Bruce Hamilton - US grants

DESCRIPTION (provided by applicant): The outcome of many genetic disorders is influenced by modifying genes. The long term goal of these studies is to identify mechanisms for modulating mutations that affect brain and behavior. Such epistatic interactions may be specific for a single mutation, applicable across genes in a biological pathway, or applicable across a class of mutagenic events. From studies on the mouse neurodegeneration mutant vibrator, we identified a genetic modifier, Mvb1, that acts on mutations caused by endogenous retroviruses. Retroviruses account for ~15% of spontaneous mouse mutations. Suppressing alleles of Mvb1 increase expression of normal gene products derived from mutant genes, including vibrator and a model of branchiootorenal dysplasia, Eya1, in which insertion into an intron has reduced gene expression. We now have complementation evidence that Mvb1 is a naturally occurring allelic difference in an mRNA export factor, Nxf1. Two amino acid substitutions that distinguish suppressing and non-suppressing alleles. This factor is recruited to spliced mRNA, but also directly binds RNA elements in unspliced retroviral genomes, in L1 LINEs, and potentially some non-repetitive cellular RNAs. Retrotranspositions, particularly L1 LINES, have been identified in several inherited human diseases. This proposal will impact human health through improved understanding of retroviral control, retroelement impact on host gene expression and creation of new classes of mouse models. This renewal has four specific aims. (1) Test the range of Mvb1-mediated genetic suppression in vivo by genetic crosses to select retroviral mutations and in cell culture by manipulation of reporter constructs. (2) Test competing hypotheses for the functional difference between allele using amino acid replacement alleles and quantitative measurements of key protein-protein interactions. (3) Test the modulation of heterologous target gene expression by Mvb1 in cell culture and transgenic mice. (4) Test the hypothesis that suppressing Mvb1 alleles arose through recent positive selection will be tested by comparing nucleotide diversity and extended haplotype homozygosity at Mvb1 compared to other loci.

This core unit provides crucial molecular resources for each of the research projects, including: preparation and cataloging of DNA sample preparation and storage, genotyping, physical mapping and genetic marker development, informatic support, DNA sequencing, and identification of polymorphisms at targeted loci as candidate functional changes. 1. Preparation and storage of genomic DNA (gDNA). Consistent and reliable DNA samples for genotyping (in analyses of linkage, association, and candidate functional sequence changes) are essential to Projects 1 and 2. We collect 50 ml of EDTA-anticoagulated whole blood from each individual in the clinic. We prepare gDNA from the white cells in small amounts and the remaining blood is stored frozen for future use. 2. DNA Sequencing. Strategy. Each of Projects 1-5 has suggested biological candidate genes as potential contributors to genetic variation in autonomic control of blood pressure. Testing these candidate genes requires linkage, association, and haplotype analysis of known markers and more particularly of single point and haplotype alleles enriched in hypertensive subjects and their first-degree relatives. 3. Genotyping. Strategy and Methods. As described under Sequencing above, we use a two-tiered strategy to maximize efficiency. (1) The first tier involves direct PCR-based resequencing of candidate loci to improve the density and appropriateness of polymorphism selection for larger-scale genotyping. (2) In the second tier of the strategy, derived marker combinations are selected and used to allow efficient discrimination of haplotypes. Obtained genotype data is subsequently analyzed for statistical significance and population parameters in the informatics core, Core B (Informatics and statistical genetics), including additional population-based controls for likely accuracy. 4. Allele-specific expression analysis. Strategy. To detect the effects of allelic variants define in this program on expression level, this core will provide expression analysis based on allelic detection from genotyped samples and heterologous promoter fusions.

[unreadable] DESCRIPTION (provided by applicant): Disruptions in the normal development of the cerebellum result in clinically significant malformations whose mechanistic bases are poorly understood. This proposal uses a combination of established and emerging genetic and genomic methods to dissect molecular mechanisms of cerebellar vermis malformation in a new mouse model resembling Dandy-Walker malformations. Homozygotes for the nur12 mutation show show nearly complete agenesis of the vermis and choroid plexus, cystic dilation of the fourth ventricle, and anterior malrotation of cerebellar structures within the posterior fossa. As preliminary data for defining molecular mechanisms relevant to features shared between this mutant and human patients, the applicant has identified a null mutation of a zinc finger transcription in nur12 mutants. The applicant has begun to identify developmental sequelae to this mutation, including profound effects on proliferation of neural progenitor cells. The three specific aims will (1) establish an allelic series at the locus and investigate sources of interindividual variation in the severity (hemispheric involvement) of null allele; (2) define cellular and developmental mechanisms of the nur12 malformation, including tests for the roles of BMP/SMAD and EBF signaling pathway, through a combination of in situ labeling, marker gene analyses and signaling response measurements in culture; and (3) identify molecular targets of ZFP423 activity in cerebellum development by transcriptional profiling at sequential stages of development and by identifying overlaps in promoter occupancy among ZFP423, BMP-activated SMADs and EBF factors using a highly parallel platform for genome-wide location analysis for transcription factor binding. Relevance: This proposal will identify mechanisms relevant to the Dandy-Walker malformation - a severe defect found in 1/25,000 -1/30,000 births. Preliminary results suggest that the mechanisms acting here affect the ability of precursor cells to continue dividing, which may have therapeutic application for both prenatally diagnosed malformations and pediatric brain cancers. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal describes a predoctoral genetics training program on the La Jolla Mesa including the University of California, San Diego (UCSD) School of Medicine, the UCSD Division of Biology, and The Salk Institute for Biological Studies. The goal of this program is to train graduate students for future careers as academic or industrial scientists investigating genetic phenomena, or using genetic methods to understand biological problems important for human health. Our training faculty have joined together to create a common vision of contemporary genetics training in which we first build a foundation of basic biological and biomedical science. This foundation will then support a tripod of integrated educational principles of genetics and genomics including: 1) rigorous education in the classical principles and intellectual methods of genetics; 2) research training in the newest methods of classical and molecular genetics including genomics; and 3) development of an appreciation of the problems, outlooks, and ethical issues associated with modem clinical and medical genetics. The development of this unique program is tied to continued expansion of our graduate programs based on substantial new faculty recruitment in genetics and genomics and as a companion to the integration of previously distinct training efforts at UCSD. Thus, we are requesting financial support for 20 trainees in the first year phasing into 26 trainees after four years to be educated in genetics laboratories at UCSD and the Salk Institute.

DESCRIPTION (provided by applicant): Neural stem cells (NSCs) are a multipotent cell population capable of both self-renewal and differentiation into neurons and glia. Engraftment of NSCs has been proposed as a therapeutic approach for a variety of human neurological disorders, however, genetic control of the production, potency and capacity for renewal of this cell population are not fully understood. This proposal is based on unexpected observations in the PI's laboratory that deletion of a particular zinc finger protein impairs the propagation of NSCs from forebrain subventricular zone, both in vivo and in vitro. The aims of this proposal will test the hypotheses (1) that this transcriptional regulator controls self renewal and potency of NSCs and multipotent progenitors;(2) that absence of this factor results in premature differentiation at the expense of renewal;and (3) that this factor acts by titrating the availability of differentiation-promoting transcriptional regulators through the formation of heteromeric complexes in the absence of differentiation signals.

PROJECT SUMMARY The UCSD Genetics Training Program (GTP) is designed to provide advanced training in Genetics and Genomics to predoctoral students beginning in their second graduate year. While several degree-granting umbrella programs at UCSD include genetics or genomics research, GTP is uniquely the point of integration across programs and builds both lateral and vertical cohorts of PhD students interested in the history, practice and future applications of Genetics and Genomics in life and health sciences. Mentor laboratories encompass a broad range of basic science and clinical/translation research and span a range of organisms?including microbial, plant, experimental animal, and human subjects?but share a focus on genetic, epigenetic and genomic mechanisms and approaches. Students who have committed to thesis research in one of these laboratories enter the GTP and may be selected for support by this training grant. The GTP curriculum cuts across traditional graduate programs in participating schools, divisions and departments. GTP students take graduate core courses in Genetics and Quantitative Methods and participate in a weekly journal club during graduate years 2-4. The journal club includes rotating topics in contemporary genetics or classic, landmark papers relevant to the intellectual development of the field. GTP students select quarterly topics in consultation with participating faculty. Journal club papers and discussion are used to assess methods and logic, experimental design, data analysis, and responsible conduct of research in additional to the quarterly research topics around which they are selected. A program-wide annual retreat, organized by year 5 students and a standing faculty committee, includes invited outside keynote speakers, research presentations by program faculty and students. GTP has more than 30-40 students training in any given year, for whom we are requesting 16 training grant slots.

DESCRIPTION (provided by applicant): Most human disease has a genetic basis in cause, susceptibility, or resistance. However, the genetic architectures for most disease phenotypes are only poorly understood and architectures of genetic plasticity - the ability to modify genetic networks to favor one trait without adversely affecting other traits that rely on the same underlying genes - are understood almost not at all. We have previously demonstrated through positional identification of a novel wild-derived modifier gene in mice one instructive example. The Mvb1 locus attenuates the effects of retroviral insertion mutations without apparent disruption of the host gene expression program. Indeed, the variant we identified shows hallmarks of positive selection in wild populations, consistent with a role in innate immunity to a pathogen. This competing renewal application continues our work to understand the mechanisms of genetic suppression mediated by alleles of the Nxf1 gene at the Mvb1 locus. This renewal supports development of new mouse models for human disease and therapy based on modulating gene expression levels to reflect therapies that increase or replace expression of genetic defects, analysis of the genetic networks of normal gene expression that might be affected by manipulating this pathway, and tests for effects of feedback regulation within the proposed network architecture. PUBLIC HEALTH RELEVANCE: Understanding mechanisms through which gene expression networks have been modified by selective pressures or can be manipulated therapeutically to favor host gene expression programs over those of pathogens, including RNA viruses, and molecular parasites, including retrotransposons, has potential applications in infectious disease and cancer. Understanding the network architecture and properties of component proteins such as Nxf1 has applications for animal models of a wide range of genetic disease and genetic susceptibility to disease as well as providing new insight into basic mechanisms of RNA processing downstream of transcriptional initiation.

DESCRIPTION (provided by applicant): The ciliopathies comprise a spectrum of disorders unified by defects in primary cilia. Clinical presentations range from primary involvement of a single organ (most often hindbrain, kidney, liver or eye) to more severe presentations, such as Meckel syndrome, with severe and pleiotropic developmental phenotypes in several organs. Several genes have been and continue to be identified for ciliopathy disorders, with the overwhelming majority encoding structural components of primary cilia. Regulatory genes that control cilium-dependent signaling and modifier genes that control the outcome of ciliary defects are only beginning to be tied to pathogenic mechanisms. This project focuses on the synthetic lethal interaction between a transcriptional regulator that control ciliary phenotypes, Zfp423, and an unknown modifier gene. Zfp423 encodes a 30-zinc finger transcription factor required in several signal transduction pathways and in multiple organ systems. Animals that lack Zfp423 have prominent brain malformations with a high frequency of hydrocephalus as well as defects in several peripheral tissues. The distribution of phenotypes in surviving animals is dependent on both modifier genes and apparently stochastic processes. However, on the most commonly used strain background, no mutant animals survive. Genetic mapping identifies a single major locus linked to embryonic and perinatal lethality. The aims of this proposal will identify this synthetic lethal modifier locus, elucidate its mechanism and place it in the context of other genes in the ciliopathy network.

? DESCRIPTION (provided by applicant): This proposal will examine the role of Zfp804a in mouse brain development and behavior as a potential model for biological basis of psychiatric illness. Schizophrenia and other psychiatric illnesses have been recognized to have a familial component for several decades, but genes that underlie these disorders have only recently begun to be identified. In addition to a very few genes with high penetrance in rare families, consortium studies have begun to identify with high confidence larger numbers of genes whose common variants are associated with schizophrenia and bipolar disorder in the general population. While these associated variants individually have only limited predictive power for diagnosis, they nonetheless mark biological bases for the disorder. Understanding the basic mechanisms through which these genes impact brain and mental function may therefore provide key insights into risk and intervention. One of the best-supported and most-replicated associations with schizophrenia and bipolar disorder is ZNF804A, which encodes a protein with a single zinc finger motif and other conserved sequences whose functions are not known. This exploratory proposal aims to create animal models that will (1) test whether the ZNF804A homolog in mouse is required for brain development or normal behavior and (2) facilitate molecular studies to place ZNF804A in cellular and molecular context.

Project Summary: This project focuses on cellular, genetic, and molecular mechanisms that underlie developmental abnormalities in ZNF423-realted ciliopathy. The ciliopathies comprise a spectrum of disorders unified by defects in primary cilia. Clinical presentations range from primary involvement of a single organ (most often hindbrain, kidney, liver or eye) to more severe presentations, such as Meckel syndrome, with severe and pleiotropic developmental phenotypes in several organs. Several genes have been identified for ciliopathy disorders, with the overwhelming majority encoding physical components of primary cilia. Regulatory genes that control cilium-dependent signaling and genetic modifiers that control the outcome of ciliary defects are only beginning to be tied to pathogenic mechanisms. This project focuses on the role and mechanisms of ZNF423, a constitutively nuclear transcriptional regulatory protein mutated in Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) patients. ZNF423 is thought to comprise an integrative node among several transcriptional complexes that respond to classical developmental signals. As ZNF423 expression is also developmentally dynamic, the extent to which phenotypes are cell autonomous, rather than defects in reciprocal intercellular signaling, remains unclear. Aim 1 will use recently developed genetic tools (MADM) to assess cell autonomy by creating a simple platform for inducing and marking mitotic clones in situ. Because patient mutations are individually rare, often found on only one allele, and found in subjects with a range of presentations, it remains unclear what fraction of patients is attributable to ZNF423 and which ZNF423 mutations are truly pathogenic. Aim 2 will use genome editing in a sensitive and well-validated mouse model to test phenotypic effect of patient-derived mutations. It remains unclear whether specific targets of ZNF423 activity might be able to modulate phenotype. Aim 3 will determine whether decreasing activity of newly identified ZNF423-repressed genes, whose expression is increased in both knockdown cell and mutant animals, can improve cilium-dependent functions and emergent phenotypes in the Zfp423 mouse model.