Barbara J. Wold - US grants

RNA physiology in the eukaryotes is an intriguing and complex problem. We are studying gene products that are thought to mediate aspects of gene expression and RNA biogenesis. These are members of two families, one comprised of the proteins of the HnRNP complex and the other consisting of cMyc and its relatives. Myc gene products are also implicated in the regulation of cellular growth control, but the connection between this property and its capacity to influence gene expression is not yet understood. The experimental system of choice for this work is Xenopus because it provides the opportunity to study these molecules in the context of oogenesis and embryogenesis, and also because it affords some important technical advantages. Initial experiments proposed here will provide molecular level descriptions of gene and protein structure and the patterns of expression during development and differentiation. These studies set the stage for subsequent experiments which focus increasingly on the function and regulation of RNP and myc genes and proteins. Another major goal is to continue development of antisense nucleic acid analysis. This is a potentially general technique for establishing and studying cells or individual organisms that are depleted in a specific gene product. The goal is to bridge the gap between knowledge of gene structure and expression on the one hand, and an understanding of the function of the gene product on the other hand. In some cases antisense analysis may also reveal regulatory circuits governing a particular gene or protein. This aspect of antisense nucleic acid studies is considered further in one st of experiments. A final aim is to apply antisense analysis to the RNP and Myc gene products that are already being studied by more conventional means. Several different parameters can be quantiated to describe phenotypic changes caused by altered expresion of these gene products. This is an important feature that makes them better targets than are genes whose function can only be evaluated by gross phenotypic changes.

DESCRIPTION: The central goal of the proposed research is to illucidate the molecular events involved in the sequential cellular decisions that lead from the multipotential mesodermal precursor cells to the differentiated skeletal muscle. The experimental focus is on two biologically interrelated problems. I) Significance of the four member MRF family. In most in vitro biochemical assays and tissue culture transfections assays, the four MyoD family members are remarkably similar in function but their individual patterns of expression during development are significantly different from each other. Moreover, recent experiments in the investigator's lab (MRF4 knockout) and others have shown that individual disruption of each produces a different phenotype. What remains entirely unclear is whether distinct functions uncovered in vivo are intrinsic to individual family member proteins or are dependent instead on the timing and amount of expression. The investigator will address this problem directly by generating selected transplacements of one MRF family member for another: 1) MyoD transplaced into the myogenin locus and into the MRF4 locus with concomitant elimination of myogenin coding sequences. This tests whether the apparent uniqueness of myogenin for muscle differentiation can be taken over by the erstwhile dispensable determination member MyoD. 2) Myogenin transplace into the myf5 locus with concomitant deletion of myf5 coding sequences. From this they will learn whether expression of myogenin at the earliest times leads to premature differentiation or acts instead to complement the deficiency of myf5. II) Defining pre-MRF steps in embryonic myogenic lineages. It is now clear that expression of even the earliest MyoD family member is a relatively late event in the myogenic pathways in vivo. Therefore a major goal is to define the molecular and cellular basis for the steps that lead to the activation of the MRFs. The new approach proposed here begins with developing a system for nondestructively identifying, tracing, enriching and even isolating and culturing precursors cells of interest from the embryo. The investigator has chosen to begin with Pax3. Its expression marks precursors of axial and limb myogenic cells over an extended period prior to the onset of expression of myf5 , including cells that emigrate from the somite to found the limb musculature. Marking will be done by transplacing a vital marker and a selectable marker into the Pax-3 locus. This will permit the investigator to further define of the precursor cell population, assay and ultimately identify signals that induce or suppress their transition to myoblasts.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Description: All three research projects in this Program depend very heavily on the use of a facility which can successfully and efficiently produce transgenic mice by conventional means and mutant animals by homologous recombination in embryonic stem cells. The director proposes to establish a gene manipulation Core built on an existing transgenic/ES cell facility at Caltech. The facility will be responsible for producing genetically engineered animals and will serve as a common central repository from which unmodified animals can be obtained. A centralized cell culture facility will be established to standardize the necessary set of techniques needed to produce animals with inactivated genes. This facility will serve as a producer of mutant cell lines (and animals) and also serve as a training resource for individuals who wish to learn and master this technology. To preserve transgenic mice and animals with targeted gene mutations, a cryopreservation facility is proposed.

protein structure function; striated muscles; myogenesis; transcription factor; recombinase; phenotype; gene expression; developmental genetics; alleles; gene targeting; embryonic stem cell; western blottings; genetically modified animals; laboratory mouse; polymerase chain reaction; genotype; nucleic acid probes;

DESCRIPTION: The focus of this amended application is cell specification and pattern formation in the mammalian musculoskeletal system. The proposed experiments rely heavily on the use of cutting edge and developing technologies, the recombinase mediated molecular lineage tracing (RBLT) and magnetic resonance imaging (MRI) for cellular lineage tracing. The third technology that is more routine is the use of homologous recombination to inactivate genes. The investigators will apply the MRI based lineage tracing to analyze development of craniofacial muscle and skeleton. The rationale for this is that such an atlas of development will prove to be useful in analyzing the impact of mutations on these structures. RBLT is a complementary technology that will allow tracing the fate of individual cells during development. This technology will interface with the experiments dealing with myogenic regulators. Strains of mice will be developed with alterations in myogenic regulatory genes. In addition, the fate of those cells that express myogenic regulators early in development will be traced. There have been several changes in the proposal since the original application was considered by the Review Committee in November 1993. First, Project 4 (the original application consisted of four projects and one Core unit), which was viewed by the Committee to be not closely tied in with the theme of the program project, has been deleted. Second, for Project 3 of the original application, one major criticism was the lack of detail and the potential lack of resolution of the method used. The investigators have included new preliminary data and acknowledged that more conventional methods could be employed if needed. Finally, the issue of the centralization of the Caltech transgenic mouse facility has been clarified. Both Drs. Wold and Anderson have a direct role in establishing this facility. They have interacted effectively in the past, although this interaction has not been formalized by joint publications. P01AR426710001 Description (adapted from the applicant's abstract): Four closely related transcription factors, MyoD, myogenin, myf5 and MRF4/herculin/myf6 are thought to function as major, positive acting regulators of skeletal myogenesis. The current view of their function in muscle determination and differentiation came initially from studies in cell culture models and from the observation that their expression is restricted to skeletal muscle and its immediate progenitors. This is presently being both supported and substantially modified by the ongoing analysis of mice in which myogenin, MyoD or myf-5 have been disrupted. A successful disruption of MRF-4 has not yet been achieved and published. This proposal will focus on two major questions concerning mammalian myogenic lineages: What are the functions of MRF-4 during development, and what roles do different MyoD family members play in restricting cells to a myogenic lineage? To address these questions, the investigators propose to construct and study strains of mice in which MRF-4 has been disrupted by the standard ES cell mediated design. Previously, when they cloned and characterized mouse MRF-4, they found that several properties distinguish it from the other three MyoD family members. Chief among these is the fact that MRF4 is by far the quantitatively dominant MyoD family member expressed in muscle of adult mice. This led the investigators to propose that MRF-4 plays the key role in maintaining muscle phenotype, and perhaps in mediating responses to activity level. The knockout will permit them to test directly this hypothesis and others concerning phenotypic interactions among different MyoD family members, autoregulation and cross-regulation in the HLH network and possible roles in specifying subsets of the skeletal lineage. As part of the Program Project, the investigators propose to help develop recombination based lineage tracing (Projects 1 and 2) and then use it to address several distinct questions about myogenic lineages and the roles played by MyoD family members in their specification. This novel analysis will permit them to examine the origin and determination of lineages leading to satellite cells and to the appendicular musculature. Each of these myogenic settings offers different features that will balance two goals of the Program. One is to probe the biological significance of MyoD family expression, whether transient or continuous, in a given lineage over a specific developmental time window. The second goal is to provide a well-controlled and experimentally accessible series of tests for Recombinase mediated lineage tracing in the mouse.

myogenesis; muscle metabolism; gene expression; aging; regulatory gene; gene targeting; phenotype; musculoskeletal regeneration; cell differentiation; muscle function; genetic regulatory element; DNA binding protein; nucleic acid sequence; transfection /expression vector; embryonic stem cell; genetically modified animals; laboratory mouse;

This proposal requests partial funding for the Gordon Research Conference on Myogenesis for the year 2001. The Myogenesis Gordon Conference meets every third year. By informal community agreement, it alternatives with Keystone/Asilomar and EMBO meetings in a three year cycle that provides one major international meeting for scientists in the area per year. Within this framework the Gordon Conference emphasizes skeletal and cardiac muscle develop during embryogenesis and regeneration. This Gordon Conference has been very successful and influential. One way that it has achieved its success is to select each year one or two especially timely topics to introduce to its audience, in addition to provide a forum for new discoveries within its core topic areas. For 2001 the two new areas of emphasis will be stem cell biology and genomics. Stem cells from various source tissues in embryos and adults are a hot topic cutting across many biological disciplines for reasons of fundamental interest and for its implications for cell-based therapeutics. Dr. Guilo Cossu, whose work opened the way into a reconsideration of stem cell sources with myogenic potential is meeting vice-chair, and he will lead this session. We have also recruited Prof. David Anderson, a leading international figure in stem cell biology of the nervous system, to give the keynote talk. He is also the leader in studying genes that are the nervous system equivalents of MyoD. The second new session for 2001 will inform this community about resources, methods, and discoveries in genomics and functional genomics. This is very timely, since the human genome draft sequence will be complete and the mouse draft should then be nearing completion. An important current challenge for the investigators in this field in how they will use these data and the new methods that are being developed for whole genome biology in the context of myogenesis. This is the expertise of the 2001 meeting chair, Dr. Wold.

DESCRIPTION (provided by applicant): This project is to use high-throughput genomic sequencing analysis of histone marks and transcription factor binding sites to track the succession of regulatory changes that underlie multipotent progenitor differentiation into T lymphocytes. The project will characterize the transitions from one stage to the next in terms of genome-wide changes in histone modifications ("epigenetic marks"), changes in RNA polymerase II loading and activity, and changes in comprehensively measured transcript accumulation. The feasibility of this project is based not only on access to extensive Solexa/Illumina sequencing and quantitative informatics analysis, but also on the advent of efficient methods for expanding and isolating primary mouse hematopoietic cells at a series of discrete phases across the transition from stem cell to committed T cell precursor. This system provides unusually advantageous access to cell states in a real, precisely coordinated developmental continuum. As defined by previous work of the applicants, the T-cell developmental sequence includes opportunities to observe both the mechanisms through which T lineage-specific genes are first activated and the mechanisms through which stem cell pluripotency genes and self-renewal genes are programmed for long- term silencing. The project thus offers a novel opportunity for the epigenomics field to test the significance of patterns of epigenetic marking at sites throughout the genome, in light of known trajectories of changing expression of these loci over time. PUBLIC HEALTH RELEVANCE: The genome that is the blueprint for life includes much more than sequences that code directly for proteins. It also includes sequences that are control sites where levels of RNA and protein expression can be regulated. As stem cells develop into mature cell types, for example the blood cells that are produced every day throughout a human life, thousands of genes must be re-tuned in their expression levels to create the new cell identity. This regulation can be extremely sensitive, as small variations can lead to disease. Recent advances help to map where regulatory sequence sites are likely to lie in mammalian genomes, but knowledge of the rules that govern when they are acting have so far lagged behind. What is needed is to bring these new technologies to bear on a well characterized set of mammalian cells caught at various stages in the process of development from a stem cell to a differentiated fate, where the changes across the genome can be followed across time. In this project, we therefore use advanced technology for tracking the action at regulatory sequences as differentiation progresses to understand in detail the genome-wide events that cause a stem cell to develop into a T lymphocyte of the immune system. This system is profoundly important to human health. In this project, we will determine how different types of regulatory events occurring across the genome can explain which type of cell the precursors become, as well as how they avoid being sidetracked into leukemia. .

DESCRIPTION (provided by applicant): This proposal is to support a collaboration between the laboratories of John Allman and Barbara Wold to investigate gene expression in laser micro-dissected cell populations in autopsy brains of well-phenotyped autistic individuals versus age and sex matched controls using a recently developed techniques, RNA-Seq. RNA-Seq will be interpreted in the context of SNP and copy number variation (CNV) genotyping. FI (Fronto Insular Cortex) and ACC (Anterior Cingulate cortex) are functionally implicated in social interaction and reciprocity, in empathy, and in the awareness and regulation of bodily functions. These functions are crucially affected in autism. Unlike other homeostatic systems, including those in the hypothalamus, the FI/ACC system appears to have direct access to consciousness and motivation. Analysis of initial RNA-Seq on FI samples produced two coherent gene networks that differ between autistics and neurotypical controls. Immunostaining for several network members has begun to show how networks map onto the cellular circuitry. Specifically, at the cell level, FI and ACC contain large bipolar cells (Von Economo neurons, VENs) that are distinctive features of these structures in apes and humans. We found that VENs express receptors for the cytokines interleukin 4 (IL4R) and interleukin 6 (IL6R) in normal and autistic subjects, and RNA-Seq identified a prominent gene network related to inflammation which is strongly up-regulated in a subset of our autistic cases (autism-A). This network is centers on IL6 and includes ATF3, SOCS3, and GADD45B, which are selectively expressed in VENs. Microglia, the immune cells of the nervous system, are numerous and are in the activated state in autism-A, making a likely signal source. Our remaining autistic cases comprise an autism-B group, which is characterized by increased expression of genes in the presynaptic terminal including NRXN1 (neurexin 1), which provides Velcro-like binding to neuroligins in the post-synaptic membrane. NRXN1 is one of the genes most strongly and consistently associated with autism. NRXN1 has many splice variants which could provide specificity in formation or strength of synaptic connection. To probe these networks more deeply;to assign gene expression and splice isoforms to their proper cells;and to discover remaining differences in autistics, we propose generation-2 RNA-seq on laser-dissected cells. Thus VENs and other key cell types are rare (<5% of cells in FI). This reduces their transcriptome completeness: genes under- expressed in autism relative to controls will be especially affected. Successful laser capture can overcomes this hurdle. We also propose deep RNA sequencing for NRXN1 isoforms and other complex families that may be uncovered. PUBLIC HEALTH RELEVANCE: We seek to understand the cellular bases of autism by using a new technology, RNA-Seq, to determine differences in gene expression in autopsy brains of subjects with well described autism versus age and sex matched neurotypical individuals. We have investigated two specific cortical areas involved in self-awareness and social reciprocity which are abnormal in autism and have found increased expression in a network of genes related to inflammation in autism group A, whereas the remaining cases, autism group B, have increased expression in a network of genes related to synapses. We propose to use laser micro-dissection to investigate gene expression in specific neuronal and non-neuronal populations in the cortical areas of interest in the autism-A, autism-B and control groups.

DESCRIPTION (provided by applicant): Cis-acting regulatory elements control gene expression and are involved in all aspects of development, behavior and physiology; but no cis-regulatory element map yet exists for any metazoan genome. We therefore propose to identify genome-wide cis-regulatory elements in C. elegans. Among existing model organisms, C. elegans offers a strong combination of biological properties, transgenic technology, comparisons to genomes in four sibling species, and critical computational and bioinformatic infrastructure. We intend to find genomic regulatory elements in genes with widely varying expression patterns, gene functions, and cis-element content that drive expression in diverse developmental stages, cell types and physiological conditions. A pipeline of genomic predictions followed by efficient transgenic reporter assays will allow us to generate and analyze 10 DNA constructs each week. In year 1 we plan to identify hundreds of regulatory elements, with higher numbers in following years as we become better at predicting functional elements. Predicted elements will be assigned statistical scores based on the quality of their supporting computational and experimental data. We will also use chromatin immunoprecipitation analyzed by intense sequencing (ChIP-seq) to find regulatory modules, and compare its accuracy in finding functional sequences to that of predictions based on comparative genomics. The first round of our results from direct tests of predicted elements and ChIP-seq will be combined with external data from modENCODE to improve our predictive algorithms for later cycles of genome-wide element prediction. Our data will be released promptly to WormBase, and all our computational tools are freely available with full source code.

PROJECT SUMMARY/ABSTRACT with changes for year5 extension (underlined) It is said that if the genome represents the set of things that the cell could say, then the transcriptome represents the set of things that the cell is saying right now. The transcriptome is the primary read-out of the genome and is composed mainly of mRNAs, long noncoding RNAs, and microRNAs. At all stages of life, in health and in disease, differences in the transcriptome causally define and distinguish each cell type and cell state. The mission of the ENCODE Consortium is to discover, map and define all genes and their regulatory elements. RNA-Seq data and the resulting transcriptomes have therefore been a prominent component of prior phases of ENCODE, and our groups have contributed over 350 released poly(A) mRNA and microRNA datasets for the project. Valuable and high quality as those data are, new technologies will allow us to produce a new generation of transcriptomes that are much more information rich and definitive. Specifically, they resolve the multiple different cell types that comprise complex tissues and they resolve the multiple molecular isoforms. They document long-range single molecule splicing and end processing and we provide companion quantification of microRNAs, plus discovery of their often-undocumented precursor RNAs. Finally, we map RNA secondary structure across the transcriptome in vivo. We propose to complete contributing at least 200 higher precision long-read transcriptomes and companion measurement types, and their integrated analysis. We also add short- read ribo-minus RNA-seq across the NHGRI approved consortium biosamples. A portion of this study focuses on aging humans and mice, a whole life cycle stage that has not previously been studied in ENCODE. The corresponding disease component contrasts cognitively normal with Alzheimer?s dementia.

Any human being has on average 5 million single-nucleotide variants and 13 million nucleotides of insertions, deletions, and other regions present in variable copy numbers compared to the human reference genome. Together these variants must account for all of the genetic contributions to every phenotype of that person, whether it is their height or their familial predisposition to complex diseases. While we can quickly measure the presence of these variants in a genome, we lack the framework to understand which of the variants impact genomic function or how they interact with each other in living, breathing organisms. A core mission of the IGVF Consortium is to identify variants that impact the expression of genes using single-cell techniques and computational modeling. Applying a single-cell genomics approach to selected diverse mouse strains can make a powerful contribution to the mission and to resources of the Consortium. Our Center for Mouse Genomic Variation at Single Cell Resolution will first use 38 mouse Collaborative Cross recombinant inbred lines that possess similar levels of sequence diversity to humans to identify variants that influence gene expression levels and chromatin accessibility at the single-nucleus level in 8 distinct tissues. We will sequence simultaneously a subset of single-nuclei with both short-read sequencing and long-read sequencing to identify variants that impact the expression of different transcript isoforms in different cells across the different mouse strains. We will also measure the relationship of variants in these CC Lines in the response of macrophages in these tissues in response to LPS stimulation. The resulting resource catalogs of cell-type expression QTL, chromatin accessibility QTL, splicing QTL, and response QTL maps will be useful for IGVF modeling groups; for characterizing important variants; and for use by the wider community studying the function of these tissues as well as for designing better pre-clinical models of human diseases.