Frank L. Conlon - US grants

DESCRIPTION (provided by applicant): We are studying the molecular mechanisms that are involved in the induction and patterning of the early heart tube. To begin to identify the molecular pathways involved in heart development, we are studying the endogenous role of genes implicated in human congenital heart disease, the most common form of heart disease in childhood occurring in about 1% of live births and up to 10% of stillbirths. Despite the high incident of congenital heart disease only in a few instances has the genetic basis for any one type of disease been identified. Recently, it has been shown that patients with the Noonan syndrome often have mis-sense mutations in the Shp-2/SH-PTP2 gene. Shp-2/SH-PTP2 encodes for a nonreceptor phosphatase required for FGF/MAPK signaling. A second example is the Holt-Oram syndrome (HOS), a disease associated with mutations in the coding region of the transcription factor TBX5. Several of the clinical features of Noonan syndrome and HOS can overlap. For example, patients with either syndrome often display atrial septal defects suggesting that the two proteins may function in a similar pathway. Consistent with this hypothesis, we have shown a direct link between the FGF/MAPK signal transduction pathway and TBX5 transcriptional activity. We have also shown that TBX5 is post-translationally modified through phosphorylation, and that mutation of an evolutionarily conserved FGF/MAPK site results in alterations of TBX5 function. To test the hypothesis that FGF/MAPK/SH-PTP2 pathway is critical to Tbx5 activity and heart development in vivo, we plan to determine the cellular and molecular relationship between the FGF/MAPK/SH-PTP2 and Tbx5. The specific aims of this proposal are: to establish the molecular and cellular relationship between FGF/MAP kinase signaling pathway and Tbx5 in heart development, determine the role for SH-PTP2 with respect to TBX5 activity and early heart development, to identify and characterize the role of FGF/MAP/SH-PTP2 in TBX5 post-translational modifications.

[unreadable] DESCRIPTION (provided by applicant): To identify the molecular pathways involved in organ development, we are examining the endogenous role of genes initially identified based on their implication in human disease. Recently, it has been shown that patients with Noonan syndrome frequently have mis-sense mutations in the nonreceptor phosphatase Shp-2, a gene required downstream of FGF in the MARK signaling cascade. Clinical studies of Noonan patients, as well as analysis of mice mutant for Shp-2 demonstrate a fundamental role for the gene in both limb and heart development. A second example is Holt-Oram syndrome (HOS), a disease often associated with mutations in the coding region of the transcription factor TBX5, a gene also required for limb and heart development, leading to the suggestion the two genes may function in the same pathway. Consistent with this hypothesis, we have shown a direct link between the FGF/MAPK signal transduction pathway and TBX5 induction and transcriptional activity. The overall aim of this proposal is to integrate a transgenic and genetic approach in Xenopus with ongoing cellular and biochemical approaches to determine the precise endogenous role for SHP-2 and TBX5 in early organ development in the diploid frog Xenopus tropicalis (X. tropicalis). The long-term goal of these studies is to couple mutations and transgenic lines with biochemical and expression screens in Xenopus to identify the cellular and molecular pathways by which these factors function in early organ development. The specific aims of this proposal are: One, to establish Tbx5, Tbx20 and Eomesodermin cardiac specific reporter lines in X. tropicalis. Two, determine the role of Shp-2 and Tbx5 in organ development through the generation of an allelic series of Shp-2 and Tbx5 in X. tropicalis. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The identification and characterization of the molecular pathways involved in the early steps of cardiac cell proliferation are absolutely critical to understanding the pathologies and treatment of congenital heart disease. However, to date the early molecular pathways that control the progression of the embryonic cardiac cell cycle remain largely unknown. To address these issues, we cloned and characterized the Xenopus T-box gene Tbx5, the gene mutated in the human congenital heart disease Holt Oram syndrome (HOS). We have shown that TBX5 is both necessary and sufficient in vivo for the cardiac G1/S-transition of the cell cycle. From these and other studies, we hypothesize that TBX5 functions to maintain proliferation of cardiac progenitor populations. Xenopus offers an unparalleled opportunity to address this hypothesis due to the access of unlimited embryonic cardiac tissue, the development of cardiac explant assays, the availability EGFP-transgene reporter frogs that mark gene expression domains and mark specific phases of the cell cycle in living cardiac tissues, and our recent description of an extensive panel of antibodies that mark cell cycle components in the developing Xenopus heart. Here we propose to use TBX5 as a starting point to elucidate the molecular networks which control the proliferation of cardiac progenitor cells. This will be accomplished by determining if TBX5 directly regulates cyclin D1 and cyclin E2 in the embryonic heart, characterizing the biological significance of the SIN3B-TBX5 protein-protein interaction in cardiac cell cycle regulation, and through the identification of the endogenous cardiac mitogens which function through TBX5 to regulate the G1 to S transition of the cardiac cell cycle. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE The ability to isolate and propagate cell populations that can differentiate into cardiomyocytes in vivo offers the opportunity to treat a wide range of cardiac diseases. This proposal focuses on the characterization of the transcription factor TBX5, the gene mutated in the congenital heart disease Holt Oram syndrome, and its endogenous role in cardiac proliferation. Our immediate goal is to define and characterize the molecular pathways by which TBX5 functions with the overall goal, to use TBX5 as a starting point in an effort to begin to elucidate the pathways and molecular networks which control the survival and proliferation of cardiomyocyte progenitor cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We are studying the molecular pathways involved in craniofacial patterning and heart development and their role in the pathology of congenital disease. A large number of congenital malformations affecting infants involve either craniofacial structures or the heart, leading to substantial morbidity and mortality. Interestingly, many congenital syndromes result in abnormalities both in craniofacial development and cardiac development suggesting that the molecular signals involved in the development of these two different organ systems are shared. Yet, the identities and the biological roles of many of these signals are still not well defined. Here, we will develop genetic approaches using both a forward and reverse screen in Xenopus that will be integrated with ongoing cellular and biochemical approaches in our two labs to investigate the genetic control of craniofacial pattern formation and heart development.

DESCRIPTION (provided by applicant): An understanding of the molecular mechanisms that are required for cardiomyocyte cell fate decisions is critical for uncovering the pathologies and treatments for congenital heart disease. To address these issues, we have cloned and characterized the vertebrate orthologues of the zinc finger transcription factor, Castor (Cst). We have gone on to show that in Xenopus Cst is in required for cardiomyocyte differentiation; in the absence of Cst, cells at the ventral midline retain early cardiac progenitor fate but are blocked from differentiating into cardiomyocytes. The role of Cst is further emphasized by recent genome-wide association studies showing a genetic link between Cst and high blood pressure and hypertension. The overall goal of this proposal is to elucidate the cellular and molecular mechanism by which CST functions. To address these issues we have generated a set of unique alleles of Cst in mouse and will now use these alleles to determine the requirement and fate of Cst expressing cells in cardiac development. In addition, to address the molecular mechanisms by which CST functions in heart development, our lab has undertaken a set of approaches to identify the CST transcriptional complex. From these studies we have demonstrated that CST directly interacts with the congenital heart disease associated protein (CHD5), a protein initially cloned and identified from the minimal region containing the gene responsible for congenital heart disease in Down Syndrome patients. Moreover, we have used a directed proteomic-based approach to show that CST and CHD5 directly associate with the Nucleosome Remodeling and Deacetylase (NuRD) complex including histone deacteylase-1 and 2 (HDAC1/2). Based on our findings, we hypothesize that Cst functions as a transcriptional repressor which is required for early cardiac cell fate decisions. To test this hypothesis we will a) determine the fate and requirement of Cst-expressing cells to the developing heart, b) define the core components of the Cst-NurD transcriptional complex and c) determine the role of CHD5-CST interaction in regulating CST activity.

DESCRIPTION (provided by applicant): ABSTRACT The goal of this proposal is to develop and optimize the technologies and reagents for use of directed and quantitative proteomic approaches in Xenopus. Specifically, we propose a series of feasibility studies based on three proteins, the T-box transcription factors TBX5, the zinc finger transcription factor CASZ1, and the non-receptor protein phosphatase SHP2. We have focused on these proteins based on their evolutionary conservation in sequence, expression, and function, their cellular localization, and the observation that these proteins are causative, or have association with human disease states: patients with Holt-Oram syndrome (HOS), a disease which also affects the heart and limbs, is associated with mis-sense mutations in Tbx5, genome wide association studies have implied a role for Casz1 in hypertension and high blood pressure, and patients with Noonan syndrome, a disease that also affects heart and limb development, frequently is associated with mis-sense mutations in Shp-2. Despite the crucial role for TBX5, CASZ1 and SHP-2 in development and human disease, the molecular mechanisms by which these proteins act in vivo remains to be established. To address these issues and to establish a set of general technologies for the use of directed proteomics approaches in Xenopus we propose to 1) establish a binary transgenic system in Xenopus for the isolation of protein and protein-DNA complexes and 2) establish the technologies, reagents and protocols for directed and quantitative proteomic approaches in Xenopus.

? DESCRIPTION (provided by applicant): Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the US and Europe [1, 2]. Of the congenital malformations, congenital heart disease (CHD) is the most common [1, 2]. Clinical and genetic studies have provided direct evidence for the role of mutations in T-box transcription factors as causes of a number of CHDs. Central to this proposal mutations in the T-box gene Tbx20 are causative in a range of cardiac abnormalities including dilated cardiomyopathy, atrial septal defects, or mitral valve disease, while upregulation of TBX20 expression has been reported in patients with Tetralogy of Fallot (i.e., pulmonary outflow tract obstruction, ventricular septal defect, overriding aortic root and right ventricular hypertrophy) [3-7]. While TBX20 is an essential transcription factor for heart development and its disease relevance is well established, there are many critical questions unanswered about the mechanism of how TBX20 functions. We do not understand what proteins complex with TBX20 during different stages of cardiac development and homeostasis, how these interactions regulate TBX20's choice of distinct transcriptional targets at different times, or how these interactions function to activate and/or repress target gene transcription. To this end, our labs recently initiated a directed proteomic-based approach to identify proteins that function in association with TBX20. These studies demonstrate that TBX20 functions through distinct complexes that associate with TBX20 at defined periods of cardiomyocyte differentiation. Collectively, this work led to the central hypothesis that TBX20 function and thus its suites of target genes are regulated during cardiac development through changes in the components of the TBX20 interactome.

? DESCRIPTION (provided by applicant): The specific aim of this proposal is to obtain partial funding for the co-organization of the 2016 Weinstein Cardiovascular Development and Regeneration Conference to be held on May 19-21, 2016 in Durham, NC. The 2016 Weinstein Cardiovascular Development and Regeneration Conference originated from a series of annual meetings in which investigators funded by three separate RFAs on Cardiac Development in 1986, 1988, and 1990 came together under the direction of Dr. Constance Weinstein and colleagues at the NHLBI. Ever since its inception, the 3-day Weinstein meeting has been the premier opportunity for clinicians and researchers in the field of cardiovascular development to meet and share data and ideas on various topics including experimental cardiovascular embryology, cardiac function, genetics of human cardiovascular disease, cardiac stem cell biology, and transcriptional control of gene expression. It is impossible to overestimate the importance of the Weinstein Meeting for the development and growth of the field of cardiovascular developmental biology. The Weinstein program is entirely research community driven, with several features that make it unique among scientific conferences. The objectives of each meeting are to bring together basic and clinical scientists working in the areas of cardiovascular biology, to provide an interactive forum for students, postdocs and junior investigators to present their work and interact with senior investigators, and to actively support diversity in science. Last year, in response to the growing interest and studies in the area of potential translational cardiac repair, the Weinstein Cardiovascular Development Conference was renamed the Weinstein Cardiovascular Development and Regeneration Conference. In this proposal financial support is requested for the 2016 Weinstein meeting to be held in Boston. Funds will be used 1) to reduce student registration fees, 2) to provide financial assistance for under-represented groups, and 3) to support general meeting obligations. Narrative: Congenital heart defects and heart disease are among the most widespread and costly health issues in the United States and worldwide. The Weinstein Conference annually brings together clinical and basic scientists to discuss the latest findings about the underlying causes and potential cures for cardiovascular abnormalities and disease. This proposal seeks support from NIH to partially fund the 2016 Weinstein Cardiovascular Development and Regenerative Conference in Durham, NC.

ABSTRACT Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the US and Europe. Of the congenital malformations, congenital heart disease (CHD) is the most common. Mutations in the T-box transcription factor TBX5 have been found to be causative to a range of human cardiac abnormalities including Tetrology of Fallot and Holt Oram Syndrome (HOS), disease states associated associated with cardiac septal defects. While TBX5 is an essential transcription factor for heart development and its disease relevance is well established, there are many critical questions unanswered about the mechanism of how TBX5 functions. We do not understand what proteins complex with TBX5 during different stages of cardiac development and homeostasis, how these interactions regulate TBX5's choice of distinct transcriptional targets at different times, or how these interactions function to activate and/or repress target gene transcription. To this end, our labs recently initiated a directed proteomic-based approach to identify proteins that function in association with TBX5. These studies demonstrate TBX5 interacts with the transcriptional repression machinery of the Nucleosome Remodeling and Deacetylase (NuRD) complex. We further demonstrated that TBX5 human disease mutations disrupt this interaction, leading to ectopic expression of non-cardiac genes normally repressed by TBX5 and septal defects associated with Holt Oram syndrome. Collectively, this work led to the central hypothesis that TBX5 function and thus its suites of target genes are regulated during cardiac development through changes in the components of the TBX5 interactome. To address this hypothesis, we will used an integrated systems based approach to determine the mechanisms by which Tbx5 regulates distinct gene programs in the heart by defining the endogenous cardiac TBX5 transcriptional complexes, establish the mechanisms of TBX5 repression and activation and by determining the potential role of co-factors in TBX5 transcriptional regulation.

Abstract Congenital Heart Disease (CHD), malformations of the heart present at birth, is the most common class of life-threatening birth defect. Atrioventricular Septal Defects (AVSDs) are a common and serious form of CHD in humans, comprising 5-10% of all CHD and a greater proportion of cases requiring surgical intervention. Gaining a mechanistic understanding of atrioventricular (AV) septation is an essential goal for improving clinical approaches to structural heart disease. This R01 is based on a paradigm shift in the understanding of AV septation. Whereas the canonical view of AV septation was based on intracardiac events centered at the endocardial cushion, recent work demonstrated that the T-box transcription factor Tbx5 is required in the Second Heart Field, outside of the heart proper, for AV septation. In this proposal, we harness this new understanding of AV septation to address the molecular and biochemical mechanisms underlying AV septation, using Tbx5 as an entry point. We propose to (1) define the molecular basis of TBX5 regulation of Wnt2, required for lung development and thereby AV septation, (2) identify the proteins that interact with and function with Tbx5 in the SHF for AV septation; and (3) define the biochemical mechanisms underlying the function of Tbx5 in the SHF. The ultimate aim of the proposed work is to understand the molecular and biochemical basis of AV septation. This proposal will increase understanding of the molecular ontogeny of human AVSDs. This work is an essential step towards defining the causes of human CHD and improving the natural history of patients with CHD.

ABSTRACT Congenital heart disease is the leading cause of death in the western world affecting approximately 1.3 million Americans. The heart initially forms in vertebrates as a bilaminar tube comprised of an inner endocardium and outer myocardial layer. At later stages of development a third layer is added to the heart from the epicardium which forms from a dynamic precursor structure, the proepicardial organ (PEO) which forms on the septum transversum, a structure adjacent to the heart. As the embryo matures, cells from the PEO migrate onto the heart surface ultimately giving rise to several essential cell types in the adult heart including cardiac fibroblasts and the smooth muscle cells of the vasculature. The aim of this proposal will leverage our series of unique technologies, reagents, and animal models to elucidate the molecular and cellular pathways required for the formation and function of the epicardium. In doing so, we will provide a platform for studying heart formation and hemostasis, and thus proved mechanistic insight into human congenital heart disease.

ABSTRACT The Training Program in Cellular Systems and Integrative Physiology (CSIP) at UNC-Chapel Hill is a multidisciplinary predoctoral training program that uses a systems approach to provide comprehensive, biomedical graduate education to our trainees. The CSIP program is designed around evidence-based training activities, including didactic coursework, seminar courses focusing on scientific communication and grant-writing, career building activities, and novel doctoral research. Our rationale is that by combining these training approaches, the UNC CSIP Training Program will produce a diverse pool of well-trained scientists and leaders with the skills necessary to transition into successful careers in the biomedical research workforce. The multidisciplinary and interdepartmental CSIP Training Program draws its mentoring faculty and leadership members from 8 basic science departments (Biochemistry and Biophysics, Biology, Biomedical Engineering, Cell Biology and Physiology, Genetics, Microbiology and Immunology, Pathology, and Pharmacology) and 7 clinical departments (Medicine, Neurology, Nutrition, Oral and Craniofacial Health Sciences, Pediatrics, Psychiatry, and Surgery) that span the UNC School of Medicine, the College of Arts and Sciences, and the School of Public Health. The CSIP Training Program will transcend these individual departments to provide hands-on training and mentorship from our collaborative faculty and extracurricular, structured training opportunities across the CSIP departments. Our distinguished faculty emphasize rigor, transparency, integrity, and creative scientific reasoning that provide a broad, integrated biological foundation centered on organ systems and human diseases and have the vast resources of UNC at their disposal. By going beyond the minimum requirements of the Ph.D. granting departments and curriculums, the CSIP Training Program will serve as a model for graduate student training and education. In support of the CSIP Training Program, we request 5 years of funding to train predoctoral students for 2 years in the cellular and physiological processes of higher organisms across various scales (from molecular to whole-organism) and provide meaningful career development activities designed to prepare a diverse student population for a variety of biomedical career paths. The University of North Carolina is a national leader in developed programs to support ethnic, cultural, and physical diversity and the CSIP Training Program will strive to continue and build upon this strong tradition. .

Abstract Congenital heart disease (CHD) remains the most common congenital malformation. Therefore, attaining a mechanistic understanding of cardiomyocyte formation is crucial for improving outcomes to structural heart disease. Though much emphasis in the last few years has been placed on transcription factor networks that control cardiomyocyte differentiation, these studies have mainly focused on transcriptional activation. However, there is growing recognition that alterations in transcriptional repression also lead to CHDs. Transcriptional repression involves not only cardiac transcription factors but also broadly expressed multiprotein machines that modify and remodel chromatin. Prominent among these is the Nucleosome Remodeling and Deacetylase (NuRD) complex. In this proposal we address the role of chromodomain helicase DNA-binding protein 4 (CHD4), the catalytic core component of the NuRD complex. The critical nature of CHD4 is highlighted by mutations in Chd4 being causative to CHDs. The goal of the current application is to test the central hypothesis that CHD4 functions with the NuRD complex to regulate cardiac chromatin architecture. This will be achieved by: 1) Determine whether CHD4 both represses and activates cardiac gene expression. 2) Delineate how CHD4 and NuRD are recruited to cardiac loci. 3) Establish how the human Chd4 missense mutations lead to cardiac disease.