Stephen Tapscott - US grants

The existence of intermediate filament (IF) protein insoforms with characteristic cell-type distributions suggests either a cellular function or mechanism of regulation that is specific for each protein. Studies of IF protein function and regulation have been limited, however, by the resistance of IF to depolymerization and biochemical manipulation. Recent advances in recombinant DNA technology have shown that the introduction, into the nucleus or cytoplasm, of nucleic acid sequences coding for the gene (sense message), or for a sequence complementary to the gene (anti-sense message), can either enhance or inhibit, respectively, the amount of translation competent mRNA available to the cell, thereby simulating a dominant "mutant" phenotype that can be used to analyze gene or protein function. Our aims are (i) to use recombinant DNA techniques to enhance or inhibit the synthesis of individual IF proteins in single cells and cell populations, and (ii) to use these genetically engineered cells to study the function and regulation of IF proteins in nerve and muscle. PBR322 plasmids engineered to direct IF protein transcription, or to direct anti-sense transcripts of IF oligonucleotides will be introduced into chondrocytes, presumptive myoblasts, and neurons either by microinjection or transfection. These cells will then be used for biochemical and immunohistochemical analysis of cytoskeletal protein synthesis and distribution, as well as morphological and ultrastructural analysis. Areas of particular interest will be the contribution of desmin to myofibril integrity; the ability of mature neurons to transport and degrade vimentin; the role of neurofilaments in neurite stability; and the role of IF in cell viability and differentiation. Many studies have reported the abnormal accumulation of IF proteins in human pathology: e.g., desmin in inherited myopathies, neurofilament proteins and vimentin in the neurofibrillary tangles of Alzheimer's disease, and neurofilaments in motor neuron disease; however, the current ignorance of normal IF regulation and function makes these findings difficult to interpret. For this reason, the emphasis of this proposal on the function and regulation of IF proteins in neurogenesis and myogenesis should prove valuable for future studies of development and degenerative neurologic disorders that may relate to IF protein dysfunction.

DESCRIPTION (provided by applicant): The combination of microarray analysis of gene expression and molecular studies of transcription-factor activity are beginning to reveal the circuitry of gene expression networks. We have shown that the myogenic transcription factor, MyoD, functions in an instructive chromatin context and directly regulates genes that are expressed throughout the myogenic program, achieving promoter-specific regulation of its own binding and activity through a feed-forward mechanism. This gives us the opportunity to deconstruct the myogenic program into discrete molecular regulatory phases, similar to the cellular compartments identified in hematopoietic cell development. The broad and long-term objective of the parent application is to apply the knowledge we have gained from our studies of gene regulation in myogenesis to identify the molecular defect(s) in the differentiation program of rhabdomyosarcomas. We have used our model systems of MyoD-mediated regulation of gene expression to demonstrate that a dynamic balance between MyoD and MSC arrest the rhabdomyosarcoma cells in an unstable transitional state that normally occurs during myoblast growth and differentiation. This leads to a new broad hypothesis regarding transitional cell states: The regulative growth phase is an unstable state maintained by a dynamic balance between antagonistic factors that bind an overlapping network of genes, such as MyoD and MSC in skeletal myogenesis. The goal of this Revision application to the parent is to identify the systems wide regulatory interactions between MyoD and MSC. This will provide new insight into the network mechanisms that regulate transitional states in mammalian cell differentiation.

DESCRIPTION: Myotonic dystrophy (DM) is caused by the expansion of a CTG repeat. The repeat is in the 3' end of the dystrophia myotonia protein kinase gene (DMPK) and immediately 5" of the dystrophia myotonia associated homeobox protein (DMAHP) gene. It is still unknown whether the altered expression of either of these genes contributes to the DM phenotype. While a large amount of work from other labs has demonstrated that the CTG expansion affects the processing of the DMPK transcript, work in my lab has demonstrated that expression of the adjacent DMAHP gene is suppressed by the expanded repeat. Th investigator's have demonstrated that a well-defined hypersensitive site is positioned between the repeat and the promoter of the DMAHP gene. They have shown that the hypersensitive site contains transcriptional enhancer elements and the activity of these elements correlate with expression of the DMAHP gene suggesting that expansion of the repeat both eliminates the hypersensitive sit and suppresses expression of the adjacent DMAHP gene. These data support the broad hypothesis that the hypersensitive site is critical for the normal regulation of gene expression at the myotonic dystrophy locus, and that suppression of factor access to this region contributes to the myotonic dystrophy phenotype. The broad goals of this application are to characterize the regulatory and structural elements at the myotonic dystrophy locus and determine whether they have a role in the pathogenesis of myotonic dystrophy. The specific aims of the application are to (1) characterize the regulatory elements of hypersensitive site enhancer and DMAHP promoter; (2) characterize the elements at the locus that establish nucleosome phasing and maintain the hypersensitive site; (3) determine whether inactivation of the DMAHP gene or the hypersensitive site enhancer contributes to the phenotype of myotonic dystrophy. The significance of the proposal is that this application will characterize the elements that regulate gene expression at the myotonic dystophy locus and determine the role of these elements in the pathophysiology of myotonic dystrophy, ultimately leading to the rational design of therapies.

DESCRIPTION (Adapted from Applicant's Abstract): Members of the neurogenic basic-Helix-Loop-Helix (bHLH) gene family are expressed at different periods o neuronal determination and differentiation. The broad hypothesis of this application is that specific physical properties of each neurogenic bHLH protein confer a distinct function during neuronal development. The long term goal is to understand the molecular mechanism of generating specific neuronal phenotypes. The work proposed in this application will identify the special role of neuroD2 in neuronal differentiation and the physical mechanism of fulfilling that role. The specific aims will: (i) identify the mechanism of regulating the activity of the neuroD2 transcription factor; (ii) determine whether regulation of the activation domain or regulation of DNA binding controls neurogenesis by neuroD2; and (iii) identify the role of neuroD2 in Purkinjie cell differentiation. The significance is that understanding the molecular basis of neuronal diversity will provide the foundation for the rational manipulation of cell phenotype for the treatment of human disease. Th health relatedness of this work is that the ability to generate neurons and neuroendocrine cells will be critical for the treatment of numerous human diseases.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Myogenesis as part of the Gordon Research Conference series to be held in I1 Ciocco, Italy, May 16-21, 2004. The broad and long term goal of the conference is to increase our understanding of the fundamental mechanisms of normal muscle biology and disease, with an emphasis on skeletal muscle but with strong comparisons to cardiac and smooth muscle. The specific aims of this meeting will be to convene 45 speakers that represent critical areas of muscle research with a total of 135 participants for a five day conference in a relatively isolated setting. The program will have a keynote address and nine sessions that broadly address current issues in somitogenesis, the generation of muscle cell types (skeletal, cardiac, and smooth), regeneration of muscle by muscle stem cells and satellite cells with critical comparison to the regeneration of complex body parts, signal transduction and cell cycle in normal muscle development and disease, and finally a direct discussion of human disease therapy. In addition, two evening poster sessions will permit broad contribution to these topics. The significance of this application is that the Gordon Research Conference on Myogenesis, scheduled to convene every three years, is a critical component of the yearly series of conferences that propel research in the international community of muscle researchers. The health relatedness of this application is that the discussions will define the questions that require experimental resolution in areas that affect human development, skeletal muscle development and disease, cardiac development and disease, smooth muscle development and disease, and cancer of these muscle types.

Abstract: This proposal requests partial support for an international meeting on Myogenesis as part of the Gordon Research Conference series to be held in Il Ciocco, Italy May 13-18, 2007. The broad and long term goal of the conference is to increase our understanding of the fundamental mechanisms of normal muscle biology and disease, with an emphasis on skeletal muscle but with strong comparisons to cardiac and smooth muscle. The specific aims of this meeting will be to convene 45 speakers that represent critical areas of muscle research with a total of 135 participants for a five day conference in a relatively isolated setting. The program will have a keynote address and eight sessions that broadly address current issues in embryonic development, the generation of muscle cell types (skeletal, cardiac, and smooth), regeneration of muscle by muscle stem cells and satellite cells, signal transduction in normal muscle development and disease, a session devoted to human muscle disease, and a session devoted to cancers and muscle. In addition, two evening poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference on Myogenesis is a critical component of the yearly series of conferences that propel research in the international community of muscle researchers. The health relatedness of this application is that the discussions will define the questions that require experimental resolution in areas that affect human development, skeletal muscle development and disease, cardiac development and disease, smooth muscle development and disease, and cancer of these muscle types.

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) in both humans and dogs is a fatal, X-linked, recessive muscle disease caused by lack of dystrophin due to deletions or mutations in the dystrophin gene. Adeno- associated virus (AAV)-mediated delivery of micro-dystrophin to skeletal muscle has been successful in mice, however, recent studies indicate that the efficacy of AAV-mediated therapies might be limited by an immune response to viral capsid proteins in humans. By direct intramuscular injection of AAV vectors in wild type and cxmd dogs, we demonstrated robust cellular immune responses to AAV capsid proteins, suggesting the likelihood of cellular immunity to AAV vectors in humans. We further demonstrated that the immune response generated following intramuscular injection of AAV vectors can be averted by a brief course of intense immunosuppression. The broad, long-term objective of this project is to develop AAV- mediated gene therapy strategies in cxmd dogs that can be applied to human patients with DMD. We will test the hypotheses that (1) novel, more effective transient immunosuppression with less toxicity permits sustained transgene expression and subsequent improvement of dystrophic muscle;and (2) that the commonly used AAV serotypes induce a similar immune response. These studies will provide necessary information for future human trials of AAV-mediated gene therapy in humans with Duchenne muscular dystrophy. PUBLIC HEALTH RELEVANCE: The significance of the application is that the methodologies developed in the canine cxmd model can be used to treat human patients with DMD. Gaining a better understanding of the immunogenicity of AAV and developing even better and less toxic immunosuppression regimens will increase the likelihood of achieving the goal of effective gene therapy for human Duchenne muscular dystrophy

DESCRIPTION (provided by applicant): We propose to generate the first high-resolution, comprehensive view of the epigenetic landscape of subtelomeres, the most variable and recombination-prone regions of the human genome. Subtelomeric genes vary markedly in copy number, location, and sequence context among individuals. To fully understand the biological significance of these fast-evolving regions, we will characterize the epigenetic context in which subtelomeric genes operate. In addition to their genomic plasticity, these regions are likely to have unusual chromatin structure due to their proximity to telomeric heterochromatin and abundance of other tandem repeats. We will survey the epigenetic state of many chromosomal ends, but give more attention to subtelomeres with highest relevance to human function and disease. One such region is 4qter, where recurrent contraction of a tandem array, in the context of just one of two structurally variant subtelomeric alleles (4qA), leads to facio-scapulo-humeral dystrophy (FSHD), the third most common inherited muscular dystrophy. This deletion is believed to induce epigenetic changes that cause inappropriate gene expression in 4q or elsewhere, but it is not known how this occurs or what genes are affected. We aim to identify the genetic and epigenetic features of normal and FSHD-causing 4qA alleles in order to provide a link between the 4q deletion and aberrant muscle function. This work should lead to better diagnosis and counseling of recurrence risk for FSHD patients. We will also focus on regions surrounding WASH genes, which exist in grossly different allelic and paralogous contexts in human subtelomeres. We recently discovered that WASH genes encode a highly conserved, widely expressed, yet previously unrecognized new subfamily of WASP proteins, which reorganize the actin cytoskeleton in response to extracellular signals. To accomplish our goals, we will characterize in detail genomic differences among 4qter alleles and among selected chromosomes carrying WASH, as these regions have very limited representation in the current human genome assembly. For epigenetic profiling, we will develop a microarray covering sequences in and near subtelomeres, regions lacking from other arrays. We will use these arrays to analyze subtelomeres in various cells for chromatin modifications characteristic of repressed and active chromatin. In parallel, we will use FISH to analyze larger-scale epigenetic features of specific WASH or 4q copies, such as nuclear location, chromatin condensation, and matrix association. Finally, we will apply tools capable of distinguishing SNPs and paralogous sequence variants (PSVs) to directly relate epigenetic characteristics with transcriptional activity of specific copies. This research will illuminate the significance of subtelomeric genomics and epigenetics in normal phenotypic variation among humans, as well as in inherited disorders, cancer, and aging.

? DESCRIPTION (provided by applicant): The overall theme of the PPG will be identifying the pathological mechanisms associated with repetitive element de-repression and the concomitant new opportunities for therapeutic development. The major projects will be: Project 1, pathways and mechanisms repressing D4Z4 repeats (Silvere van der Maarel), will identify the molecular mechanisms of repeat element repression. Project 2, repeat derepression and RNA-mediated toxicity in FSHD (Robert Bradley), will determine the molecular consequences and RNA-toxicity associated with de-repression of repetitive elements in the genome. Project 3, targeting the D4Z4 sequence to enhance repeat repression (Stephen Tapscott), will identify mechanisms of enhancing repeat-mediated epigenetic repression as a therapy for FSHD. The Bioresources Core, resources for FSHD research and clinical trials (Rabi Tawil), will provide biological resources necessary for each project and to prepare for clinical trials through development of outcomes measures, including biomarkers and patient assessments. The Administrative Core (Stephen Tapscott) will coordinate the activities and communications among the investigators and provide budgetary and administrative oversight, and coordinate the scientific oversight provided by the External Advisory Board. Together these three Projects and two Cores will address the mechanisms and pathways that converge to epigenetically silence D4Z4 in the repeat-mediated silencing pathways, determine the pathophysiologic consequences of inefficient silencing of repetitive RNAs and accumulation of aberrant RNAs, exploit new opportunities for therapeutic development, and provide the resources necessary for moving studies toward clinical trials.

PROJECT 2 r RNA Regulation in FSHD I We have demonstrated that multiple regions of D4Z4 are transcribed in the sense and antisense direction and are processed to small -21 nt fragments. We also have preliminary data demonstrating that RNA processing and possibly protein production might be developmentally regulated during the transition from ES cells to differentiated cells. This leads to the hypothesis that developmentally regulated transcription and RNA processing produces biologically functional RNA or proteins from the D4Z4 region, including but not limited to the full-length DUX4 protein, which contribute to the pathophysiology of FSHD. Therefore, the long-term goal is to identify the RNA, RNA fragments, and/or protein expressed from the D4Z4 region in FSHD that causes muscular dystrophy. Aim 1 will characterize the biological function of the small RNA fragments produced from D4Z4 RNAs and determine whether small RNAs contribute to the pathophysiology of FSHD; Aim 2 will test the hypothesis that the D4Z4 repeats regulate DUX4 expression and have a biological role in early embryonic development; and Aim 3 will determine whether repressive chromatin can be re-established in the D4Z4 units on the disease-associated pathogenic allele, either in a deleted pathogenic allele or in a non-deleted phenotypic FSHD2 allele.

DESCRIPTION (provided by applicant): The broad and long-term goal of this application is to identify the molecular mechanisms that cause facioscapulofaciohumeral dystrophy (FSHD). The broad hypothesis is that deletion of a subset of the D4Z4 units on 4qA161 results in increased or aberrant transcription from the remaining D4Z4 units on the deleted allele. Our preliminary studies have identified multiple sense and anti-sense transcripts from the D4Z4 unit. We have characterized one of the polyadenylated D4Z4 sense transcripts and developed assays to demonstrate its biological function. The specific goal of this application is to determine whether the protein product of a D4Z4 transcript causes FSHD. Aim 1 will characterize sense and anti-sense transcripts from pathogenic and non-pathogenic D4Z4 alleles, their potential protein products, and their expression relative to FSHD. Aim 2 will test the hypothesis that the polyadenylated RNA transcripts from the D4Z4 locus encode several distinct proteins, including the full-length DUX4 and splice-isoforms of DUX4, each with specific developmental biological functions in myogenesis, and that these functions contribute to FSHD pathology. Aim 3 will characterize the IRES element of the DUX4 RNA and test a new hypothesis regarding transitions between RNA translational programs during myogenic differentiation. Together, these studies will advance our knowledge regarding the pathophysiology of FSHD and muscle cell biology. PUBLIC HEALTH RELEVANCE: The human health relatedness of this proposal is that it will identify possible molecular causes of FSHD and provide a basis for future therapeutic development.

CORE B: ADMINISTRATIVE CORE The overall goal of the Administrative Core is to provide administrative, fiscal and scientific management of this POl Program Project Grant. Key components of this Core will be to provide overall fiscal and administrative coordination. Additional objectives of this Core include organizing scientific interactions communications, and to coordinate interaction with NINDS.

DESCRIPTION (provided by applicant): Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 repeat that results in expression of the DUX4 retrogene. Mutations in SMCHD1 result in decreased D4Z4 epigenetic repression through its direct repressor activity at the D4Z4 repeat and cause FSHD. The broad and long-term goal of this application is to develop therapies for FSHD based on increasing SMCHD1 activity or protein level, or decreasing the activity of factor(s) that counter-act SMCHD1 epigenetic activity at the D4Z4 repeats. The specific goal of the research design is to determine the positive and negative modulators of SMCHD1 activity and their epistatic relationship to the epigenetic repression of the D4Z4 macrosatellite repeat and DUX4 expression. Aim 1 will identify the molecular components and the biological functions of the pathways that regulate the post-transcriptional and post-translational production and activity of SMCHD1, and provide a rational basis for the therapeutic modulation of SMCHD1 in FSHD muscle. Aim 2 will test the hypothesis that FSHD2-associated SMCHD1 variants partially inhibit the activity or stability of the wild-type protein and provide a rationale to target the variant isoforms as a therapeutic approach. Aim 3 Identifies components of the SMCHD1 repressive complex and determines their role in D4Z4 epigenetic repression. Together, these studies will provide the basis for future therapeutic development based on increasing the epigenetic repression of D4Z4 in FSHD individuals.

PROJECT 3: Targeting the D4Z4 sequence to enhance repeat repression Abstract FSHD is caused by mutations that decrease the efficiency of D4Z4 repeat-mediated epigenetic repression and result in the low-level variegated mis-expression of DUX4 in skeletal muscle. The broad and long-term goal of this project is to identify components and modulators of the pathways that epigenetically silence D4Z4 repeats that can be exploited to develop a therapy for FSHD, and potentially other human diseases. The major hypothesis of this project is that enhancing the efficiency of repeat-mediated epigenetic repression will suppress DUX4 expression in FSHD muscle and decrease, or reverse, the progression of disease. The specific goal of the project is to identify compounds, their mechanisms of action, and their pre-clinical efficacy in suppressing DUX4 expression in skeletal muscle. This will be accomplished by: Aim 1, Identify and characterize drug-like compounds that enhance the epigenetic repression of D4Z4 and suppress DUX4 expression; Aim 2, Determine the pathways and sequences necessary for D4Z4 epigenetic repression; and Aim 3, perform preclinical development of oligonucleotides to enhance repression of D4Z4 and DUX4 expression. Together these aims will identify compounds, their mechanisms of action, and their pre-clinical efficacy in suppressing DUX4 expression in skeletal muscle. The significance of these studies is that they will identify compounds, their mechanisms of action, and their pre-clinical efficacy in suppressing DUX4 expression in skeletal muscle. The health relatedness is that these studies will provide the basis for future human clinical studies in FSHD.

PROJECT 3 ROLE OF CTCF IN CHROMATIN AND NUCLEAR ORGANIZATION AT THE FSHD 4qD4Z4 LOCUS The chromatin insulator protein, CTCF, has been shown to protect genes from epigenetic silencing. CTCF binding is generally inhibited by DNA methylation and CTCF prevents spreading of DNA methylation. Recent evidence suggests that CTCF can also act to bring spatially separated chromatin domains in close proximity, both in cis and in trans. In collaboration with Dr. van der Maarel, we have mapped CTCF binding sites in the D4Z4 unit and demonstrated both enhanced binding of CTCF to the partially deleted pathogenic D4Z4 allele in FSHD cells and decreased DNA methylation at these CTCF binding sites. Moreover, enhanced CTCF binding to D4Z4 was also observed in undifferentiated ES cells. These findings lead to the hypothesis that inappropriate CTCF binding in the D4Z4 units in FSHD might interfere with the establishment or maintenance of developmentally regulated epigenetic repression and result in inappropriate transcription or nuclear localization. Therefore, our long-term goal is to determine whether CTCF binding on the disease associated allele regulates regional chromatin structure and/or RNA transcription. In our Aim1, we will determine whether the CTCF sites in the D4Z4 units function as insulators, transcriptional regulators, or chromatin boundary elements, and whether CTCF is necessary for regulating D4Z4 chromatin structure and transcription. These studies will be done using siRNA-mediated knock-down of CTCF in control and FSHD human muscle cells and D4Z4 mouse models. In Aim 2, we will detennine whether human IPS cells generated from control and FSHD fibroblasts accurately recapitulate the chromatin structure of D4Z4 in ES cells before and during in vitro differentiation, and whether CTCF binding and/or deletion of D4Z4 subunits prevent a developmentally regulated chromatin-mediated repression of D4Z4 transcripts. Finally, in Aim 3, we will use chromatin conformation capture in combination with chromatin immunoprecipitation techniques to determine whether CTCF, chromatin context, and/or developmental state mediate intra- or inter-chromosomal interactions at the 4q D4Z4 locus and whether these interactions are altered in FSHD cells.

ADMINISTRATION CORE: Abstract The Administrative Core will have overall responsibility for administrative management, reporting and communication between components of the Program Project Grant, the External Advisory Committee, NIH and the FHCRC, and members of the community. The long-term goal of the Administrative Core is to ensure that the scope of work described in the application is accomplished in an efficient and compliant manner. This will be accomplished by: (Aim 1) Organizational and administrative management of the overall program; (Aim 2) Planning and coordination of research activities.