Lisa Stubbs - US grants

DESCRIPTION (provided by applicant): Kruppel-type zinc finger (ZNF) loci comprise one of the largest of all human gene families. In mammals, the majority of ZNF genes are of a single subtype, encoding proteins in which DMA-binding zinc finger arrays are attached to a chromatin-interacting domain, called KRAB, that confers a potent transcriptional repressor activity. Although certain KRAB-ZNF loci are highly conserved, ongoing segmental duplication events have created largely unique gene sets in each mammalian lineage. More than one-third of the 404 human KRAB- ZNF loci are primate-specific, and even the most recent duplicate genes have diverged in ways that indicate a selection for novel proteins with distinct DNA recognition sites. This single gene family comprises one-fifth of all predicted human transcription factor loci, and available data suggest broad roles in regulating processes that are critical to human health. However, since regulatory targets and pathways are known for only a handful of KRAB-ZNF proteins the functions of most family members remain a matter of conjecture. We hypothesize that this dynamic gene family has played a significant role in shaping human health-related biology, including both deeply conserved and primate-specific traits. The proposed research program is designed to address this hypothesis through functional analysis of 25 KRAB-ZNF genes. As a primary focus we will analyze proteins encoded by 25 genes involved in the most recent primate-specific duplications with emphasis on paralogous proteins that are most amenable to experimental analysis. Specifically, we will (1) determine genes and pathways that are regulated by each ZNF protein by manipulating gene expression in human cells;(2) define genomic regions to which the ZNF proteins bind in human chromatin using chromatin immunoprecipitation techniques;and (3) identify and validate consensus DNA motifs defining favored recognition sites for each protein using combined bioinformatics and experimental approaches. This study will provide a first in-depth look at functions for this large family of human transcriptional repressors, permitting us to examine their roles in regulating in human immunity, reproduction, development, cancer susceptibility and other processes in which KRAB-ZNF genes have been implicated. The data we generate will provide new guidelines to predict the functions of additional family members and to assess the potential impact of the gain, loss, mutation and dsyregulation of KRAB-ZNF genes on human health.

DESCRIPTION (provided by applicant): The prostate arises from the urogenital sinus relatively late in mammalian development, with most of the growth and differentiation of the organ occurring after birth. Throughout prostate development and into adulthood, paracrine signals from the surrounding mesenchyme regulate the growth, differentiation, and maintenance of the prostate epithelium in response to androgens. In turn, the differentiated epithelium provides signals to regulate activities within th mesenchyme and a continuing, balanced cross-talk between mesenchymal and epithelial components is critical to maintenance of a healthy, functioning prostate throughout adult life. Significant progress has been made in identifying the signaling pathways involved in mesenchymal- epithelial (M-E) interactions in the prostate. However, relatively little is known of the network of transcription factors (TFs) that regulate the earliest stages of prostate development, or that serve to maintain a balance of cell types and signaling pathways in adults. Our preliminary studies identify the T-box protein, Tbx18, as a vital member of this prostate regulatory network, active in the urogenital mesenchyme from the earliest stages of prostate development and continuing to play an important role in prostate maintenance into adulthood. Tbx18 null mutants die at birth, complicating the analysis of phenotypes in late-developing tissues. However, we have identified a relatively long-lived regulatory mutation of Tbx18, called 12Gso, and our preliminary studies have revealed a striking prostate phenotype in these mice. Using siRNA knockdown and chromatin immunoprecipitation (ChIP) in mouse cell lines, we have identified direct regulatory targets of Tbx18, many of which play key roles in prostate development. However, nothing is known about the in vivo targets of the Tbx18 protein, or of the upstream TFs that control its complex pattern of developmental expression. The goals of this project are (1) to elucidate Tbx18 target genes in the developing prostate; (2) to identify regulatory elements and upstream TFs that control Tbx18 prostate expression; and (3) to further characterize the developmental time-course and cellular manifestations of prostate phenotypes in Tbx18 mutant mice, and to tie development of these phenotypes to interacting TFs and target genes. In pursuit of these goals, we will be aided by a collection of valuable mouse genetic and other tools that are already available in our laboratory, including transgenic reporters, a conditional null (floxed) allele, the 12Gso regulatory mutant, and a custom Tbx18 antibody developed by our group. We hypothesize that Tbx18 is a critical missing link in a regulatory network that controls prostate development, appearing in the UGM at the earliest developmental stages and continuing to act in both stroma and epithelium in adults.

? DESCRIPTION (provided by applicant) Many types of neuropsychiatric conditions, including Autism spectrum disorders (ASD), bipolar disorder, and schizophrenia, have a significant genetic component, although they are generally thought to involve a complex mix of interacting genes. However, some of these disorders can also be driven by mutations in single genes, suggesting that these loci have central and pivotal roles in the neurodevelopmental underpinnings of the disease. One such essential genetic factor, AUTS2, was originally discovered as disrupted in a pair of autistic twins, and AUTS2 mutations have since been more generally linked to syndromic forms of intellectual disability as well as epilepsy, schizophrenia and other disorders. Genome-wide association studies (GWAS) have further linked AUTS2 to the inheritance of bipolar disorder, major depression, alcohol consumption, and susceptibility to heroin addiction. This single gene is thus implicated in an exceptionally broad range of neuropsychiatric disorders with profound societal impact. However, the cellular and developmental functions of this important gene are not understood. We have discovered a mouse mutation, called 16Gso, which dysregulates certain transcripts arising from the Auts2 gene during critical times in development; despite this partial loss-of-function, 16Gso mutants display morphological, behavioral, and brain structural abnormalities that model human AUTS2-linked phenotypes strikingly well. Our developmental studies have revealed the loss of critical neuronal populations in 16Gso mutants during early postnatal life; the loss of these neurons could well explain many aspects of the 16Gso phenotype, and similar neuronal abnormalities may underlie human AUTS2-linked symptoms as well. Thus, clues provided by analysis of the 16Gso mutant could suggest new avenues for diagnosis and treatment of human AUTS2-linked disease. This proposal is focused on developing this mouse model, and specifically, defining the role of Auts2 in the 16Gso phenotype using genetic complementation tests. To achieve this goal we will obtain Auts2-/- targeted mutants, and compare their morphology, behavior, and molecular phenotypes to 16Gso homozygotes as well as 16Gso/Auts2- compound heterozygotes. Together these mutant animals will provide important tools for analysis of Auts2 neurodevelopmental function in a mammalian model system.

PROJECT SUMMARY / ABSTRACT Many types of neuropsychiatric conditions have a significant genetic component, although they are generally thought to involve a mix of interacting genes. However, some disorders can also be driven by rare mutations in single genetic loci, highlighting genes with basic and pivotal roles in neurodevelopment. One such locus, AUTS2, was originally discovered as disrupted in a pair of autistic twins. However, AUTS2 mutations have since been linked to a wide range of neurological disorders, including epilepsy, schizophrenia, bipolar disorder, addictive behaviors to name a few. This single genetic region is thus implicated in an exceptionally broad range of neuropsychiatric disorders with profound societal impact. However, the way that AUTS2-region mutations predispose to these diseases is not well understood. Complicating the genetic picture, most human AUTS2 mutations are genomic rearrangements that could impact the functions of other neighboring genes. Of particular interest in this regard is WBSCR17, which is linked to AUTS2 in a conserved topographically associating domain (TAD), suggesting co-regulation of the genes. While Auts2 mouse ?knockout? mutations express certain phenotypes that could be considered parallel to certain neuropsychiatric traits, they have not provided a compelling model for AUTS2-linked disease. This project is focused on a novel mouse mutation, called 16Gso, which disrupts the Wbscr17-Auts2 TAD and dysregulates both genes. Despite this complex genetic effect, 16Gso mutants display morphological, behavioral, and brain structural abnormalities that model human AUTS2 phenotypes strikingly well. We hypothesize that Wbscr17 contributes to 16Gso and AUTS2-linke human neurological phenotypes by interacting with Auts2 in a basic cellular pathway required for the extension, survival, and connectivity of neuronal processes in the developing and adult brain. Further we propose that disturbance of this pathway leaves affected individuals susceptible to a wide range neuropsychiatric disease. This proposal is focused on addressing these hypotheses by defining the contributions of Wbscr17 to 16Gso phenotypes, and the genetic interactions between Wbscr17 and Auts2. We will investigate the cellular functions regulated by the two loci in a cellular model, and define the regulatory mechanisms that control these linked genes. Together these data will provide novel explanations for genotype:phenotype correlations in a genetic region linked broadly to susceptibilities to human neurological disease.