Larry Gerace, Ph.D. - US grants

Nuclear pore complexes (NPCs) cary out signal-mediated transport of proteins and RNAs between nucleus and cytoplasm, a process that is fundamentally important for control of gene expression and cell metabolism. At the present time, nuclear transport mechanisms are just beginning to come under molecular scrutiny. Developing a detailed understanding of these processes is likely to provide the potential for new therapeutic approaches to a wide spectrum of human diseases caused by microbial pathogens and genetic disorders. The long term goal of this project is to understand mechanisms of nuclear protein import at a biochemical and molecular level. This project period will involve two NPC proteins, Tpr and the p62 complex, which are linked to ligand docking at the NPC and the central gated channel, respectively. The studies also will concern several cytosolic factors required for nuclear import, including the GTPase Ran, which may be a key regulator of nuclear transport, and cytosolic factors that bind to O-linked NPC glycoproteins, including the p62 binding factor NTF2. High resolution EM analysis will be carried out on Tpr and the p62 complex, and the 3 uncharacterized subunits of the p62 complex will be molecularly cloned. Binding partners in the NPC for Ran and p62 will be identified and molecularly characterized, as will cytosolic transport factors that interact with Tpr and O-linked NPC glycoproteins. With a combination of antibodies, binding competitors and dominant negative mutants, the functions of this array of components in nuclear protein import will be examined using an in vitro assay for nuclear import involving permeabilized cells, and in vivo cultured cell systems manipulated by microinjection. Furthermore, the locations of these proteins and their associated transport steps within the 3-D structure of the NPC will be analyzed. Together, these investigations should substantially expand understanding of the series of steps responsible for signal-mediated transport through the NPC.

The nuclear lamina, a filamentous protein meshwork lining the nuclear envelope (NE), contains a polymer of lamins and associated transmembrane proteins of the inner nuclear membrane. The importance of the lamina in cellular functions is underscored by the findings that mutations in the genes for lamins A/C and lamina-associated transmembrane proteins cause over 15 inherited human disorders ("laminopathies"), including many dystrophies that affect heart and skeletal muscle. The long-term objectives of this project are to promote an understanding of the lamina's role in cell organization and functions, and its involvement in human diseases. Recently, 67 novel putative NE transmembrane proteins (NETs) have been identified in a comprehensive proteomics analysis of the NE. Expression profiling has revealed that 6 of these NETs are significantly up-regulated during myoblast differentiation and are expressed at high levels in adult skeletal muscle relative to other tissues, indicating an important role in muscle development. This proposal will focus on a 3 of these NETs, which have been validated to be authentic NE proteins, and which have putative roles in signaling based on their homologies to known proteins. The specific aims are to: 1) Carry out RNAi- mediated gene silencing in a myoblast differentiation model, and molecularly analyze the phenotypic defects that may arise related to differentiation, signaling responses related to muscle regulation, or NE structure. 2) Biochemically analyze specific NET functions suggested by their sequence homologies, and immunolocalize the endogenous NETs at the light and EM levels. 3) Implement MudPIT proteomic analysis of NET- containing protein complexes solubilized from cells to identify other NE proteins that interact with the NETs, and use in vitro binding studies to extend the results of the proteomics. This work is expected to provide a new level of insight on the NE as a signaling platform in the nucleus, and to promote an understanding of how mutations in NE and lamina proteins cause human diseases, particularly diseases affecting striated muscle.

The long term goal of this project is to contribute to understanding how structures and components involved in cell reproduction are regulated by the cell cycle engine. This research is based on the use of MPM-2, a monoclonal antibody recognizing a phosphorylated epitope found on a large number of proteins during M phase, which are putative targets of cell cycle regulation. In the previous project period, cDNA clones encoding portions of 11 novel MPM-2 antigens were isolated. In this period, detailed analysis is planned for three of these proteins. These antigens are localized to the centrosome (MPP3), DNA replication centers (MPP7) and the Golgi apparatus (MPP9), which are important regulatory targets of the cell cycle machinery in interphase and/or mitosis. cDNA clones yielding full length coding sequences of MPPs3, 7 and 9 will be isolated and sequenced, and used to prepare antibodies for biochemical, localization, and functional studies. Phosphorylation and expression of MPPs3, 7 and 9 through the cell cycle will be analyzed by metabolic labeling and peptide mapping, and cdk-cyclin complex(es) responsible for phosphorylation of these components at discrete periods of the cell cycle will be analyzed with in vitro and in vivo studies. Functions of MPPs3, 7 and 9 in the centrosome, DNA replication centers and Golgi will be investigated by in vivo approaches including microinjection of antibodies and antisense oligonucleotides, and expression of antisense RNA, ribozymes and putative dominant negative mutants in cultured cells. In vitro functional studies will include cell-free assays for microtubule nucleation by the centrosome, DNA replication and membrane trafficking. Finally, interaction partners of these proteins will be determined by biochemical methods and a yeast two- hybrid system to provide a basis for further functional understanding. Together, these studies are expected to provide important insight on how some major features of cell organization are regulated by the cyclin-cdk system.

The long-term objectives of this work are to understand the molecular and structural organization of the nuclear lamina and to determine how this is important for nuclear function. The lamina consists of a polymer of intermediate-type filament proteins called lamins, which directly bind to chromatin, and a number of more minor lamina-associated polypeptides. Work in this project period will be focused on analysis in mammalian cells of two lamin-binding integral membrane protein of the inner nuclear membrane, LAP1 and lAP2. These are good candidates for proteins involved in attachment and assembly of lamins at the inner nuclear membrane. The project also will involve detailed functional analysis of the interaction of lamins with chromatin. A first aim is directed at dissecting the molecular interactions of LAP1 and LAP2. This ill involve characterization of the region of LAP2 that interacts with chromosomes, the chromosome component that binds to LAP2, and the regions of lamins and LAP1 that associate. A second aim will examine the roles of lAP1 and LAP2 in nuclear envelope and chromosome organization by functional studies in cultured cells. This will involve expression of mitotic phosphorylation site mutants of lAP2, microinjection of antibodies to LAP1 and lAP2, downregulation of LAP1 and lAP2 expression by antisense approaches, and expression of putative dominant negative mutants of lAPs. A third aim will examine possible roles of LAP1 and lAP2 in modulating nuclear lamina structure by in vitro assembly studies with purified lamins and lAPs. A fourth aim will involve in vivo functional analysis of the lamin-chromosome interaction by injection of antibodies into cultured cells and long-term expression of lamin mutants deficient in chromatin binding that may affect genome stability. Together this set of approaches is expected to significantly advance current understanding of the nuclear lamina, and contribute to knowledge of how aberrations in nuclear organization can promote human disease states such as cancer.

Nuclear import of proteins that is mediated by classical nuclear localization sequences (NLSs) involves the formation of a complex containing the substrate and importin alpha/beta in the cytoplasm, followed by the stepwise movement of this complex through the nuclear pore complex (NPC) to the nuclear interior. The small GTPase Ran plays an important but poorly understood role in this process. The long term goal of this work is to understand the molecular basis for signal mediated import through the NPC, and how this is coordinated with other cellular processes. This project will involve a detailed analysis of a number of key issues related to NLS-mediated nuclear import. First, the functions of Ran in nuclear import will be analyzed by electron microscopy of staged in vitro import assays involving gold-coupled substrate, as well as by biochemical and functional analysis of the interactions of Ran during discrete transport steps. Second, the interactions of importin beta during movement of a transport complex through the NPC will be analyzed by in vitro binding studies with discrete nucleoporins, by investigation of transport arrests obtained with importin beta mutants, and by biochemical analysis of the binding partners of importin beta under different conditions of transport arrest. Third, the functions of specific nucleoporins in the nuclear import pathway will be examined in permeabilized cell assays with domain-specific inhibitory antibodies and with cells expressing dominant negative mutants of nucleoporins. Since nuclear transport is integrally involved in gene expression, this project has direct relevance to a number of human health issues including cancer, viral pathogenesis, immunity and aging.

DESCRIPTION (provided by applicant): Adenoviruses are non-enveloped DNA viruses with an -36 kb genome. In humans, adenoviruses cause a significant number of gastrointestinal and respiratory infections. They also are a major cause of viral conjunctivitis, including epidemic keratoconjunctivitis (EKC), a condition that can threaten long-term visual function and for which there is no effective treatment. In addition, adenoviruses are being intensively investigated as vectors for human gene therapy because of their broad tissue tropism. Although significant insight has been obtained on how adenovirus penetrates the cell to reach the cytoplasm, little is known about the molecular mechanism of nuclear import of the adenovirus genome, which is critical for virus reproduction. This proposal is directed at obtaining detailed molecular insight on adenovirus DNA import. The aims are: 1) The mechanism for docking of adenovirus to the nuclear pore complex will be investigated, focusing on an analysis of the adenovirus hexon protein and its interaction with specific nucleoporins. 2) The role of protein VII in the transport of adenovirus DNA through the nuclear pore complex will be analyzed, and the possibility that protein VII can be used as a nonviral method for achieving efficient gene transfer will be investigated. 3) The role of cytosolic factors, including hsc70 and its cofactors, in virus uncoating at the pore complex and in DNA import, will be analyzed. Considered together, this work will provide a valuable model for understanding the nuclear import of the genomes of pathogenic DNA viruses. The work also could potentiate the development of new therapies for EKC in humans. Finally, it could provide the basis for developing efficient means for nonviral gene transfer, which would be useful for gene therapy and functional studies of cells.

Replication of the human immunodeficiency virus type-1 (HIV-1) in the nucleus requires the virus-encoded regulatory protein Rev. Because of its key role in the viral life cycle, Rev is an attractive therapeutic target for small molecules. Rev binds to a structured RNA element, termed the Rev Response Element (RRE), which is found in the unspliced and singly spliced HIV mRNAs that encode the Gag, Pol and Env proteins. The binding and multimerization of Rev at the RRE recruits the nuclear export receptor Crm1, and as a consequence induces export of the HIV mRNAs to the cytoplasm to allow their translation. It is known that Crm1 and the small GTPase Ran are involved in Rev-mediated viral mRNA export, and a number of other potential export factors have been described as well. However, the composition and assembly of the nuclear export complex based on the Rev/RRE interaction remain poorly understood. The goals of this project are to investigate these issues in detail, using a combination of biochemical and functional approaches. The specific aims are: 1) Proteomic and in vitro binding assays will be used to analyze the nuclear export complex formed on the Rev oligomer associated with the RRE; 2) A permeabilized cell assay will be used to analyze the soluble factors involved in nuclear export of an HIV-derived mRNA containing the RRE; 3) In vivo functional studies will be carried out to analyze potential factors involved in mRNA export mediated by the Rev-RRE complex. The other investigators in this program will study in detail the Rev-RRE interaction and Rev multimerization, and will attempt to identify small molecule inhibitors of these associations. In turn, we will test promising compounds for their effect on HIV mRNA export in cells. This work is expected to lead to a greater understanding of how Rev mediates HIV mRNA export, and could help to identify promising lead compounds for drug development.

[unreadable] DESCRIPTION (provided by applicant): Trafficking of proteins and RNAs between the nucleus and the cytoplasm is a fundamental process of eukaryotic cells, which is required to segregate and coordinate the functions of the nuclear and cytoplasmic compartments. Nuclear transport is mediated by nucleocytoplasmic shuttling receptors of the karyopherin (importin/exportin) superfamily. Karyopherins interact with nucleoporins to translocate protein and RNA cargoes across the nuclear envelope, and are regulated by the small GTPase Ran. Up to now, only one class of small molecule inhibitors of the nuclear transport machinery has been described, which targets the exportin Crm1. The goal of this project is to develop and validate a high-throughput screening (HTS) assay involving permeabilized cells, to discover additional small molecule inhibitors that target the nuclear import machinery. The focus will involve the prototypical nuclear transport receptor importin beta, and two of its cargoes whose import involves different conformations of this receptor. The screen will use automated data acquisition and processing to measure the accumulation of fluorescently labeled cargoes in the nucleus of permeabilized cells, which are cultured in 96-well plates. It is expected that transfer of this assay to a 384-well format, with robotic handling of all reagents, will be straightforward. Since nuclear import requires multiple protein-protein interactions involving importin beta and Ran, the permeabilized cell screen is high content. Nonetheless, straightforward protocols for validation of hits and target identification are feasible and will be described. In a pilot study, the assay will be used to screen approximately 5000 compounds from a combinatorial library of peptidomimetic small molecules, to evaluate whether peptidomimetic libraries can yield useful inhibitors of nuclear transport. Transport of protein and RNAs between the nucleus and the cytoplasmic is critical to the proper functioning of all human cells, and can be perturbed in pathological conditions. This work is expected to provide new pharmacological tools to investigate the functioning of the nuclear- cytoplasmic transport machinery, and to contribute information that will promote the development of new avenues for treatment of human diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cholesterol plays a key role in humans and many other organisms, by serving as a structural element of membranes and by providing a precursor for steroid hormones. Since elevated LDL-associated serum cholesterol is linked to greatly increased risk of cardiovascular disease in humans, understanding the molecular basis for cholesterol homeostasis is of major importance. The SREBP-2 pathway controls cholesterol synthesis and uptake by cells. Core components of the cholesterol sensing machinery in the endoplasmic reticulum (ER) that regulate the activation of SREBP-2 have been characterized in detail. In contrast, little is known about certain other aspects of their regulation, including whether these components are regulated by protein/lipid nanodomains in the ER. Erlin-1 and erlin-2 are closely related ER proteins that fractionate with cholesterol-rich, detergent-resistant membranes. Erlins previously have been linked to ER-associated degradation of the IP3 receptor. Based on recent biochemical and functional results linking erlins to the SREBP-2 pathway, this project will investigate the hypothesis that erlins organize cholesterol-rich, membrane raft-like nanodomains in the ER that are important for regulation of cholesterol biosynthesis. The work will generate erlin-2 mutants that are predicted to be deficient in organizing these nanodomains. These mutants will be analyzed for their ability to partition into a detergent-resistant raft fraction, and to complement the loss of sterol-sensitive regulation of SREBP-2 that is induced by the silencing of endogenous erlins. Additional work will analyze the ability of recombinant wild-type erlin-2 and erlin-2 mutants deficient in raft formation to directly bind cholesterol and ceramide, and will investigate the components of the SREBP-2 machinery that are nearest neighbors of erlins by crosslinking and mass spectrometry. Together, these studies are expected to delineate major features of erlins that underlie their role in organizing ER rafts and in regulating the SREBP-2 pathway. Thus, the work should shed light on a previously unrecognized mechanism involving ER nanodomains that may be crucial for regulation of cholesterol biosynthesis. PUBLIC HEALTH RELEVANCE: Although cholesterol plays a key role in the structure and functions of cells, elevated LDL- associated serum cholesterol is linked to greatly increased risk of cardiovascular disease in humans. This project will analyze new cellular mechanisms implicated in regulating cell cholesterol. This work is relevant for understanding and controlling cholesterol-related cardiovascular diseases.

DESCRIPTION (provided by applicant): A broad spectrum of human diseases (laminopathies) is caused by mutations in components of the nuclear lamina, a protein meshwork that lines the nuclear envelope. Many of these disease mutations affect heart and skeletal muscle. The prototype laminopathy is Emery-Dreifuss muscular dystrophy (EDMD), which causes dilated cardiomyopathy with conduction defects (DCM-CD) and dystrophy of skeletal muscles, and is caused by mutations in either lamin A or in inner nuclear membrane (INM) protein emerin. While the molecular basis for laminopathies is unknown, proximal causes are speculated to involve defects in cell signaling. EDMD models show elevated ERK1/2 signaling in heart, a pathway known to be linked to pathological cardiac hypertrophy. The broad, long-term objectives of this work are to understand how components of the nuclear lamina regulate signaling and contribute to normal and pathophysiological processes in heart and skeletal muscle. The current project is aimed at analyzing two INM proteins associated with the nuclear lamina that were recently shown to regulate signaling in myoblast differentiation, Lem2 and Net37. Lem2 (gene name, Lemd2) is involved in attenuation of ERK1/2 signaling, and Net37 is needed for secretion of IGF-II, a factor essential for autocrine signaling in myogenesis. The work will use cultured myoblasts to analyze how these INM proteins control signaling at the molecular level, and mouse models to understand their functions in heart and skeletal muscle and their potential relevance to diseases. Aim 1 will dissect the regions of Lem2 required for ERK attenuation, identify potential ERK regulators that interact with these regions by proteomics, and functionally analyze these components in myoblast differentiation. Aim 2 will evaluate whether Lemd2 can functionally compensate for loss of Emd in mouse, by analyzing animals with a hemizygous Lemd2 gene trapped allele in an emerin-null background. Also, a floxed allele of Lemd2 will be constructed for tissue-specific knockouts in adult heart and skeletal muscle, to evaluate the importance of Lemd2 in cardiac maintenance and in skeletal muscle function and repair. Aim 3 will dissect the regions of Net37 required for IGF-II secretion and myoblast differentiation, analyze how the putative glycosidase activity of its luminal domain is involved in IGF-II folding and secretion, and determine whether Net37 and IGF-II have distinctive functions in muscle regeneration in mouse after silencing the endogenous genes by AAV vectors. It also will investigate whether the binding of signaling factors to the nucleoplasmic side of Net37 regulates the activity of its luminal domain. Overall this project is expected to shed significant new light on the molecular functions of Lem2 and Net37, and the results are predicted to provide insight on their functions in normal heart and skeletal muscle and in pathophysiological processes in these tissues.

? DESCRIPTION (provided by applicant): The nuclear lamina is a protein meshwork lining the nucleoplasmic surface of the nuclear envelope (NE) that contains a polymeric core of nuclear lamins and associated transmembrane (TM) proteins. The lamina has a fundamental role in organizing chromatin structure and in controlling signaling and gene expression. Over 20 human genetic disorders are caused by mutations in lamina proteins, mostly in lamins A/C and in TM proteins associated with the lamina. These laminopathy diseases predominantly affect muscle, bone and adipose tissue, which are formed from mesenchymal progenitors. A systematic analysis of the NE/lamina proteome of cells of bone and adipose tissue has not yet been carried out. Osteoblasts and adipocytes differentiate from mesenchymal stem/stromal cells (MSCs). The choice between these alternative cell fates is specified by signaling pathways that induce distinctive transcription factor programs. Many of the transcription factors that promote one pathway antagonize the other, including the central regulators of osteoblast differentiation-Runx2 and Osterix, and the core factors for adipogenesis-PPAR? and C/EBP family members. Based on preliminary studies and the results of previous proteomics, a distinctive profile of nuclear membrane proteins arises in a cell type-specific manner during differentiation. This project will test the hypothesis that TM proteins of the NE that are differentially induced in osteoblast vs. adipocyte differentiation are integral parts of the signaling networks for generatio of each these lineages. Aim 1 will use proteomics analysis to systematically compare the profile of NE TM proteins of adipocytes and osteoblasts that emerges during differentiation. Aim 2 will use RNAi screens to investigate the role of lineage-selective NE proteins in adipocyte vs. osteoblast differentiation, and to identify different transcription factor pathways that are linkedto specific proteins. This work is expected to delineate how the changing composition of the nuclear lamina during differentiation of mesenchymal progenitors promotes the alternative osteoblast and adipocyte fates. This insight could enhance an understanding of laminopathies involving skeletal tissue. More generally, the work will be relevant to understanding skeletal maintenance in pathophysiological conditions and aging, since continued differentiation of mesenchymal progenitor cells into adipocytes, chondrocytes and osteoblasts is an ongoing process throughout adult life.

? DESCRIPTION (provided by applicant): Intranuclear compartments make essential contributions to chromosome structure and function. One such compartment is organized by the nuclear lamina (NL), a protein meshwork that lines the nucleoplasmic surface of the nuclear envelope (NE) in metazoan cells. The NL contains a polymeric assembly of nuclear lamins associated with a large number of transmembrane and peripheral proteins. In addition to serving as a structural framework for the NE, the NL modulates numerous signaling and transcriptional pathways, and provides a tethering and regulatory site for chromatin at the nuclear periphery. The nuclear lamina and associated peripheral heterochromatin can be viewed as comprising a distinctive nuclear compartment, herein termed the nuclear lamina/peripheral chromatin (NL/PC) compartment. Although various components of the NL have been described, the analysis has not been comprehensive. Moreover, approaches are underdeveloped for defining its compositional changes during cell differentiation, and for elucidating the protein-protein interactions that specify the NL network and its chromatin interactions. To address these fundamental issues, this project aims to develop methods to isolate sub-nuclear fractions enriched in the NL/PC compartment from mesenchymal stem cells (MSCs), and from myocytes and adipocytes differentiated from these cells. With the use of these fractions, systematic mass spectrometry (MS) methods will be developed to characterize the protein composition of the NL/PC compartment and to quantitatively chart its changes during differentiation. Chemical crosslinking (XL) combined with MS will be deployed to map the interactions of constituent proteins, and biochemical methods in tandem with super-resolution light microscopy will be used to validate these associations. From implementation of this complementary set of approaches, the project will provide a comprehensive reference map of the NL and the changes that it undergoes during differentiation of defined MSC lineages. This will potentiate the formulation of more general concepts on the functions of the NL/PC compartment, and will identify new components and nodes that can be targeted in future functional studies. This is expected to substantially advance our understanding of the role of the NL in cell-type-specific regulation of peripheral heterochromatin and gene expression.

The nuclear lamina (NL) is a protein meshwork lining the nuclear envelope (NE) that contains a polymer of nuclear lamins and associated proteins, including transmembrane proteins of the inner nuclear membrane (INM). The NL provides a scaffold for the NE, regulates its mechanical and dynamic properties, and modulates the activity of signaling and transcriptional pathways. The NL has well-recognized importance in the biology of higher eukaryotes, as underscored by the association of over 15 human genetic diseases with mutations in NL proteins. Although major proteins and general properties of the NL have been characterized in animal and cultured cell models, a detailed molecular understanding of how specific NL components control nuclear functions is only beginning to be developed. This project builds on the finding that the NL protein Lem2, encoded by the Lemd2 gene, regulates three major MAP kinase (MAPK) families in cells, ERK, p38 and JNK. Work demonstrated that Lemd2 is vital for mouse embryogenesis and heart development, that disruption of Lemd2 in adult liver results in metabolic derangements, and that knockout in skeletal muscle causes a dystrophic phenotype. The project is focused on obtaining a molecular understanding of how Lem2 controls MAPK signaling in cells and adult tissues of mouse. One Aim is directed at understanding the mechanisms by which Lem2 regulates the activity of ERK, p38, and JNK MAPKs using Lemd2-null mouse embryo fibroblasts (MEFs) reconstituted with Lem2 mutants. Potential effectors of ERK regulation by Lem2 will be identified by candidate and discovery-based approaches, and the validity of candidate effectors will be examined with a battery of functional tests. The findings on ERK regulation will be extended to an analysis of the related p38 and JNK MAPKs. A second Aim involves studying the functions of Lem2 in the biology of liver and skeletal muscle using mouse strains with tissue-specific knockouts of the Lemd2 gene. The metabolic defects with a liver-specific disruption of Lemd2, which are correlated with elevated p38, will be characterized in detail. These results will be applied to mechanistic analysis of Lem2 functions related to glucagon signaling in primary hepatocytes from Lemd2 liver-knockout animals. In separate studies, the dystrophic phenotype in a mouse strain with a skeletal muscle-specific disruption of Lemd2 will be characterized by analysis of muscle physiology and signaling pathways. The work will be directed at understanding how aberrant signaling caused by loss of Lem2 gives rise to muscle defects. Altogether, these studies should afford refined molecular insight on how signaling is controlled by a specific NL protein- Lem2- and how defects in this control become manifest in different mouse tissues.

Sphingolipids (SLs) are essential structural components of membranes and provide reservoirs for critical bioactive lipids, including ceramide and sphingosine 1-phosphate (S1P). Whereas ceramide is associated with apoptosis and growth arrest, S1P commonly promotes survival and proliferation. The best- characterized targets of S1P are S1P receptors localized to the plasma membrane. S1P also can act in the nucleus by binding to and inhibiting class I histone deacetylases (HDACs), which are major regulators of gene expression. SL signaling is characterized by highly compartmentalized generation and targeting of the bioactive molecules. Substantial insight on localized S1P production and targeting at the plasma membrane has been obtained, but little is known about regulation of SL metabolism in the nucleus. This project involves investigation of proteins with potential roles in generating bioactive SLs in the nucleus. The work includes analysis of a neutral sphingomyelinase localized to the nuclear envelope (NE) that is required for myoblast differentiation, which may be integral to nuclear SL metabolism. This component and other enzymes with potential links to nuclear SL metabolism will be analyzed in a cultured myoblast differentiation model. Subcellular fractionation and lipidomics will be used to characterize the role of these proteins in regulating the dynamic SL landscape of the nucleus during myoblast differentiation, and in producing bioactive SLs including S1P. In addition, the potential functions of S1P in activating expression of myogenic genes as an inhibitory ligand for class I HDACs will be evaluated. Overall this project will investigate the hypothesis that the nucleus houses a distinctive SL metabolism that has a major role in myogenic differentiation. The work has clear relevance to muscle pathology in humans, since HDAC inhibitors including S1P have been shown to ameliorate muscular dystrophies in animal models.