Brian L. Black - US grants

DESCRIPTION (appended verbatim from investigator's abstract): The long term goal of these studies is to define the transcriptional programs controlling the determination and differentiation of skeletal, cardiac, and smooth muscle. Efforts to initiate or inhibit muscle cell growth and differentiation following injury, disease, or as a result of congenital abnormalities require a greater understanding of basic genetic control within these lineages. The identification of the transcription factors regulating muscle specific gene expression and defining how these regulators function is critical to understanding the mechanisms controlling myogenesis in vivo. The hypothesis for these studies is that transcriptional regulation of myogenesis is controlled by multiple sets of transcription factors which function combinatorially to drive gene expression in a unique temporospatial pattern within each distinct muscle lineage. For example, transcription factors of the myocyte enhancer factor 2 (MEF2) and MyoD families are essential cofactors for one another and function combinatorially to induce myogenesis in skeletal muscle. Evidence suggests that MEF2 proteins also function combinatorially to induce differentiation of cardiac and smooth muscle. However, to date no "MyoD equivalents" serving as coregulators for MEF2 have been isolated from other muscle lineages. The studies proposed in this application are designed to identify transcriptional regulators in cardiac and smooth muscle that are comparable to MyoD in that they induce myogenesis and gene expression through combinatorial interactions with MEF2 factors. The specific aims of this proposal are: 1) To define the transcriptional regulation of HRC, a gene which is expressed in all three muscle lineages in mammals during development and in adults. Preliminary data suggests that MEF2 factors collaborate with previously unidentified DNA binding proteins to direct expression of HRC in cardiac and smooth muscle lineages. 2) To identify MEF2 coregulators comparable to MyoD in smooth and cardiac muscle.

[unreadable] DESCRIPTION (provided by applicant): Cardiovascular defects represent the largest class of congenital anomalies in the United States and efforts to prevent these defects require a greater understanding of the genetic pathways controlling the formation of the heart and vascular systems. Likewise, efforts to regenerate cardiac tissue, or to selectively inhibit or induce the growth of the vasculature require a deeper understanding of the embryonic programs that control the formation of these lineages. The long-term goal of these studies is to define the genetic pathways that control the formation of the cardiovascular system. This proposal focuses on the transcriptional requlation and function of the transcription factor MEF2C in cardiac and vascular development. Mef2c is expressed in the heart and vascular endothelium shortly after the initial specification of myocardial cells in the precardiac mesoderm and in the vasculature shortly after the initiation of vasculogenesis. Mef2c is an early factor involved in differentiation and may be a direct transcriptional target of lineage specific determination factors. Targeted disruption of mef2c in the mouse results in embryonic lethality at midgestation and these mice display profound cardiac and vascular defects. However, it is not clear whether both of these defects are due to a primary requirement for mef2c in that tissue. Using a conditionally targeted allele of mouse mef2c, the work proposed in these studies will remove mef2c function specifically in endothelial cells using Tie2-Cre and will use a novel anterior heart field transcriptional enhancer defined in this application to direct Cre-mediated excision of mef2c in the anterior heart field. The work proposed here will also define the regulation of two novel transcriptional enhancers from the mouse mef2c gene, one of which is exclusively expressed in the vascular endothelium and the other that is restricted only to the anterior heart field, using transgenic approaches in mice. The specific aims of this proposal are: 1) To determine the requirement of mef2c function in the anterior heart field and in the vascular endothelium. This proposal is designed to test the hypotheses that the cardiac abnormalities observed in mef2c null mice are due to primary defects in anterior heart field development and that mef2c function is required in endothelial cells for vascular development. 2) To define the requlation of mef2c in the anterior heart field and the vascular endothelium. These studies will identify the transcriptional regulators that confer spatial restriction to the mef2c anterior heart field enhancer and will identify the upstream regulators of a vascular endothelial enhancer from mef2c. 3) To determine the regions of the adult heart derived from the mef2c-expressing cells in the anterior heart field. [unreadable] [unreadable]

Muscular dystrophies are debilitating diseases that affect about one out of every 3500 live male births. Several therapeutic strategies for muscular dystrophy are aimed at the capacity of satellite cells or other muscle associated stem cells to repair damaged muscle fibers. Importantly, recent evidence has shown that the regulatory pathways functioning during muscle regeneration share many common features with the myogenic program that functions during skeletal muscle development. Thus, the long term objective of the studies proposed here is to define fully the transcriptional pathways governing skeletal muscle development, regeneration, and repair. One family of transcription factors that plays a key role in skeletal muscle development is the myocyte enhancer factor 2 (MEF2) family. MEF2 proteins function as part of a combinatorial transcriptional complex with members of the MyoD family of basic helix-loop-helix (bHLH) proteins, which are essential for muscle specification and differentiation during development and also play critical roles in satellite cell proliferation and differentiation. The studies proposed here will define the transcriptional regulation and genetic function of the mef2c gene in skeletal muscle. Because MEF2 transcription factors play an essential role in muscle differentiation, it is important to define the regulation and function of these genes during muscle development. A better understanding of these developmental pathways is essential for the development of therapies based on increasing regenerative capacity in skeletal muscle. This study will address the following two specific aims: 1. To determine the function of mef2c in skeletal muscle. Using conditional targeting approaches in mice, the mef2c gene has been specifically ablated in skeletal muscle. The development, function, and regenerative capacity of skeletal muscle without mef2c will be analyzed using histology, molecular markers and analyses of muscle function. Downstream targets will be identified by microarray analyses. 2. To define the regulators of mef2c transcription in skeletal muscle. Recently, a small enhancer and promoter fragment from the mef2c gene that is sufficient to direct expression exclusively to skeletal muscle in transgenic mice was identified. The studies proposed here will define c/s-acting elements that are required for expression in transgenic mice and will identify the trans-acting factors that regulate mef2c.

DESCRIPTION (provided by applicant): Craniofacial anomalies account for nearly one-third of all birth defects and are a severe cause of morbidity and mortality in infants. In spite of their prevalence, the underlying genetic and molecular mechanisms causing most craniofacial defects remain largely unknown. The long-term goal of the proposed studies is to define the molecular and genetic pathways that control craniofacial development for the purpose of regeneration and repair, tissue engineering, and the diagnosis of and intervention into congenital craniofacial birth defects. Specifically, this application is focused on the identification of the transcriptional pathways and mechanisms involved in craniofacial development. Recent work has identified a novel genetic model for Pierre Robin Sequence (PRS) in mice. PRS occurs in about 1 in every 800 live births and is characterized by small lower jaw, improperly positioned tongue and posterior cleft of the palate, which can result in upper airway obstruction and feeding difficulties. Previously, it was thought that PRS occurred as a result of environmental factors that restrict outgrowth of the mandible, but it is now quite clear that this sequence also has an undefined genetic component(s). Mice that carry one mutant allele of Dlx5/6 locus and one mutant copy of the Mef2c allele die at birth from craniofacial defects resembling PRS. The Dlx5/6 locus encodes two Distal-less related homeobox transcription factors, while the Mef2c locus encodes a MADS box transcription factor. In addition to their genetic interaction, MEF2C and Dlx5 cooperate to induce a robust synergistic transcriptional response. The hypotheses underlying this proposal are that Dlx5/6 and MEF2C form a transcriptional complex downstream of endothelin receptor signaling, that this complex is essential for the activation of a subset of genes that are required for craniofacial development, and that mutations or aberrant expression of Dlx5/6-MEF2C target genes contributes to craniofacial anomalies such as PRS. To address these hypotheses, three specific aims are proposed. Aim 1 will define the physical and functional interaction between Dlx5 and MEF2C and how this results in transcriptional activation. The goal is to define the transcriptional mechanisms that control gene expression during craniofacial development. Aim 2 will analyze the craniofacial defects in Mef2c-Dlx5/6 double heterozygotes in detail. The goal is to identify possible cellular mechanisms underlying the mandible and palate phenotypes in double heterozygotes to understand how these processes control palate closure and jaw growth. Aim 3 will identify upstream regulators of Mef2c transcription in craniofacial mesenchyme and will determine whether Mef2c is a direct target of the endothelin signaling pathway, using a transgenic mouse approach. The goals are to place Mef2c into a transcriptional pathway and to identify the immediate transcriptional effectors of endothelin signaling in craniofacial development. PUBLIC HEALTH RELEVANCE: Craniofacial anomalies, which account for nearly one third of all birth defects and are estimated to occur in 1 every 300 live births, are a severe cause of morbidity and mortality in infants. In spite of the prevalence of craniofacial birth defects, the underlying genetic and molecular mechanisms causing these anomalies remain largely unknown. The proposed studies will contribute to the understanding of the molecular and genetic mechanisms controlling craniofacial development, which is essential for determining how to reactivate or modulate these programs for the purpose of regeneration and repair, tissue engineering, and the diagnosis of and intervention in craniofacial birth defects.

Cardiovascular disease is the most common cause of mortality in adults, and congenital heart defects are the most common form of birth defects in the US. AMP-activated protein kinase (AMPK) is a master regulator of energy balance and homeostasis and plays a central role in the switch from fatty acids to glycolysis during cardiac hypertrophy and heart failure. AMPKa2 is the predominant isoform ofthe catalytic subunit expressed in the heart and is encoded by the Prkaa2 gene. Although AMPK and its role in metabolism and heart failure have been extensively studied, the transcripfional control ofthe genes encoding AMPK subunits is completely unknown. The MADS box transcription factor MEF2C is required for cardiac development and functions as a signal responsive transcription factor that interacts with a variety of cofactors to either negatively or positively regulate transcription. The most potent transcriptional coactivator for MEF2C is the SAP domain transcripfional regulator Myocardin. MEF2C specifically interacts with Myocardin-93S, which is highly enriched in the heart, yet the transcripfional targets and mechanisms of synergy facilitated by the Myocardin-MEF2 complex have not been identified. Preliminary studies found that the Prkaa2 gene is a direct transcripfional target ofthe MEF2C-Myocardin complex via a novel cardiac- specific enhancer. This enhancer contains two bona fide MEF2 binding sites that function together in a multiplicative fashion in response to Myocardin-93S and MEF2C. Although transcripfional synergy is frequently observed in many contexts and is a well-described phenomenon, the role of cis-acting elements, their position, spacing, and sequence in facilitating synergy have been far less well described. Using transgenic mouse, cell culture, biochemical, and mass spectrometry approaches, this work will define determinants of MEF2-Myocardin transcripfional synergy, will identify additional interaction partners for MEF2C and Myocardin through interactome mapping, and will identify in vivo targets ofthe Myocardin-MEF2 complex. This work will also determine the upstream regulation of Prkaa2 for the first time. This work may provide additional strategies for manipulating AMPK expression during heart failure.

DESCRIPTION (provided by applicant): The field of vascular biology has experienced an explosion of information in recent years. In light of this wealth of information there is an increased need for discussion and interdisciplinary exchange of ideas. Vascular biology has evolved over the past decade as a major cross-disciplinary field that impacts on a wide number of major human diseases, including atherosclerosis, hypertension, cancer, neurological disorders, diabetes, stroke, and hematological disorders. More recently, inflammation has emerged as a critical topic in vascular biology with major implications for vascular disease and with important intersections with vascular development and genetics, including in the emerging field of vascular regeneration. Vascular inflammation is an emerging topic in vascular biology. Vascular diseases with a large public health impact such as hypertension and atherosclerosis have significant inflammatory and immune components. In addition, vascular inflammation impacts on angiogenesis, stem cell biology, regeneration, and vascular differentiation. Therefore, the sixth NAVBO Developmental Vascular Biology Workshop, now including a genetic component, will be held concurrently with the first ever NAVBO Vascular Inflammation Workshop. This joint conference will bring together scientists with the common interest of understanding the process of blood vessel formation in development and how this information pertains to pathological states. The program was developed to include the latest unpublished information in traditional topics of interest in both o these fields and to integrate novel, emerging themes in each area and at the intersection of these previously diverse areas. In this application we request funds to partially support both of these interdisciplinary, international workshops, which will bring together investigators from the academic, and private sectors, post-doctoral fellows, and graduate students from diverse fields of study for four days of intense discussion and study. The conference will be held at the Asilomar Conference Grounds in Pacific Grove, CA from October 19-23, 2014. We have assembled a roster of speakers that reflects the leaders in developmental biology, genetics, and inflammation as those issues pertain to the vasculature. In addition, approximately half of the speakers at the workshops will be chosen from submitted abstracts in order to capture the latest unpublished work in these disciplines. The majority of abstract presentations will be from early stage investigators and trainees. It is our aim to foster a high-level exchange of ideas among a diverse group of investigators, including investigators directly involved in translational research. We have included joint sessions on translational biology and vascular therapeutics to facilitate interactions among researchers involved in basic and clinical/translational research. The conference seeks insight into possible new therapeutic approaches for amelioration of a broad spectrum of pathological states, including developmental defects, hypertension, cancer, myocardial infarction, and stroke.

Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiac muscle cells, increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance cardiomyocyte recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration is limited in adult mammals, but occurs naturally in adult zebrafish and neonatal mice through the activation of cardiomyocyte division. Whereas the research community has identified several factors important for heart regeneration over the past decade, we still know little of the regulatory mechanisms needed to activate regeneration programs in injured cardiac tissue. In particular, questions of whether regulatory enhancer elements are employed, and how specific they are to regeneration, are virtually unexplored. This is a critical deficiency, as the identification and manipulation of such elements could both expand our understanding of regeneration and have applications in regenerative medicine. In a recent collaboration between our groups, we found evidence for tissue regeneration enhancer elements (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs including the heart. Here, we propose a multi-PI project exploiting the strengths of the zebrafish and mouse model systems to delineate regulatory sequences that control regeneration programs, and to create TREE-based factor delivery constructs to optimize heart regeneration in higher and lower vertebrates. 1) We will define the cis-regulatory motifs and binding factors necessary for activity of a TREE that is linked to the zebrafish leptin b gene. 2) We will use this TREE to define effects of enhancer-delivered pro-regenerative factors after cardiac injury in zebrafish and mice, focusing initially on provision of mitogenic and angiogenic molecules. 3) We will use open chromatin profiling approaches to identify new TREEs that activate expression in endothelial and/or endocardial cell types during zebrafish heart regeneration. We will perform sequence comparisons in mice, and we will initiate a program to generate transgenic reporter and TREE deletion animals to test the sufficiency and requirements for these sequences in directing regeneration programs. With these approaches, we will test the hypothesis that cardiac injury activates regeneration enhancer elements to facilitate heart regeneration.

Vascular Biology 2017 Developmental Vascular Biology and Genetics Workshop (Organizers: Brian Black, UCSF and Victoria Bautch, UNC-Chapel Hill) and Vascular Matrix Biology and Bioengineering Workshop (Organizers: Craig Simmons, University of Toronto and Jessica Wagenseil, Washington University) October 15-19, 2017 Asilomar Conference Grounds, Pacific Grove, California Principal Investigator: Brian Black Project Summary/Abstract The field of vascular biology has experienced an explosion of information in recent years. In light of this wealth of information, there is an increased need for discussion and interdisciplinary exchange of ideas. Vascular biology has evolved over the past decade as a major cross-disciplinary field that impacts on a wide number of major human diseases, including atherosclerosis, hypertension, cancer, neurological disorders, diabetes, stroke, and hematological disorders. Many of the key cellular events that lead to these pathologies recapitulate processes that take place during vascular development, guided by genetics, vascular cells, the extracellular matrix, and biophysical forces. This important intersection between developmental biology, genetics, matrix biology, and bioengineering not only has major implications for vascular disease, but also for stem cell biology, vascular differentiation, and regeneration. The seventh NAVBO Developmental Vascular Biology and Genetics Workshop will be held concurrently for the first time ever with the NAVBO Vascular Matrix Biology and Bioengineering Workshop, now in its sixth presentation. This joint conference will bring together scientists with the common interest of understanding the process of blood vessel formation in development, how this information pertains to pathological states, and how it can be exploited for regeneration. The program was developed to include the latest unpublished information in topics of interest in both of these fields and to integrate novel, emerging themes in each area and at the intersection of these previously separate fields. In this application we request funds to partially support both of these interdisciplinary, international workshops, which will bring together investigators from the academic and private sectors, postdoctoral fellows, and graduate students from diverse fields of study for five days of intense discussion and study. The conference will be held at the Asilomar Conference Grounds in Pacific Grove, CA from October 15-19, 2017. We have assembled a roster of speakers that reflects the leaders in developmental biology, genetics, matrix biology, and bioengineering as they pertain to the vasculature. In addition, approximately half of the speakers at the workshops will be chosen from submitted abstracts to capture the latest unpublished work in these disciplines. The majority of abstract presentations will be from early stage investigators and trainees. It is our aim to foster a high-level exchange of ideas among a diverse group of investigators, including those directly involved in translational research. We have included joint sessions on bioengineering and vascular therapeutics to facilitate interactions among researchers involved in basic and clinical/translational research. The conference seeks insight into possible therapeutic approaches to ameliorate a broad spectrum of pathological states, including developmental defects, atherosclerosis, hypertension, cancer, myocardial infarction, and stroke.

7. PROGRAM SUMMARY Cardiovascular disease remains a major cause of morbidity and mortality in the U.S. and is increasing worldwide. Application of modern cell biology, genetics, 'omic and other technologies is producing remarkable progress in our understanding of basic processes related to cardiovascular diseases, and the need for broadly trained scientists who can adopt innovative technologies, assemble tools from different disciplines, and bridge basic and clinical science is greater than ever. The overall goal of UCSF Training Program in the Molecular and Cellular Basis of Cardiovascular Disease is to train investigators who will be at the cutting edge of cardiovascular research. Toward this end we: 1) Capitalize on the outstanding research environment of the CVRI and UCSF to provide multidisciplinary training in areas of signaling and cell biology, developmental biology, regeneration and congenital anomalies, ion channels and arrhythmias, vascular biology and atherothrombosis; metabolism, obesity and metabolic diseases; myocyte biology and heart failure; and genetics, biomarkers and disease prevention. (2) Attract graduates of top Ph.D. and M.D.-Ph.D. programs to cardiovascular research and (3) Provide opportunities for M.D.s in clinical fellowships at UCSF to obtain rigorous research training. The Program brings together a diverse and outstanding group of mentors with a common interest in cardiovascular biology within the Cardiovascular Research Institute at UCSF, a multi- departmental and multi-disciplinary research organization. It places trainees in remarkably productive and interactive laboratories. Multi-disciplinary affinity groups, largely co-located in the Smith Cardiovascular Research Building, provide collaborations and co-mentoring to promote acquisition of the knowledge and skills required for success. A major outpatient clinic for patients with cardiovascular diseases in the Smith building helps drive integration of laboratory- and patient-based research.

PROJECT SUMMARY/ABSTRACT PROJECT 3 Cardiovascular disease, including heart failure, is the most common cause of mortality in adults, and congenital heart defects are the most common form of birth defects in the US. An important concept that has emerged in recent years is that disruptions of cardiac transcription factor networks play important roles in congenital heart defects and in heart failure in adults. MEF2C is one of the core cardiac transcription factors and is required for cardiac development and for postnatal cardiac gene expression and homeostasis. MEF2C functions as signal responsive transcription factor that interacts with numerous co-regulator proteins to control gene expression, yet much remains to be determined about how MEF2C functions in the heart. The most potent transcriptional coactivator for MEF2C described to date is myocardin. MEF2C specifically interacts with a long isoform of myocardin (myocardin-935) to synergistically activate cardiac transcription. Preliminary studies identified a novel bridging mechanism whereby two myocardin-935 molecules interact with MEF2C and with each other via a leucine zipper (LZ) dimerization motif to cooperatively activate paired MEF2 sites, supporting a central role for myocardin dimerization for activation of MEF2-dependent cardiac genes. Furthermore, in silico analyses of cardiac enhancers suggests that paired MEF2 sites are prevalent and occur more frequently than predicted by chance specifically in cardiac enhancers. However, whether these enhancers are bona fide targets of the MEF2C-myocardin complex and how myocardin dimerization influences activity of these enhancers and gene expression in vivo remains to be determined. Additional, unpublished preliminary studies have identified a family with congenital heart defects likely caused by an in-frame microdeletion that results in loss of the myocardin LZ dimerization motif. Similarly, unpublished work shows that mice with an analogous deletion the leucine zipper domain of myocardin die with congenital heart defects. This project will test the hypothesis that the MEF2C-myocardin complex recruits a larger transcriptional coregulatory complex, which is influenced by upstream signaling and myocardin dimerization to regulate cardiac gene expression. To test this overall hypothesis, this project will define the interaction partners of MEF2C and myocardin in the embryonic and adult heart and will identify the phosphorylation sites and other modifications on MEF2C and myocardin. This will provide critical insight into the post-translational regulation of these key cardiac transcription factors. This project will also utilize RNA-seq, ChIP-seq, and other genome- wide approaches from embryos and endogenous tissues to identify transcriptional targets of the MEF2C- myocardin complex and will determine how myocardin dimerization influences the complex and downstream gene expression. Finally, this project will determine the requirement for myocardin dimerization for heart development in vivo by examining the lethal heart development phenotype in myocardin leucine zipper mutant mice and will identify gene expression changes associated with loss of myocardin dimerization.

PROJECT SUMMARY/ABSTRACT A major translational hurdle for hematopoietic stem cell (HSC) biology is the in vitro generation of bona fide, transplantable hematopoietic stem cells. As adult bone marrow HSCs are relatively quiescent, and not physiologically required to expand in large numbers, attempts at in vitro expansion have resulted in limited self- renewal, inability to provide multi-lineage engraftment, and cell death. An alternative approach has been to reprogram cells with early embryonic attributes to HSCs, but many times the cells generated are too immature and unable to provide long term engraftment. To improve current attempts of generating HSCs in vitro, it is imperative we understand how HSCs are initially formed in the embryo. It is now well accepted that the precursor to an HSC is a specialized hemogenic endothelial cell, that undergoes an endothelial to hematopoietic fate transition. While the first HSCs arise from an endothelial-to-hematopoietic (EHT) transition within a subset of endothelial cells, during a narrow window of development, it is still unclear why certain endothelial cells undergo the transition and not others, despite similar gene expression patterns and anatomic location; and it is also unclear why the process only occurs during a narrow developmental time window. While the factors regulating EHT are beginning to be elucidated, the production of HSCs solely from endothelium has yet to be achieved, suggesting there may be other cell requirements yet to be discovered. One major well studied hemogenic vascular site is the dorsal aorta, where in addition to hemogenic endothelial populations, there exists an extensive mesenchymal cell population underlying the hemogenic vasculature. Due to the previous lack of restricted markers for the mesenchymal layer, studying the contribution of this compartment to hemogenic endothelium and HSC emergence has not been previously possible. Recently we have identified a mural cell marker that is specifically expressed in mesenchymal layer during the hemogenic window. Capitalizing on this new insight, we plan to fully examine the role of the sub-aortic mesenchyme in the development of HSCs from the endothelium. We hypothesize that the underlying mesenchyme plays a regulatory role in controlling hemogenic endothelial cell number and conversion to hematopoietic fate. This work will shed light on the critical steps in HSC emergence and specification, laying the groundwork for large- scale in vitro generation of HSCs.