Rachel Brem, PhD - US grants

DESCRIPTION (provided by applicant): Transcription from loci encoding no known functional elements is widespread in the human genome, and in many model systems. A key challenge in genome biology is to determine which such "dark matter" transcripts are functionally relevant. This search for functional RNAs is motivated in part by the potential of RNAs as targets for treatment of human disease, and as therapeutic agents themselves. The investigator proposes to develop methods to infer function of un-annotated transcripts on a high-throughput scale, using yeast as a model. Previously, the investigator pioneered the genetic analysis of mRNA expression differences between genetically diverse individuals. On the basis of this demonstrated expertise with experimental genomics, software development, and molecular genetics, the investigator now proposes to develop a related strategy for un-annotated, putative noncoding RNAs in yeast. The principal goal is to harness the co-regulation of known genes and un-annotated transcripts to infer function of the latter. The project will map DNA differences between yeast strains that cause variation in levels of RNAs-both annotated and un-annotated. Software for genetic mapping will identify polymorphisms in master regulators, each of which affects the expression of multiple downstream targets in trans. In such a regulon, functional genomic analysis will find common pathway membership among known genes, leading to the inference that un- annotated transcripts also function in the same pathway. Mapping software will also identify polymorphisms in cis-regulatory elements, each of which affects levels of a transcript encoded nearby;this will allow the discovery of promoters and other cis-acting regulatory regions for novel RNAs. Molecular methods will provide experimental confirmation of the predicted function and regulation of individual RNAs. Discoveries of these RNAs, and the software tools that enable them, will serve as a springboard for future work in metazoans. PUBLIC HEALTH RELEVANCE: The treatment of human disease with small molecule drugs requires laborious testing of compounds for specificity and potency, and is largely restricted to the targeting of proteins with small ligand binding sites. Synthetic oligonucleotide therapeutics that interact with RNAs are emerging as a potentially revolutionary alternative, but the field of RNA-based drugs is still in its infancy. This proposal aims to develop approaches and tools that infer function of novel RNAs on a high-throughput scale, ultimately widening the landscape of targets for therapeutics.

DESCRIPTION (provided by applicant): The somatosensory system mediates pruritus, or itch, the unpleasant sensation that evokes a desire to scratch. Acute pruritus serves an important protective function by warning against harmful agents in the environment such as insects, toxic plants or other irritants. Pruritus can also be a debilitating condition that accompanies numerous skin, systemic, and nervous system disorders. While many forms of itch are mediated by histamine signaling, recent work by us and others makes clear that additional key neural pathways are at play. Mast cells release a variety of puritogens that mediate allergy-evoked itch, psoriasis and eczema, and anti-histamines are not always effective in treating the full spectrum of allergic disorders. Likewise, most chronic itch conditions are insensitive to antihistamine treatment. For many itch disorders, therapeutic targets for treatment have yet to be identified. In light of the need for novel drug targets, the goal of this proposal is to identify genes and biomolecules that underlie itch, focusing on signaling mechanisms in primary afferent neurons and spinal cord modulatory interneurons. Somatosensory afferents are activated by itch-producing compounds that are released by a variety of cells in the skin. Pruritogens trigger somatosensory neuron activation by binding to G-protein coupled receptors and opening transduction channels that depolarize the nerve terminal and promote action potential firing; these neurons then signal to itch-specific neurons in the spinal cord. While recent studies have begun to delineate the basic characteristics of the itch circuit, the molecular mechanisms underlying itch have yet to be identified: the receptors, transduction channels and downstream signaling factors are largely unknown, in both primary afferents and spinal neurons. This grant proposal describes the development of new genetic approaches to meet this challenge. We are two biologists with experience and expertise in sensory neurobiology, genetics, and genomics who seek to identify the genes that drive itch behaviors. We will analyze the natural variation between genetically distinct mouse strains in itch-evoked behaviors and identify sequence and gene expression differences that underlie such phenotypic change. In contrast to traditional genetic screening approaches, which are not easily applicable to live-animal phenotypes in the mouse, the genetic mapping paradigm has the potential to survey a genome's worth of genetic perturbations and uncover novel determinants of itch. Identification of candidate itch factors will provide new targets for development of drugs and therapies to treat intractable itch. ! PUBLIC HEALTH RELEVANCE: Chronic itch results from of a number of skin diseases and systemic conditions, such as eczema, kidney failure, cirrhosis and some cancers. While itch from allergies or bug bites is readily treatable with anti-histamines, most forms of chronic itch are resistant to antihistamine treatment. Understanding the neural mechanisms that evoke acute and chronic itch may lead to the development of much needed, novel drugs and therapies.

Project Summary Coccidioides spp. are major fungal pathogens endemic to Southern California, Arizona, Central America, and South America. In recent years, the incidence of coccidioidomycosis has continued to rise, resulting in hospitalization costs greater than $2 billion. Coccidioides infects, colonizes, and kills immunocompetent individuals when they inhale spores from soils. The ability of Coccidioides to cause disease depends on an elaborate developmental transition from saprophytic soil form to host form, which can be triggered in the laboratory by incubating fungal spores at elevated temperature and carbon dioxide conditions. Specifically, the hyphal form of the organism produces arthroconidia, which disperse easily and can be inhaled by mammalian hosts. Once inside the host lung, arthroconidia germinate, enlarge, and undergo nuclear division and segmentation to form large spherules filled with vegetative endospores. Rupture of the spherules allows release of endospores and dissemination of the fungus to other sites. Given the critical role of spherule development in disease progression, the focus of our proposal is the genomic and genetic dissection of this process, also known as spherulation. We will take advantage of two complementary approaches, high-resolution transcriptomics and genome-wide association studies (GWAS), to perform an innovative molecular dissection of spherulation in Coccidioides. Principal Investigator Sil has extensive experience working with Biosafety Level 3 pathogens and is well equipped to apply her expertise in transcriptional profiling of thermally dimorphic fungi to Coccidioides. PI Brem is an evolutionary and statistical geneticist with a track record of applying GWAS to fungi to identify genes that play a critical role in biologically important traits. Together, we will harness the tools of systems genetics to discover new gene functions on a genomic scale in Coccidioides. In Aim 1, we will identify a core set of spherulation-enriched transcripts by performing a high-resolution time-course analysis of the transcriptome of three Coccidioides strains undergoing spherulation. In Aim 2, we will apply GWAS analysis to identify genes that underlie variance of spherulation phenotypes, using 150 clinical isolates of Coccidioides posadasii. Taken together, these approaches will provide a rich dataset of spherulation-associated genes that will allow us to begin to elucidate critical molecular events that take place during spherule development in the context of infection.

PROJECT SUMMARY/ABSTRACT Over the four billion years that life has evolved on this planet, organisms have acquired amazing phenotypes. Some, like lions' manes and butterflies' wings, capture our attention by their sheer beauty. Others get us excited in a very different way?their relevance to biomedicine. Ecologists have catalogued remarkable disease and stress resistance traits in the plant and animal worlds, which have arisen to solve problems similar to those in human patients. We'd love to know the molecular basis of these natural resistance phenotypes, so that we can design drugs to mimic them in the biomedical context. However, most often, we know about a given trait because it is a defining feature of its respective species, acquired long ago to adapt to a unique niche. Now, millions of years later, the species usually has lost the ability to interbreed with relatives in other environments. And this reproductive isolation is a death knell for existing tools to map genotype to phenotype. The latter, which fill the pages of the modern genetics literature, rely on big panels of recombinant progeny from matings between distinct parents. These tools are no use in the study of species that can't mate to form progeny in the first place. We have developed a new strategy to break through this roadblock, and map the genetic basis of trait variation between long-diverged species. Our approach starts with a viable, but sterile, interspecific hybrid. In this hybrid, at a given gene, we introduce mutations to disrupt each of the two alleles in turn from the two species parents. These hemizygote mutants are identical with respect to background, except that at the target gene, each strain expresses a wild-type allele from only one of the parents. As such, if the hemizygotes differ with respect to a trait of interest, we infer that it must be because of functional allelic variation at the manipulated site. We have pioneered a genome-scale pipeline for this so-called reciprocal hemizygosity test, which we call RH-seq, using yeast as proof of concept. In the current proposal we describe experiments to port RH-seq to mammalian cells. We focus on a little-studied mouse species, M. castaneus, which can regrow axons of the central nervous system after injury. The genes we find in this pioneering study will serve as a springboard for drug design for stroke and brain trauma patients. And our metazoan RH-seq approach will pave the way for the genetic dissection of trait variation between species across Eukarya.