R35 proposal is designed to consolidate two R01 programs, ES005703 and ES020668 into one program with an emphasis on understanding how environment influences human health through the PAS sensor family of proteins. Our approach is to use a highly experienced team, a broad spectrum of biochemical and genetic reagents, a transdisciplinary approach, and the expertise of an array of collaborators and clinician scientists to define the roles that PAS sensors play in environmentally influenced disease states such as cancer, infertility, obesity, diabetes and inflammatory bowel disease. Our overarching idea is that PAS sensors, and their related environmental signals, are impinging on almost every aspect of human health through their capacity as sensors of circadian time, oxygen status, chemical exposure and microbiome changes. We propose that by understanding these pathways, we can not only identify important gene by environment, and environment by environment interactions, but that we can use this information to develop intervention strategies in numerous environmental scenarios likely to be causing human morbidity. Our vision is to understand these pathways through the prism of the Ah receptor (AHR) and through the overarching idea that these pathways are in fact interacting through shared partners, cofactors or ligands. We propose that the insights gained from the R35 will ultimately be useful in intervention strategies to manipulate these pathways via therapeutics or to guide/modify human behavior or the human environment in a manner that is most beneficial to sensitive populations. Over the next eight years, this consolidated R35 should give us the freedom and power to make considerable advances in our understanding of PAS sensors and how they influence human health.

PROJECT SUMMARY / ABSTRACT Genetically engineered mice and rats are widely used as models for defining the molecular mechanisms underlying cancer etiology and for evaluating new strategies for cancer treatment and prevention. The Genome Editing and Animal Models Shared Resource (GEAM), formerly the Transgenic and Mutant Animal Facility, has served as a shared resource for the University of Wisconsin Carbone Cancer Center (UWCCC) for over two decades and is one of the elite facilities within the United States in offering a comprehensive array of services related to generation and preservation of genome-edited animal models. The primary mission of the GEAM is to make state-of-the-art genome editing technologies accessible to UWCCC members. Specific Aim 1 is to provide the expertise and infrastructure required to generate novel and relevant genome-edited or transgenic animal models and genome-edited cell models for use in cancer research. GEAM staff are capable of serving UWCCC members in all aspects of experiment planning and execution, including design of efficient and specific approaches to achieve the desired loss, gain, or alteration of gene function using CRISPR/Cas9 or other genome editing and transgene-based approaches; design, production and use of the required genome editing reagents or transgene vectors; identification of animals that carry the desired genome edit or transgene; and minimization of off target edits. Our skills in reproductive biology, embryo manipulation, and animal husbandry enable us to edit the genomes of inbred mouse and rat strains that exhibit low reproductive capacity. Specific Aim 2 is to provide state-of-the-art services that allow valuable animal models to be banked and recovered as needed to preserve these animal models or reduce the costs associated with maintaining live breeding stock for novel models that are not actively being studied. GEAM staff are highly experienced and capable of cryopreserving mouse and rat embryos or sperm and recovering mouse and rat models through embryo transfer or in vitro fertilization. In addition, we are capable of rederiving mouse and rat strains to eliminate pathogens that may compromise research or prevent animal models from being imported into our vivaria or shared between investigators working at different institutions. Since its inception, GEAM has generated hundreds of transgenic, knockout, and genome-edited mouse and rat models for UWCCC members. Sixty-one unique UWCCC members have been served during the current CCSG funding cycle, an increase of 30% compared to the previous grant cycle. Support from the CCSG allows our services to be provided to UWCCC members at costs far below those of commercial vendors or similar cores at other research universities. Convenient access to our first rate, cost-effective services enhances the ability of UWCCC investigators to conduct innovative research that advances the UWCCC strategic mission.