Paul Soloway - US grants

Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is an inhibitor of the 16 matrix metalloproteinases (MMPs) that collectively degrade all extracellular matrix. Mice deficient for TIMP-1 were shown to be hyper-resistant to P. aeruginosa corneal infections by a complement-dependent mechanisms. Long infections were also cleared more effectively and inflammatory responses to three additional stimuli were altered in mutant mice. Furthermore, differences in vascular structure and/or function were observed in mutant animals using four different assays. Vascular changes may underlie the altered responses to infection and inflammation in TIMP- 1-deficient mice. The goal of this proposal is to elucidate the mechanisms underlying the altered response to infection and inflammation in timp-1 mutants. The first two Aims seek to determine which of the two mechanisms used by complement to kill bacteria is required for the phenotype and if complement activity itself is affected by the timp-1 mutation. The third Aim is designed to determine whether TIMP-1 phenotype, and identify the structural changes in the vasculature that have occurred. The fourth Aim seeks to determine if TIMP-1 loss alters sensitivity to ocular inflammation and breakdown of the blood aqueous or blood retinal barriers. Evidence id described demonstrating that the effect of TIMP-1 loss on hyper-resistance to corneal infection is indirect, mediated by one or more MMPs. Aim 5 is designed to identify the key MMP(s) responsible for the mutant phenotype. The fact that hyper-resistance to pulmonary infections is seen in timp-1 mutants may be enormous clinical significance to immunocompromised individuals, or people with cystic fibrosis or pneumonia. Each of these groups is at risk of life-threatening pulmonary infections. If loss of TIMP-1 facilitates more effective clearance of such infections, perhaps interfering with TIMP-1 function would be of great benefit to these people. A simple genetic experiment proposed in Aim 6 will confirm if this is the case. Im 7 propose to use phage display to identify TIMP-1 antagonists that may have a wide variety of therapeutic uses.

[unreadable] DESCRIPTION (provided by applicant): Methylation of cytosines within CpG dinucleotides is critical for normal development in mammals. In cancer, proper regulation of DNA methylation goes awry often leading to silencing of tumor suppressors or activation of growth-stimulating genes. These events can strongly contribute to tumorigenesis. Many loci have been identified that acquire methylation or whose expression is methylation-sensitive in the normal and cancer genome, but virtually nothing is known about how methylation is regulated. Imprinted loci are useful for identifying cis-acting DNA sequences that regulate local methylation since these loci undergo predictable patterns of allele-specific methylation in normal tissue. RASGRF1 is a GTP exchange factor that activates RAS and has transforming activity. In mice, the Rasgrf1 locus is imprinted: There is paternal allele-specific methylation within a differentially methylated domain (DMD) 30 kbp 5' of the promoter and the locus is paternally expressed. We have shown that a repeated sequence element found immediately 3' of the DMD regulates establishment of methylation of the DMD in the male germ line. The DMD behaves like a methylation-sensitive enhancer-blocking element and together with the repeat sequence, represents a binary switch that regulates allele-specific expression of the locus. The central goal of this proposal is to elaborate the mechanisms by which the repeat element regulates DNA methylation. If we understand how DNA methylation is normally regulated, this may help us understand how inappropriate methylation occurs in cancer. Furthermore, these studies may identify therapeutic targets for treating diseases characterized by aberrant DNA methylation.

Diet is a critical environmental variable that exerts a powerful influence on health and disease. Cancer, cardiovascular disease, obesity and type-2 diabetes are particularly sensitive to diet. There is a parallel rise in the incidence of these conditions and the consumption of foods that contribute to their development. While genetics and diet each unquestionably affect disease incidence, it is increasingly clear that the dynamic between genetics and diet is what determines health or disease status. Neither variable acts entirely independently of the other. Furthermore, the genetic influences maybe both quantitative - affecting frequency of tumor development, and complex- involving multiple gene-gene interactions. It is possible to use the genetic diversity of different inbred strains of mice as the basis of a systematic, genome-wide hunt for loci that control if and how diet affects colorectal cancer. Our preliminary data show that when the A/J and C57BL/6 genomes are combined in F1 mice, and the carcinogen azoxymethane (AOM) is used to induce colorectal cancer, the tumor number in those F1 animals depends on diet. In the inbred parents, no diet responsive phenotype was seen. This demonstrates that A/J and C57BL/6 harbor interacting alleles that can combine to produce a diet-responsive, complex, quantitative affect on tumor number. In a narrowly-focused single Aim, I propose to use recombinant inbred (Rl)and chromosome substitution strains (CSS) derived from A/J and C57BL/6 as tools in the next incremental step to map genetic loci controling if and how diet affects colorectal cancer. I propose to maintain the BxA Rl panel and B.A CSS lines derived from this strain combination on either a control or "western style" diet that is high in fat,low in fiber, Ca+2, vitamin D3, choline and folic acid. After two weeks on the diet, mice will be given eight weekly injections of AOM to induce colon tumors and will be maintained on their respective diet for the duration of the experiment. Twenty-five weeks after the last AOM injection we will monitor tumor burden in mice and other qualitative tumor phenotypes. Using the publicly-available strain distribution patterns (SDP) known for the Rl and CSS mice, we will perform interval mapping to identify genomic regions controlling diet- responsive tumor phenotypes. This complex quantitative trait analysis will reveal loci at the heart of gene- nutrient interactions relevant to colon cancer and lays the foundation for subsequent work to identify the genes critical for those interactions.

DESCRIPTION (provided by applicant): In this application, we seek to develop a transforming technology that will revolutionize epigenomic studies. The approach builds upon a technology we have developed in the past that allowed us to characterize individual DNA molecules in a complex mixture by fluorescence imaging at a rate exceeding 4,000 molecules per minute using femtogram quantities of DNA. These previous fluorescence-imaging studies used phage DNA markers labeled with intercalating dyes for a proof-of-principle test for high throughput single DNA molecule analysis. In three Aims, we propose to extend our existing technology in a stepwise and systematic way, to (a) analyze methylation on DNA and multiple epigenetic marks simultaneously in mammalian chromatin;(b) do this using chromatin isolated from extremely low abundance sources, such as preimplantation embryos and laser microdissected tumors;and (c) sort and recover DNAs carrying specific combinations of marks for subsequent high throughput DNA sequencing. If successful, our efforts will revolutionize epigenomic studies by enabling whole epigenome profiling of single cells;increasing the sensitivity of epigenetic modification detection by several orders of magnitude;facilitating simultaneous monitoring of multiple epigenetic modifications;and providing functional correlates between epigenetic states and gene expression competence. This technology will greatly enhance the capabilities of the Reference Epigenome Mapping Centers. PUBLIC HEALTH RELEVANCE: It has become abundantly clear during the past 20 years that epigenetic alterations to the genome can influence development and health as profoundly as mutagenesis of the genome. One of the most dramatic examples is the fact that methylation of DNA at the promoter of the p16 tumor suppressor is as effective at silencing the gene as mutations to the body of the gene itself and that both events contribute to the development and progression of colorectal cancer [1]. Importantly, unlike mutations, epigenetic silencing of p16 can be reversed pharmacologically, with potential therapeutic benefit [2]. This proposal seeks to develop a revolutionary new tool for assessing multiple epigenetic modifications, simultaneously and genome-wide, in vanishingly small quantities of material. If successful, this will vastly increase the epigenetic analyses possible in the now-forming Reference Epigenome Mapping Centers. This technology will be of enormous benefit to the discovery mission of these Centers and their impact on public health.

Epigenetic features in mammals include covalent modifications to histones, and methylation of cytosines. Their proper placement is fundamental to many biological processes. Assaying features in the epigenome is important for understanding human biology, diagnosing disease, monitoring responses to epigenome modifying drugs and facilitating development of new medicines. Such assays will also facilitate emerging therapeutics based on embryonic and induced pluripotent stem cells, which require modifying the cells to assume epigenetic states of differentiated cells. State-of-the-art assays for histone modifications use chromatin immunoprecipitation (ChIP), followed by genome wide sequencing. Methylated DNA can be identified by immunoprecipitation followed by sequencing, or by bisulfite sequencing. There are two fundamental limitations with all these approaches. First, they query only one epigenetic feature at a time. Epigenetic features arise in combinations, and those combinations rather than individual features regulate the underlying genes. Unless multiple features can be detected and measured simultaneously, it is not possible to know, with certainty, when combinations coexist on a given gene. Second, assays use populations of cells and report the average epigenetic states within the population, not the actual distribution of epigenetic states present on individual DNA molecules comprising the population. Third, ChIP often uses abundant amounts of chromatin making it impractical to assay multiple epigenetic features in rare or impossible to culture cells. In this application, we seek to develop a transforming technology, Single Chromatin molecule Analysis in Nanofluidics (SCAN) that can overcome each of these limitations and revolutionize epigenomic studies. In SCAN, chromatin molecules are bound to fluorescent probes recognizing distinct epigenetic features, then driven by voltage through nanofluidic channels where the fluorescent properties of single molecules are detected. By using multiple probes, each recognizing different features and carrying distinct fluorophores, we can directly detect their binding to individual molecules, allowing precise enumeration of multiple epigenetic features simultaneously. Our first-generation devices were operated in an analytical mode, simply counting features. Our second-generation devices were operated in a preparative mode, allowing us to sort and isolate molecules carrying defined epigenetic features. In this proposal, we seek to further develop this next generation epigenomics technology. First, we will modify the analytical device and analyte preparation to increase sample throughput by two orders of magnitude. Second, we will use the new analytical device to address selected questions in epigenomics. Third, we will use our preparative device to isolate chromatin with defined epigenetic features, sequence the DNA and compare our results to data obtained by current ChIP-seq methods.