1997 — 2000 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cardiogenesis--Molecular Mechanisms @ University of Texas SW Med Ctr/Dallas
DESCRIPTION (adapted from investigator's abstract): This proposal is concerned with the two bHLH proteins, dHAND and eHAND, and their role in cardiac morphogenesis. The investigator was responsible for their cloning and showed that expression of eHAND is seen at 9.5dpc and dHAND at 8.5dpc in the embryonic heart. They are co-expressed in a variety of neural crest derived tissues and in the pharyngeal arches. eHAND is basically embryonic specific whilst dHAND is expressed at low levels in a number of adult tissues. In mice with a disruption of the endothelin-1 gene which exhibit heart malformations, the HAND genes were downregulated in the heart but not in the non-neural crest derived tissues. The first aim of this proposal, based upon the previous studies of the investigator, involve the generation of null mutants and examination of the morphological consequences. Initially, an emphasis will be placed upon dHAND null mutants since eHAND expression occurs in the extraembryonic membranes and could lead to embryonic lethality. The second aim will delineate the domains of dHAND and eHAND that are responsible for their binding to DNA and their further characterization to identify which amino acids are necessary for the interaction. Also, after identification of the binding site, promoters of genes expressed in the lateral mesoderm and neural crest will be examined as potential targets. The second part of this aim will attempt to define the transactivation domain of the genes using their known interaction with E12 which will be fused to the GAL4 DNA binding domain. Various truncated versions of the HAND genes will be tested for activation of a CAT reporter gene downstream of the GAL4 DNA binding sites. The final portion of this aim will determine which portions of the HAND proteins are necessary for protein-protein interactions using the CAT reporter system as for the transactivation experiments. The third and final aim will define the cis-acting sequences that regulate the expression of the HAND genes in tissue culture cells and transgenic mice.
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0.939 |
1999 — 2002 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Syndromic Cardiac Outflow Tract Defects @ University of Texas SW Med Ctr/Dallas
Congenital heart defects are the result of abnormal development of mesodermal cells, which form the muscular portion of the heart, or neural crest-derived cells, which populate the cardiac outflow tract and aortic arches. Defects in the two population of cells usually occur in a segmental fashion resulting in abnormalities of distinct regions of the heart with neighboring regions being relatively normal. The long term goal of this proposal is to understand the independent molecular pathways and mechanisms which would control segmental cardiac development. This type of understanding is the first step in identifying the genes which cause heart defects in distinct regions of the heart. Specifically, we focus on elucidating the pathogenesis of isolated cardiac outflow tract defects and those which occur as part of DiGeorge/CATCH-22 (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, and hypocalcemia associated with chromosome 22 microdeletion) syndrome. In both scenarios, a high percentage of affected individuals harbor a microdeletion of one allele of chromosome 22q11.2 and are thought to have a defect in neural crest-derived cells which populate the branchial and aortic arches and cardiac outflow tract. In contrast, conditions such as hypoplasia of the right or left ventricles have been thought to be the result of flow abnormalities during cardiogenesis. Our recent targeted deletion of the basic helix- loop-helix transcription factor, dHAND, suggests that a subset of cardiac outflow tract defects and hypoplastic right ventricle may be the result of excessive programmed cell death from single gene defects. Although dHAND-null embryos have hypoplasia of the neural crest-derived branchial and aortic arches and right ventricle, dHAND does not map to human chromosome 22. However, by subtraction cloning between wild type and dHAND-null embryos, we have found that a ubiquitin fusion degradation protein (UFD1) is downstream of dHAND and maps to the DiGeorge critical region of ch.22. We have shown that UFD1 is normally expressed in the branchial arches, cardiac outflow tract and right ventricle but is down-regulated in dHAND-null embryos. The UFD family has been studied in yeast where they represent a novel proteolytic pathway for degradation of cellular proteins. Targeted deletion of UFD1 in yeast results in cell death in a dose-dependent fashion, suggesting that the down-regulation of UFD1 may mediate the apoptosis seen in dHAND mutants. Having placed UFD1 as a candidate gene for CATCH-22 syndrome from a molecular pathway and mechanistic approach, we propose three major aims for this proposal: 1) to determine, in mice, the role of UFD1 during embryogenesis and if UFD1 is one of the genes responsible for a DiGeorge/CATCH-22-like phenotype, 2) to determine if mutations and/or deletions of UFD1 in humans contribute to cardiac outflow tract defects or subsets of CATCH-22, 3) to define the role of UFD1 and dHAND in cell survival during embryonic development. The aims utilize in vivo models of yeast, mice and humans to understand the development of cardiac mesoderm and neural crest in normal and abnormal embryogenesis. In this fashion, we intend to approach the molecular basis for certain congenital heart defects, particularly those affecting the cardiac outflow tract and aortic arch in isolation and in CATCH-22 syndrome.
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0.939 |
1999 — 2002 |
Srivastava, Deepak |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Mechanistic Regulation of Segmental Cardiac Regulation @ University of Texas SW Med Ctr/Dallas
Congenital heart defects are the result of abnormal development of mesodermal cells, which form the muscular portion of the heart, or neural crest-derived cells, which populate the cardiac outflow tract and aortic arches. Defects in the two population of cells usually occur in a segmental fashion resulting in abnormalities of distinct regions of the heart with neighboring regions being relatively normal. The long term goal of this proposal is to understand the independent molecular pathways and mechanisms which could control segmental cardiac development. This type of understanding is the first step in identifying the genes which cause heart defects in distinct regions of the heart. Specifically, we focus on elucidating the pathogenesis of DiGeorge/CATCH-22 syndrome, which is a defect of cardiac and pharyngeal neural crest development, and of hypoplastic right and left ventricle syndromes. The recent discovery of the basic helix-loop-helix transcription factors, dHAND and e-HAND, provide the impetus for study in these two areas because of their expression in both cardiac mesoderm and cardiac neural crest. Furthermore, d-HAND and e-HAND, provide the impetus for study in these two areas because of their expression in both cardiac mesoderm and cardiac neural crest. Furthermore, dHAND and eHAND are expressed in unique segments of the developing heart and serve as an entrance point to dissect the upstream and downstream members of chamber-specific pathways. dHAND-null embryos have a hypoplastic right ventricle and fail to form neural crest-derived aortic arch arteries. The mechanism of dHAND action may be mediated through inhibition of programmed cell death (apoptosis) by directly regulating a potential member of the apoptotic pathway. Identification of genes downstream of dHAND have led to the discovery of cot-22, a novel gene located in the minimal DiGeorge critical region of chromosome 22 and expressed in the heart and branchial arches The three major aims of this proposal are as follows: 1) to determine, in mice and humans, if cot-22 is the gene or one of the genes responsible for DiGeorge/CATCH-22 syndrome, 2) to define the role of apoptosis in development of hypoplastic right ventricle and arch malformations in dHAND-null mice, and 3) to determine the dominant effects of dHAND and eHAND in cardiac development and post-natal cardiac disease by over-expressing the HAND genes in a cardiac and neural crest specific manner; this aim will supplement the goals of aims 1 and 2 by addressing potential mechanistic issues. The aims utilize an in vivo model which is relevant to human disease and are complemented by the studies on hypoplastic ventricles syndromes and neural crest defects proposed by other investigations in this grant. By both elucidating a molecular pathway of segmental cardiac development and positional cloning of responsible loci of specific diseases, it will be possible to identify genes responsible for distinct cardiac defects, similar to the manner in which cot-22 was discovered.
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0.939 |
2001 — 2005 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cardiogenesis-- Molecular Mechanisms @ University of Texas SW Med Ctr/Dallas
DESCRIPTION (appended verbatim from investigator's abstract): Congenital heart disease (CHD) is the most common birth defect in humans, occurring with a frequency of nearly 1 in 100 live births and 1 in 10 first trimester miscarriages, yet the etiology remains unknown. The long-term goals of my laboratory are to dentify molecular pathways regulating cardiogenesis in the interest of understanding the bases of congenital heart disease. Because CHD affects specific segments of the heart, we have searched for chamber-specific regulatory pathways that function during cardiac development. Our studies have shown that dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in a complementary fashion in the right and left ventricles, respectively, of the mouse heart. Mice lacking dHAND have a hypoplastic right ventricle and cardiac neural crest cell defects from excessive apoptosis, although early cardiac failure precluded identification of independent roles for dHAND in specific tissues. Inquiries into how segmental gene expression is established in the heart led to the discovery of a novel subclass of cardiac bHLH proteins sharing homology with dHAND. These proteins (HRTI, HRT2, HRT3) belong to the Hairy family of transcription factors that mediate Notch signaling. HRTI and HRT2 are expressed specifically in the atria and ventricles of the heart, respectively, providing an entry to understand atrial vs. ventricular-specific gene expression. This proposal seeks to define the molecular cascades regulating chamber-specific cardiac development through the study of bHLH networks during cardiogenesis. The specific aims are: 1) To define the tissue-specific functions of dHAND during embryonic development using conditional gene deletion in mice; 2) To determine the genetic interactions between dHAND and eHAND and their functional redundancy by intercrossing dHAND and eHAND mutant mice and by placing eHAND into the dHAND locus by homologous recombination; 3) To determine if the HRT proteins function downstream of Notch signaling in tissue culture and genetically altered mice; 4) To understand whether HRT-related pathways regulate segmental cardiac and vascular development through targeted gene deletion. These studies will provide insights into the mechanisms of segmented gene regulation during cardiac morphogenesis that may ultimately allow for preventive interventions for CHD.
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0.939 |
2003 — 2006 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Isolated and Syndromic Cardiac Outflow Tract Defects @ University of Texas SW Med Ctr/Dallas
DESCRIPTION (provided by applicant): Elucidating the molecular pathways and mechanisms regulating pharyngeal arch development is fundamental to understanding the pathogenesis of the numerous congenital syndromes involving craniofacial and cardiac structures. Pharyngeal arches harbor distinct compartments of endodermal, mesodermal and neural crest-derived cells, each participating in one another's development, yet contributing to independent lineages and morphologic structures. Heterozygous microdeletions of chromosome 22q11 are the most common microdeletion in humans and are characterized by defects in derivatives of the third and fourth pharyngeal arch and pouches, including craniofacial, aortic arch and cardiac outflow tract defects. This deletion syndrome provides an entry into understanding the molecular pathways regulating pharyngeal arch and cardiac outflow tract development. The T-box transcription factor, Tbx1, appears to be a major genetic determinant in the 22q11 locus that may contribute to some features of 22q11 deletion syndrome. Tbx1 is expressed in the pharyngeal endoderm and pharyngeal mesoderm but not the pharyngeal neural crest. Mice lacking Tbx1 have aortic arch patterning defects and other pharyngeal arch-derived defects. Some aspects of the Tbx1 mutant phenotype may be from cell autonomous effects but others that affect neural crest cells are likely to be non-cell autonomous. The cell lineages that are responsible for the observed defects and the signals that affect neural crest cells remain unknown. Therefore, we are generating mice lacking Tbx1 in specific cell lineages to dissect the mechanisms through which Tbx1 controls pharyngeal and cardiac development. In addition, we have found separable cis elements that regulate pharyngeal mesoderm and endoderm expression of Tbx1 in transgenic mice and reveal Tbx1 expression in the cardiac outflow tract. Finally, we have found that Tbx1 physically interacts with two interesting transcription factors, GATA3 and Msx1. GATA3 maps to a second DiGeorge locus on chromosome 10p12 and is expressed in the pharyngeal endoderm and Msx1 mutation causes craniofacial anomalies. We hypothesize that Tbx1 is regulated by independent factors in the pharyngeal endoderm and mesoderm and that Tbx1 has distinct functions in the endoderm and mesoderm through interactions with other tissue-specific transcription factors. The specific aims of this proposal are: 1) To determine the modular mechanism of Tbx1 gene regulation in transgenic mice; 2) To test the relative contributions of Tbx1 function in the pharyngeal endoderm, mesoderm or cardiac outflow tract during embryonic development; and 3) To understand the mechanisms through which Tbx1 regulates downstream target genes by studying its interaction with the transcription factors GATA3 and Msx1. These studies will reveal the upstream and downstream mechanisms through which Tbx1 functions and are essential to understanding the pathogenesis of syndromic types of pharyngeal arch and cardiac outflow tract defects.
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0.939 |
2005 — 2009 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cardiac Defects/Cross-Species Analysis/Human Mutations @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): The complementary use of human genetics and model systems to understand disease-causing genes and the mechanisms that lead to disease holds great promise. A small number of genes causing congenital cardiovascular malformations (CCVMs) have been identified using genomewide linkage analysis in affected pedigrees, including the gene encoding a transcription factor, GATA4. In contrast to the limited number of genes identified through human studies, the recognition that key regulatory programs of cardiogenesis are conserved across species has led to the identification of over one hundred genes that, when mutated in model systems ranging from fruit flies to mice, cause defects in cardiac development. Approximately half are transcription factors. However, the contribution of genes encoding transcription factors to CCVMs and the precise mechanisms through which most of the essential genes function are largely unknown. In preliminary data, the PIs have undertaken a large-scale effort designed to translate the basic discoveries of the last decade into an understanding of the molecular basis of CCVMs. A screen of 60 patients with CCVM for sequence variations in 100 cardiac developmental genes resulted in identification of significant gene mutations in nearly half of all patients studied with at least one-third having demonstrable function-altering mutations. The PIs hypothesize that many of the human mutations in cardiac transcription factors significantly affect protein function and are likely to predispose to disease. In this project, they will test the hypothesis stated above for selected transcription factor mutations that are likely to be critical for human cardiogenesis. Specifically, they will build on the existing strengths of their lab and focus on the significance of human mutations in the hairy-related transcription factor, HRT2, GATA4 and the potent transcriptional activator, Myocardin (MYCD). By utilizing numerous available assays, the PIs will determine the mechanisms through which human mutations in these genes affect protein function in vitro and cardiogenesis in vivo, using both mouse and frog systems. Such studies will not only provide insight into the relevance of the human mutations, but will also reveal important structure-function aspects regarding the biology through which the cardiac regulatory proteins operate.
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0.939 |
2006 — 2021 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cardiogenesis: Molecular Mechanisms @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Genetic mutations that cause congenital heart malformations are often heterozygous and involve a partial reduction in protein dosage or an increase in protein activity. Although developmental events are precisely controlled by signaling pathways and transcriptional networks, our studies have revealed an intertwined layer of post-transcriptional regulation that involves microRNAs (miRNAs) that titrate protein dosage. The muscle-specific miRNA miR-1 is co-transcribed with a second miRNA, miR-133, and participates in septal formation, cell-cycle regulation, cardiac conduction, and other aspects of cardiac development and homeostasis. miR-1 and miR-133 are encoded in two loci as a result of a gene duplication, with identical mature sequences of miR-1-1 and miR-1-2, as well as miR-133a and miR- 133b. Loss of two redundant copies of miR-133 results in a ventricular septal defect, similar to deletion of miR-1-2. miR-1 and miR-133 appear to function in concert in some biological settings, but have opposing functions in others. The function of miR-1 is dose sensitive, as shown by gene targeting of miR-1-2, although the full function of miR-1 awaits compound deletion of miR-1-2 and its redundant allele, miR-1-1. Several targets of miR-1 are known, including Hand2, Irx5 and Delta-like 1. However, the contribution of individual targets to miR-1's function in vivo is unknown, and most targets of miR-1 and miR-133 are also unknown. We hypothesize that miR-1 is required for cardiac progenitor development in vivo and for postnatal cardiac function and that a discrete set of mRNA targets play major roles in mediating miR-1's function. We also hypothesize that miR-1 and miR-133 converge on common targets to cooperatively regulate cellular decisions but have other targets that mediate distinct functions. To test these hypotheses, we propose three specific aims: Aim 1) To determine the dose- dependent requirement of miR-1 in cardiac development and in post-natal cardiac function by analyzing compound deletions of miR-1-1 and miR-1-2; Aim 2) To determine whether repression of individual miR-1 targets mediates major functions of miR-1 in vivo; and Aim 3) To determine whether miR-1 and miR-133 share common targets upon which they can cooperate or synergize and whether they have distinct targets that mediate opposing functions. These studies will utilize several innovative approaches and will reveal miRNA and transcriptional networks that titrate protein dosage to control critical events in cardiogenesis. PUBLIC HEALTH RELEVANCE: Quantitative disruption of the molecular networks regulating cardiac development underlies many forms of congenital heart disease. In this proposal, we integrate the precise control of protein dosage through small RNAs, known as microRNAs, with other known regulators of cardiogenesis to understand the multiple levels by which heart formation is controlled.
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0.904 |
2008 — 2012 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Signaling and Transcriptional Networks in Cardiac Patterning @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Congenital heart malformations, the most common human birth defects, occur in nearly 1% of the population worldwide. Typically, they result from abnormal lineage decisions of early progenitors or disruptions in patterning, both leading to morphogenetic defects. In addition, heart disease is the leading killer of adults in the U.S. Deciphering the secrets of heart formation might lead to novel approaches to repair or regenerate damaged heart muscle by harnessing the potential of stem cell biology. The overall goal of this PPG is to focus the efforts of multiple investigators to dissect the signaling and transcriptional pathways that dictate early decisions of cardiac differentiation in unique regions of the heart and to reveal the mechanisms that guide patterning events during cardiogenesis. In a coordinated fashion, we will test the hypothesis that specific signaling and transcriptional networks govern the decisions of distinct regions of myocardium and result in specific sublineage decisions and patterning of functional regions of the heart. The project and core leaders have been collaborating for several years, and the proposal arises from mutual and complementary interests and approaches. Project 1 will determine the mechanisms by which canonical Wnt/?-catenin signaling and its downstream transcriptional events promote cardiac proliferation and differentiation in specific domains of the mouse heart in vivo. This knowledge will be complemented by and used to manipulate embryonic stem (ES) cells into the cardiomyocyte fate. Project 2 will explore the mechanisms underlying the differentiation of cardiomyocytes in the septal region and at the atrioventricular boundary, particularly as they relate to the function of key transcription factors such as Tbx5 and Nkx2.5 during patterning of the mouse heart; this will be done in vivo and in ES cells, as in Project 1. Project 3 will determine the mechanism by which Bmp4 patterns the outflow tract myocardium to influence valve formation at the ventriculo-arterial boundary in mice and will use a Mef2-dependent valvular enhancer to dissect the signaling networks leading to domain-specific gene expression. This project overlaps with the valve interests of Projects 2 and will also integrate the Wnt signals involved in valvulogenesis and possibly Mef2c activation. Three scientific cores will support the proposed experiments, as well as an administrative core. The synergistic and mutually reinforcing projects and cores in this proposal combine expertise in mouse genetics, stem cell biology, cell biology, developmental biology and genomics to tackle the fundamental problem of patterning of the developing heart, particularly as it relates to disease.
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0.904 |
2008 — 2011 |
Srivastava, Deepak |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Mammalian Heart and Lung Microrna Deletion and Distribution Resource @ J. David Gladstone Institutes
[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Most biological analyses of gene function have focused on protein-encoding genes, which constitute a mere 1.4% of the human genome. The rest of the genome is largely unexplored. The recent discovery of conserved stretches of non-coding RNAs, including microRNAs (miRNAs), has revealed a previously unrecognized layer of genomic regulation. miRNAs 20-22 nucleotides in length, function post-transcriptionally to titrate the activity of at least one-third of the protein-coding genes in the genome. In some cases, miRNAs function as on-off switches for key pathways. In other situations, they function as a rheostat to titrate the activity of pathways in normal biology and in response to external stresses and stimuli. Although more than 450 human or mouse miRNAs have been identified, most of the knowledge of this novel class of RNAs has come from worms and flies. Targeted deletion in mice has been reported for only three miRNAs to date, with two of them having critical functions in cardiac biology. These early loss-of-function studies have revealed novel targets for intervention in human disease, and it is highly likely that disruption of additional miRNAs will be equally revealing. [unreadable] [unreadable] To catalyze discovery in this emerging and highly significant area of biology, we propose to ablate -75 evolutionarily conserved heart and lung-enriched miRNA genes in the mouse with an advanced strategy to genetically modify embryonic stem cells. These mice will be made available to the scientific community through a web-based mechanism to accelerate advances in virtually every aspect of heart and lung research. This is an ambitious effort, but we believe we have the unique tools and expertise to accomplish it. In addition, the payoff is potentially significant. We expect major breakthroughs in the understanding of this relatively unexplored portion of the genome. New mouse models of human disease could shed light on human diseases in which potential protein-encoding disease genes have been difficult to map within a given genetic locus. These include a host of cardiac and pulmonary diseases of development and post-natal maintenance and adaptation. To accomplish this project, we propose three specific aims. [unreadable] [unreadable] Specific Aim 1. To generate embryonic stem cells and mice with disruption of ~75 heart and lung-enriched miRNAs. [unreadable] [unreadable] Specific Aim 2. To complete an initial characterization of the miRNA deletion lines, including validation of disruption and analysis of endogenous miRNA expression. [unreadable] [unreadable] Specific Aim 3. To disseminate information on and coordinate delivery of miRNA-disrupted mouse lines to the scientific community. (End of Abstract) [unreadable] [unreadable] [unreadable] [unreadable]
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0.904 |
2009 — 2019 |
Boyer, Laurie A. (co-PI) [⬀] Bruneau, Benoit Gaetan [⬀] Conklin, Bruce R (co-PI) [⬀] Pollard, Katherine Snowden (co-PI) [⬀] Srivastava, Deepak Yamanaka, Shinya (co-PI) [⬀] |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. UM1Activity Code Description: To support cooperative agreements involving large-scale research activities with complicated structures that cannot be appropriately categorized into an available single component activity code, e.g. clinical networks, research programs or consortium. The components represent a variety of supporting functions and are not independent of each component. Substantial federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of the award. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
The Epigenetic Landscape of Heart Development @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Congenital heart defects (CHDs) are among the most common and most devastating birth defects in humans. Networks of transcription factors regulate cardiac cell fate and morphogenesis, and dominant mutations in transcription factor genes lead to most instances of inherited CHD. The mechanisms underlying CHDs that result from disruption of these networks remain to be identified, but regulation of gene expression within a relatively narrow developmental window is clearly essential for normal cardiac morphogenesis. In addition to transcription factors, epigenetic regulation via histone modifications, chromatin remodeling, and non-coding RNAs have key roles in modulating gene expression programs. Elucidating on a genome scale the physical and functional interactions between transcription factors and epigenetic regulators will considerably enhance our understanding of the control of heart development and will have important implications for understanding the mechanistic basis of CHDs. We propose a project as part of the NHLBI Heart Development consortium to provide an integrated epigenetic landscape for heart development, with a focus on CHD-related genes. We propose three major aims. Aim 1: Define genome-wide occupancy maps of transcription factors with known roles in cardiac development and human disease, and epigenetic regulators of transcription, in differentiating cardiomyocytes. Aim 2: Define the global function of transcriptional and epigenetic regulation in heart development and congenital heart disease. We will examine the effect of loss of function of cardiac transcription factors on epigenetic regulation, and alterations in epigenetic regulation in disease-specific induced pluripotent cells from CHD patients. We will also evaluate the global role of histone modifications in mouse heart development. Aim 3;Integrate microRNA expression and function into the regulatory networks governing cardiac development. High-resolution occupancy maps from Aims 1 and 2 will be analyzed specifically for miRNA promoter occupancy and combined with quantitative sequencing of miRNAs in differentiating cardiomyocytes. We will study the function of highly altered miRNAs, specifically those that target disease-causing cardiac transcription factors. Our studies will yield an important and transformative epigenetic atlas of heart development, which will link for the first time transcriptional and epigenetic regulators in a comprehensive network that will illuminate mechanisms underlying CHDs. RELEVANCE (See instructions): The proposed project will for the first time allow a new understanding of the gene networks that underlie congenital heart disease. Congenital heart disease is the most serious childhood illness, affecting 1% of children, and leading to significant mortality and long-term illness. However the underlying causes of these diseases are not understood. Our project will link the so-called "epigenetic regulators" that control how genes are turned on or off, to congenital heart disease, bringing new important insights into these diseases.
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0.904 |
2009 — 2015 |
Bruneau, Benoit Gaetan (co-PI) [⬀] Conklin, Bruce R (co-PI) [⬀] Srivastava, Deepak Yamanaka, Shinya (co-PI) [⬀] |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Induced Pluripotent Stem Cells in the Understanding and Treatment of Heart Diseas @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Over 1 million Americans suffer acute myocardial infarctions each year in the US, and among the survivors, 5 million are afflicted with heart failure. In addition, defects in cell lineage determination or morphogenesis underlie congenital heart malformations, the most common human birth defect. Survivors of congenital heart disease, who number over 1 million in the US, also often suffer from heart failure. Unfortunately, the heart has little regenerative capacity after injury. The recent discovery of human induced pluripotent stem (IPS) cells has opened the door for novel approaches to human disease, including the development of human cellular models for disease mechanisms and drug discovery, along with the potential for autologous cell-based therapies. We propose to assemble a team of investigators at the Gladstone Institutes and Stanford University to develop and capitalize on the potential of IPS cells in the treatment and understanding of heart disease. Methods of IPS generation avoiding genomic integration of DNA are developing rapidly, but continue to require refinement before use of iPS cells in humans; this hurdle will be addressed in this application. As methods for generating IPS cells are improved the team will work together to more efficiently generate iPS-derived cardiac cells for future therapy, capitalizing on their expertise in chromatin remodeling and microRNA (miRNA) biology and G-protein coupled receptor signaling. The team will generate iPS cell lines with fluorescent markers for progressive stages of cardiac differentiation using bacterial artificial chromosome (BAC) strategies. We will also attempt to reprogram somatic cells directly into cardiac progenitors. Survival and engraftment of cells in vivo will be examined in rodents and in large animals through our partners at Stanford. Disease-specific iPS cells will be generated to reveal novel aspects of human progenitor cell biology. This multidisciplinary team will bring broad and critical expertise to the NHLBI Progenitor Cell Consortium in an effort to aggressively capitalize on the promise and potential of iPS cells for heart disease The interaction with the Stanford group within our Hub will synergize and leverage the specific strengths of each group of investigators on the focused effort related to iPS cells. The specific aims are: 1) To develop integration-free and efficient methods of human IPS cell generation for future cell-based therapies; 2) To develop efficient directed differentiation of human IPS cells and methods of direct reprogramming; 3). To develop methods to use IPS cell-derived cardiac progenitors in animal models of cardiovascular disease and 4). To use disease-specific IPS cells for discovery of human cardiac progenitor biology and cardiovascular disease mechanisms.
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0.904 |
2012 — 2021 |
Srivastava, Deepak |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training in Developmental Cardiovascular Biology @ University of California, San Francisco
PROJECT SUMMARY This renewal proposal seeks continuation of the Training in Developmental Cardiovascular Biology program, initiated in 1966. Of the 11 fellows supported by this program during the preceding 10 years who have completed training, 6 have academic appointments at outstanding institutions and are actively engaged in federally- or privately-funded research programs. This program will continue to utilize the outstanding physiology and molecular and cellular biology laboratories at the Cardiovascular Research Institute, the Gladstone Institute of Cardiovascular Disease (GICD), and several UCSF departments to train physician- scientists and basic scientists who will be at the forefront of developmental cardiovascular research in the coming decades. Modern physiologic and molecular approaches have produced remarkable advances in our knowledge of cardiovascular biology and disease, and the fields of human genetics and stem cell biology are providing still more opportunities. These developments promise rapid scientific progress and underscore the ongoing need for well-trained investigators to both continue this work and build bridges between basic discovery and the advancement of human health. To this end we will: 1) exploit the unique training opportunities in the CVRI, GICD, the Department of Pediatrics, and UCSF in general in areas of basic biology; 2) recruit graduates of top Ph.D. and M.D.-Ph.D. programs to careers in cardiovascular research; and 3) combine clinical pediatric cardiology training with a rigorous grounding in basic research to facilitate the training of physician-scientists. This grant will support 4 postdoctoral fellows, each for 2 years, and features two tracks. Track I, the Physician-Investigator Pathway, will support M.D. trainees seeking both clinical pediatric cardiology and basic science research training. Trainees in this Track will receive support from this training grant only for the research component of their training. Track II will support Ph.D. or M.D.-Ph.D. trainees who will compete openly for postdoctoral positions in basic cardiovascular biology. A strong didactic curriculum will be provided in a rich scientific environment with rigorous graduate courses, regular trainee meetings, and attentive mentorship. Each trainee will execute a research project with one of the 22 outstanding program faculty preceptors.
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1 |
2013 — 2021 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative Core @ J. David Gladstone Institutes
The Administrative Core will be responsible for coordinating the scientific and financial activities ofthe PPG. It will establish policies for use ofthe Cores and coordinate communication between the Projects and other Cores, as well as with Advisory Committees. This Core will be responsible for scheduling and arranging monthly meetings of PPG participants and preparing periodic scientific and financial reports of progress. Maintenance and monitoring of institutional authorizations for animal use and other laboratory regulations will be performed through the Administrative Core.
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0.904 |
2013 — 2017 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Transcriptional Networks During Cardiac Differentiation @ J. David Gladstone Institutes
DESCRIPTION (Provided by applicant): Signaling, transcriptional, and post-transcriptional events regulate cardiac cell fate decisions during early cardiogenesis. Disruption of such events can lead to congenital heart malformations. In particular, human mutations in transcription factors (TFs), such as GATA4, TBX5, NKX2-5 and N0TCH1, result in heart disease in children. Embryonic pathways are reactivated under stress in adult hearts, with GATA4 and MEF2C playing central roles in the transcriptional response during cardiac hypertrophy. Recent studies highlight the importance of protein-protein interactions (PPI) in dictating the transcriptional output of cardiac DNA-binding TFs. However, the complex PPIs that titrate effects of cardiac TFs have not been systematically explored. During the previous funding period of this PPG, our discoveries focused on the effects of a variety of signaling and transcriptional events that frequently culminated in combinatorial interactions between TFs and chromatin remodeling complexes to regulate cardiac gene expression. For example, manipulation of a combination of cardiac developmental TFs, including Gata4, Mef2c and Tbx5, and chromatin remodeling proteins (e.g., Baf60c), could induce the reprogramming of non-muscle cells into cardiomyocyte-like cells and promote cardiac regeneration after injury. As genome-wide TF binding data in cardiac cells accumulate, it is imperative that we understand the complex combinatorial interactions of regulatory proteins to interpret the transcriptional consequences of DNA-binding. Here, we will leverage the expertise of several of the international experts in cardiac transcription factors, a leading systems biologist, and computational biology strengths to systematically determine the complex interactomes by which the core cardiac transcriptional machinery functions to regulate gene expression during cardiac differentiation. Three discrete projects are proposed to deeply interrogate the functional consequence of selected interactomes determined by the proposed Proteomics Core. Cardiac progenitors and cardiomyocytes derived from mouse embryonic stem cells in the Cell Production Core will be used for the proteomic studies and data will be analyzed and integrated with other enhancer and chromatin data by the proposed Bioinformatics Core.
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0.904 |
2013 — 2017 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cardiac Transcriptionfactor Interactomes Regulating Cardiac Development&Reprogram @ J. David Gladstone Institutes
Signaling, transcriptional, and post-transcriptional events regulate cardiac cell fate decisions during early cardiogenesis. Disruption of such events can lead to congenital heart malformations. In particular, human mutations in transcription factors, such as GATA4, TBX5, NKX2-5 and NOTCHI, result in heart disease in children. Embryonic pathways are reactivated under stress in adult hearts, with GATA4 and MEF2C playing central roles in the transcripfional response during cardiac hypertrophy. Recent studies highlight the importance of protein-protein interactions (PPI) in dictating the transcripfional output of DNA binding transcription factors. However, the complex PPIs that titrate effects of cardiac transcription factors have not been systematically explored. During the previous funding period of this PPG, our discoveries focused on PPIs between members ofthe Notch and Wnt signaling pathways during cardiac development. In addition, we reported that a combination of cardiac developmental transcription factors, including Gata4, Mef2c and Tbx5, could reprogram non-muscle cells into new cardiomyocyte-like cells in the adult, resulting in cardiac regeneration after injury; the addition of Hand2 further improves reprogramming. Project 1 of this PPG renewal application will test the hypothesis that Gata4, Mef2c, Tbx5, and Hand2 have complex interactions with one another and other key factors to regulate the transcripfional output during cardiac differentiation and cardiac reprogramming. The specific alms are (1) to develop a comprehensive map ofthe Interactome involving Gata4, Mef2c, Tbx5 and Hand2 during cardiac differentiation of mouse embryonic stem (ES) cells into cardiomyocytes; (2) determine the central dependency of interactomes on Gata4, Tbx5 and Hand2, and the consequences of disease-causing missense mutations In GATA4 on the interactome; and (3) determine the functional consequences of PPIs involving Gata4, Mef2c, Tbx5 and Hand2 on specific genomic loci and integrate PPIs with genome occupancy ofthe transcription factors during cardiac differentiation and reprogramming. This project will reveal mechanisms underlying cardiac gene regulation and will provide potential points of intervention to positively or negatively titrate transcriptional activity.
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0.904 |
2015 — 2019 |
Bernstein, Daniel Srivastava, Deepak |
UM1Activity Code Description: To support cooperative agreements involving large-scale research activities with complicated structures that cannot be appropriately categorized into an available single component activity code, e.g. clinical networks, research programs or consortium. The components represent a variety of supporting functions and are not independent of each component. Substantial federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of the award. The performance period may extend up to seven years but only through the established deviation request process. ICs desiring to use this activity code for programs greater than 5 years must receive OPERA prior approval through the deviation request process. |
Genetics of Hypoplastic Left Heart Syndrome @ J. David Gladstone Institutes
? DESCRIPTION (provided by applicant): Congenital heart disease (CHD) is the most common birth defect and a leading cause of morbidity and mortality in children. While the genetic cause of some types of CHD has been identified, the basis for most forms of sporadic heart disease remains unknown. Among the various forms of CHD, hypoplastic left heart syndrome (HLHS) appears to have a particularly high degree of heritability and is often associated with sub- clinical aortic valve disease in first-degree relatives. One of the major co-morbidities associated with CHD, and particularly HLHS, involves poor neurodevelopmental outcomes. While the etiology remains unclear, there is evidence to support a patient-intrinsic component to the neurologic outcome, which often is subtle and presents at school age. There is also an increased incidence of neuro-anatomic abnormalities in patients with HLHS, particularly agenesis or hypoplasia of the corpus callosum, but the genetic basis for this is unclear and it is unknown if poor neurologic outcomes are related to anatomic anomalies. In the Main Project of this proposal, we aim to test the hypothesis that HLHS arises from genetic variants with relatively strong effects and that in at least a subset of cases, the same genetic variants disrupt both aortic valve and neuronal development. By evaluating the combination of HLHS and neuro-anatomic anomalies as a unique syndrome, we may identify a common genetic etiology within this more homogeneous subset even when index cases are unrelated. In the associated Neurodevelopmental Project, we will test the hypothesis that adverse neurodevelopmental outcomes in HLHS have a genetic component and may independently be associated with neuro-anatomic defects. To achieve these goals, the we plan to do the following: 1) Enroll subjects with HLHS and collect DNA, phenotypic data, determine sub-clinical neurologic anatomies by MRI, and assess their neurodevelopmental outcomes; 2) Determine genetic variants through next-generation sequencing that are associated with HLHS with or without co-existence of anatomic neurodevelopmental anomalies and/or abnormal neurodevelopmental outcomes; 3) Experimentally determine if genetic variants that segregate with HLHS or with poor neurodevelopmental outcomes are function-altering and contribute to pathologies associated with disease by using human induced pluripotent stem (iPS) cells and state-of-the-art genome editing techniques. These aims will be pursued through two integrated projects that study the same cohort of patients and leverage data from each to discover genetic causes of HLHS and the associated poor neurodevelopmental outcomes. We have assembled a team of cardiologists, neurodevelopmental experts, geneticists, and computational biologists at UCSF, Gladstone, and Stanford to accomplish these goals, which could only be accomplished in the context of a national consortium.
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0.904 |
2019 — 2021 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 1: Regulation of Gene Networks Through Cardiac Transcription Factor Interaction With the Nuclear Membrane @ J. David Gladstone Institutes
PROJECT SUMMARY/ABSTRACT PROJECT 1 Cardiac development relies on the stepwise activation and repression of lineage-specific gene expression programs. This process is regulated by conserved cardiac transcription factors (cTFs), such as NKX2.5, GATA4, TBX5, MEF2C and Myocardin, which cooperate with one another and chromatin-remodeling complexes to establish cellular identity by controlling gene regulatory networks. The critical nature of cooperative interactions is highlighted by a heterozygous glycine-to-serine missense mutation in GATA4 (GATA4-G296S) that disrupts interaction with TBX5 and causes congenital heart disease (CHD). TBX5 and GATA4 interact with the BAF complex of chromatin-remodeling proteins (investigated in Project 2), and together, these factors promote cardiac reprogramming in embryos. Further addition of MEF2C and its co- activator, Myocardin, (investigated in Project 3) reprograms adult cardiac fibroblasts into cardiomyocyte-like cells. Our Preliminary Data revealed that TBX5 and GATA4 interact with several nucleoporins that constitute the nuclear pore complex (NPC) at the nuclear membrane. Nucleoporins can control activation or silencing of developmental genes by regulating the three-dimensional chromatin architecture, but how specific genomic regions are recruited to the nuclear membrane remains unclear. Here, we will test the hypothesis that GATA4 and TBX5 interact in a lineage-specific fashion with NPC proteins to recruit genomic loci to the nuclear membrane to regulate the transcriptional output during cardiac differentiation. We will use hiPSC-derived CMs in which disruption of individual proteins or their interaction is possible using CRISPR/Cas9-based genome engineering (supported by Core C), and validate the findings in vivo in mice. We propose that the defective interaction between GATA4 and TBX5 disrupts the stoichiometry of the protein complex with nucleoporins (supported by Core A) and, thereby, contributes to the altered cardiac transcriptional and epigenetic outcome associated with disease (supported by Core B). Our specific aims are as follows: Aim 1) determine which nuclear pore proteins interact and co-localize with TBX5 or GATA4 to establish the lineage-specific three- dimensional genomic architecture in human cardiomyocytes and reprogrammed cardiomyocyte-like cells; Aim 2) determine the nature of enhancer elements localized to the NPC through interaction with TBX5 or GATA4, and the related epigenetic and transcriptional consequences during cardiomyocyte differentiation and reprogramming; and Aim 3) determine the effects of the human disease?causing mutation in GATA4 that disrupts interaction with TBX5 on the 3D genomic architecture, the transcriptional and epigenetic states of loci recruited to the nuclear membrane, and the interdependence of these events on physical interaction between GATA4 and TBX5. These studies represent a novel investigation into the role for lineage-specific TFs in regulating gene transcription by localization of specific genomic loci to the NPC and aim to open a new field in understanding the flow of genetic information during CM lineage specification.
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0.904 |
2019 — 2021 |
Mckinsey, Timothy Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Chromatin Signaling in Heart Failure by the Brd4 Bromodomain Protein. @ University of Colorado Denver
Abstract Despite current standard of care, a diagnosis of heart failure (HF) is associated with poor quality-of-life and a 5-year mortality approaching 50%. In light of this urgent unmet need, the elucidation of novel mechanisms involved in HF pathogenesis holds promise for identifying new therapies for this prevalent and deadly disease. The PIs of this application were the first to illustrate a crucial role for a conserved family of acetyl-lysine ?reader? proteins (BET bromodomains) in the transcriptional control of HF. Importantly, these studies leveraged the use of JQ1, a first-in-class, specific small molecule inhibitor of BET bromodomains. This multi-PI renewal application seeks to vertically advance our understanding of how aberrant chromatin-dependent signal transduction (via the BET family member BRD4) drives pathologic cardiac fibrosis. Our long-term objective is to develop BRD4 inhibition as a novel therapeutic strategy in HF. Exciting preliminary studies demonstrate that BRD4 mediates cardiac fibroblast activation in vitro, and that BRD4 inhibition with JQ1 suppresses cardiac fibrosis in mouse models of HF. Mechanistically, we demonstrate that BRD4 functions downstream of pro- fibrotic TGF-? signaling by binding to regulatory enhancers that drive a gene program of myofibroblast (myoFB) activation. This proposal will test the central hypothesis that BRD4 functions as a nodal transcriptional regulator of pathological cardiac fibrosis that can be pharmacologically targeted in vivo. Guided by strong preliminary data, this hypothesis will be tested by pursuing three robust specific aims: (1) Discover the gene-specific role of BRD4 in cardiac myoFB in vivo; (2) Dissect the chromatin-dependent signaling mechanisms governing BRD4-dependent activation of endogenous cardiac myoFBs; (3) Define the roles of specific BRD4 functional domains in the control of cardiac myoFB activation. Several innovative tools that were developed during the first funding period will be employed to advance this new avenue of investigation, including floxed Brd4 mice, Brd4-3XFLAG knock-in mice, Brd4 bromodomain knock-in mice, as well as peptides and small molecules that selectively inhibit distinct functional domains in BRD4. The proposed research is significant because it will facilitate development of pharmacologic BRD4 inhibition as a novel therapeutic strategy in HF, and therefore addresses an enormous unmet clinical need. Our proposal is highly innovative because we successfully ?drug? pro-fibrotic transcription and remodeling via unprecedented approaches, we define the functions of BRD4 in cardiac fibroblasts for the first time, and we provide the first epigenomic evaluation of cardiac fibroblasts. Given the synergistic expertise of our consortium, we envision that sustained contributions from our highly-collaborative group will pave the way for the development of novel ?epigenetic therapies? for cardiovascular disease.
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0.949 |
2019 — 2021 |
Srivastava, Deepak |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Combinatorial Regulation of Gene Networks During Cardiac Development and Disease @ J. David Gladstone Institutes
PROJECT SUMMARY/ABSTRACT OVERALL COMPONENT Cardiovascular disease is the most common cause of mortality in adults, and congenital heart defects (CHDs) are the most common form of birth defects. An important concept that has emerged in recent years is that dysregulation of cardiac transcription factor (TF) networks and related chromatin remodeling machinery contributes to CHDs and heart failure. Forced expression of the core members of the TF network is sufficient to reprogram non-myocytes into cardiomyocyte-like cells for regenerative medicine purposes, suggesting a combinatorial code for determining cell fate. As genome-wide roles for critical TFs are being discovered, a conceptual understanding of their function in higher order DNA organization is emerging. Evidence that the three-dimensional organization of DNA promotes activation or repression of genomic loci has raised the question of how cooperative protein-protein interactions involving TF and chromatin remodeling complexes participate in this process. We have integrated a multidisciplinary team of developmental cardiologists, computational biologists, and systems biologists, with expertise in CRISPR/Cas9 genome engineering in human iPS cells, to investigate how the genome is regulated by TFs and chromatin remodelers to control cardiac gene expression and fate. The specific hypotheses that we test in this proposal, which involve a combination of core cardiac TFs that interact with one another to coordinately regulate cardiac gene expression, are as follows: 1) that the cardiac TFs GATA4 and TBX5 interact in a lineage-specific fashion with the nuclear pore complex to regulate the 3D genomic architecture and subsequent transcriptional output; 2) that specific BAF chromatin remodeling complexes form dynamically to coordinate regulation of distinct aspects of cardiac morphogenesis and lineage decisions through interaction with cardiac TFs; and 3) that the MEF2C-myocardin complex, which interacts with GATA4, TBX5 and BAF60c, recruits a transcriptional complex influenced by upstream signaling and myocardin dimerization to regulate cardiac gene expression. To address these questions, we have integrated unique expertise in the study of protein-protein interactions and post-translational modifications through the Advanced Proteomics Core; the ability to analyze complex datasets of PPIs, DNA-binding, and transcriptional output related to transcriptional regulators through the Advanced Bioinformatics Core; and the ability to leverage state-of-the-art genome engineering approaches through the Genome Engineering Core. The questions in this proposal will be studied in the context of human disease-causing mutations to reveal underlying mechanisms and paradigms that control normal and abnormal cardiogenesis. The integrated knowledge developed here will enable a clear reading of the transcriptional ?code? for cardiac cell fate determination and differentiation that may be leveraged for interventions in CHD and for regenerative medicine.
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0.904 |
2020 — 2021 |
Srivastava, Deepak |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Aortic Valve Disease: Mechanisms and Therapeutic Approaches @ J. David Gladstone Institutes
Aortic valve stenosis is the major cause of valve disease in the Western world, and the third leading cause of adult heart disease. It is a progressive disorder that worsens with age. Thickening of the aortic valve is followed by calcification that results in further stenosis that ultimately necessitates surgical replacement. A major risk factor for calcific aortic valve disease (CAVD) is bicuspid aortic valve (BAV), which is present in 1?2% of the population, and involves formation of a two- rather than three-valve leaflet. ~35% of individuals with BAV will develop CAVD with age, but some with BAV have thickening even in childhood, requiring intervention. There are no medical treatments available for CAVD patients, and the only clinical option is surgical valve replacement. We reported that heterozygous nonsense mutations in the NOTCH1 (N1) transcription factor cause congenital biscuspid aortic valve (BAV) and severe CAVD and evidence suggests N1 is haploinsufficient in valve endothelial cells (ECs). Induced pluripotent stem cell (iPSC)-derived ECs from several patients with CAVD and N1 haploinsufficiency showed increased expression of pro-osteogenic and inflammatory signaling. Network analysis revealed key nodes that were responsible for much of the gene dysregulation. A chemical screen for the ability to restore expression of 120 dysregulated genes by targeted RNA-sequencing revealed several drugs that restored the network close to the N1+/+ state in human iPSC-ECs, with one, XCT790, showing efficacy in vivo for aortic valve thickening, calcification and stenosis in a mouse model. XCT790 is annotated to function as an inhibitor of estrogen-related receptor alpha (ERR?). We now propose to test the hypotheses that XCT790 prevents aortic valve disease by inhibiting ERR? activity, can treat established disease, and can function in a subset of CAVD patient cells without N1 mutations. The aims are 1) to determine the mechanism of action by which XCT790 corrects disease-associated gene expression and prevents aortic valve disease; 2) to determine if XCT790 can treat established CAVD or prevent neonatal valve stenosis; and 3) to determine if gene dysregulation in primary aortic valve endothelial cells from sporadic CAVD patients with or without genetic variants in NOTCH1 pathway genes is responsive to XCT790. These studies will advance a potential therapeutic to treat a disease that represents an enormous unmet medical need, is characterized by high mortality and morbidity, and for which the only current remedy is a highly invasive surgery.
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0.904 |