Joseph R. Ecker, Ph.D. - US grants

Understanding an organism's response to stress is a fundamental problem in biology. The unifying theme in the stress response is that perturbation of the environment leads to rapid and specific activation of gene expression. In the course of evolution, plants have developed effective mechanisms for resistance to a variety of stresses. Specific induction in gene expression occurs in response to environmental stresses such as infection and wounding; the "immune response" of plants. A major goal of this proposal is to understand the molecular and cellular basis of a plants response to these stresses. Molecular and genetic approaches will be combined to dissect the mechanisms controlling the induction and expression of stress-regulated genes in a model plant system. The dicotyledonous plant Arabidopsis thaliana is ideally suited for such studies; the major advantage is that it is amenable to molecular genetic manipulations common to yeast and Drosophlia but impractical for other higher plants. The long term goal of this project is to ascertain the molecular events involved in signalling and recognition of stress. The action of stress-induced hormones is of fundamental importance in understanding these processes. This research aims to isolate and characterize mutations in genes that affect the biosynthesis, recognition and response to stress-induced ethylene and oligosaccharins and also to define the DNA sequences in stress- induced plant defense response genes which are essential for regulation by these plant stress hormones. Specifically this project aims: 1) to identify and characterize Arabidopsis mutants that are affected in the stress response to exogenous and endogenous oligosaccharide fragments; 2) to identify and "stress ethylene"; 3) to analyze the DNA controlling elements that regulate ethylene- and oligosaccharin-induced genes; 4) to use both genetic and molecular techniques to identify genes/proteins that directly or indirectly mediate the induction of ethylene- and oligosaccharin-regulated genes and 5) to construct and characterize "mutations" in genes regulated by ethylene and oligosaccharins by expression of antisense and sense RNAs for these genes.

This proposal concerns the development of technologies which will facilitate the isolation and analysis of large segments of a plant genome. At present, the cloning of plant genes about which nothing more is known than genetic map position is technically difficult. Current restriction fragments length polymorphism (RFLP) maps of most plat genomes contain large gaps which makes standard chromosome "walking" techniques difficult, if not impossible. Our approach to this problem is to develop methods for the cloning and introduction into plant cells of large segments of plant DNA. These studies will involve the development of "large DNA" methods for chromosome walking, gene isolation and transfer using a model plant system, Arabidopsis. An important aim of this work will be the construction of a low resolution physical map of the Arabidopsis genome using large segments of cloned DNA. Genomic libraries of large Arabidopsis DNA-containing yeast artificial chromosomes (YACs) will prepared and cloned into Saccharomyces cerevisiae. YACs will be analyzed by pulsed-field gel electrophoresis (PFGE) using contour-clamped homogeneous electric field (CHEF) apparatus. Overlapping YACs will be identified and linked using both molecular and yeast genetic methods. Two unrelated but equally important chromosome walking/linking projects will be initiated. We propose to: 1) link up macro-regions of the Arabidopsis genome using YACs and PFGE and 2) clone the entire centromere region of an Arabidopsis chromosome.

Arabidopsis 2010: A Sequence-Indexed Library of Insertion Mutations in the Arabidopsis Genome. With the availability of the entire Arabidopsis genome sequence, one of the next challenges is to uncover the functions of the more than 25,000 genes in this reference plant. Given the scope of the NSF 2010 program, to identify the function of all Arabidopsis genes in the next decade, an efficient and cost effective approach is necessary to identify mutations in all genes. The goal of this program is to create a sequence-indexed library of mutations in the Arabidopsis genome. The Salk Institute Genome Analysis Laboratory (http://signal.salk.edu) will use high-throughput genome sequencing methods to identify the sites of insertion of Agrobacterium T-DNA in the Arabidopsis genome. T-DNA transformed plants from the Alonso/Crosby/Ecker collection will be grown, genomic DNA will be prepared, T-DNA flanking plant DNA will be recovered and sequenced. Insertion site sequences will be aligned with the Arabidopsis genome sequence and gene annotation will be added. The data will be made available via a web accessible graphical interface-T-DNAExpress-(http://signal.salk.edu/cgi-bin/tdnaexpress) that will provide both text and DNA searches of the insertion sequence database. All DNA sequences will be deposited into GenBank (www.ncbi.nlm.nih.gov) and also provided to The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org). Seeds from the T-DNA insertion lines will be deposited with the Arabidopsis Biological Resource Center (ABRC) at Ohio State University:http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/ABRCHOME.HTM. The ABRC will propagate and distribute seeds to the community. The creation of a searchable database containing the insertion site information and the availability of the corresponding mutant lines in public stock centers will provide researchers with ready access to mutants in their genes of interest, allowing the testing of hypotheses about gene function at an unprecedented rate.

This research program pertains to the development of tools and resources for the plant biology community. Computational studies of the genome sequence of the reference plant Arabidopsis thaliana have identified approximately 30,000 genes. In order to carry out a variety of functional genomics and proteomics applications, it is essential to identify all of the genes and determine their transcription unit structures. This project will utilize a newly developed single chip whole genome tiling array to experimentally map the transcription units in the Arabidopsis genome. The transcription unit mapping information will be used to amplify and clone, in recombination-based vector, 6,000 full-length (FL) cDNA and open-reading-frame (ORF) clones. The DNA sequences of each clone will be determined to high accuracy and this information can be used to improve the genome annotation. The construction of an error-free ORF clone for each protein-coding gene will enable a variety of functional genomics and proteomics studies. All cDNA/ORF clones will be deposited with the Arabidopsis Biological Resource Center and the clone sequences will be will be deposited in GenBank. DNA sequence and tiling array hybridization data will also be displayed on the SIGnAL project web site: http://signal.salk.edu.

Broader Impacts: The beneficiaries of this research program include the entire plant biology community. The transcription unit DNA sequence information and cDNA/ORF clones produced by this project will provide investigators with essential information necessary to elucidate the functions of the Arabidopsis proteome. The collection of Arabidopsis ORF clones will enable the construction of whole genome protein arrays, the development of protein-protein interaction maps and the ability to rapidly create plants that ectopically express any ORF using any promoter of choice. The long-term impact of these enabling tools and technologies on agriculture is expected to be profound, providing fundamental knowledge for the construction of plants with superior agronomic traits. Importantly, all of the ORF clones, array data and DNA sequences will be made freely available to the research community. Finally, an important feature of the program is the training of high school and undergraduate students in bioinformatics and functional genomic methodologies.

The proposed study pertains to the development of research tools and resources for the plant biology community. Analysis of the genome sequence of Arabidopsis thaliana revealed the existence of approximately 25,000 protein-coding genes. It should now be possible to undertake systematic genome-wide functional screens that assess the contribution of every gene to a biological process. However, due to the absence of a complete set of gene-indexed homozygous mutants unbiased genome-wide functional screens are not yet feasible. To fulfill these needs, the aim of this 2010 project is to experimentally identity two homozygous insertion mutants for 25,000 Arabidopsis genes, thereby creating a "phenome-ready" genomics resource. Unbiased genome-wide screens will enable annotation of the genome with new high-quality information about the regulation and biological functions of any gene.

The resources developed by this project will be available to all researchers and will provide the basis for a variety of projects that rely upon whole genome information. The data from this project can be found at: http://signal.salk.edu, and seed will be provided to the Arabidopsis Biological Resource Center (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm) for distribution. Another important feature of the project is the training of undergraduate students in bioinformatics and genomic methodologies. The long-term impact of these mutants on agriculture is to enable construction of plants with superior agronomic traits.

[unreadable] DESCRIPTION (provided by applicant): This proposal aims to identify functional, non-coding elements in eukaryotic genomes by surveying genome-wide DNA methylation. Cytosine DNA methylation controls developmental gene expression in mammals during genomic imprinting and X-chromosome inactivation, and also silences transposons and other repeated DNA sequences. Hypermethylation of specific tumor suppressor genes contributes to cancer, showing the relevance of this work to human health. Here we propose using whole-genome microarrays (WGAs) to find all methylated sequences in a eukaryotic genome; the "methylome". We will test our methods with the model plant Arabidopsis thaliana, demonstrating their usefulness for the human genome. Arabidopsis is ideal for studying DNA methylation, because it has facile genetics, a small genome, and orthologs of every human DNA methyltransferase. In contrast to the lethality of mouse DNA methyltransferase mutants, Arabidopsis can tolerate mutations that virtually eliminate methylation. Our Arabidopsis thaliana arrays tile the complete genome with 25-mer oligonucleotides, allowing us to precisely measure DNA and RNA hybridization. We will use several different reagents to identify methylated DNA sequences, each coupled with high-throughput analysis using WGAs: bisulfite treatment of genomic DNA, restriction digest with methylation-sensitive enzymes, anti-methylcytosine antibodies, and proteins that bind specifically to methylated DNA. By using complementary and independent methods, we hope to detect DNA methylation with up to single nucleotide resolution. Arabidopsis DNA methyltransferase mutants have well-characterized DNA methylation defects at several endogenous loci. We will use mutants in every major Arabidopsis DNA methyltransferase to test and verify DNA methylation detection methods. RNA silencing mutants will determine how much DNA methylation is guided by small intefering RNAs (siRNAs). To further define functional non-coding elements in the genome, we correlate changes in DNA methylation with transcription and with the presence of antisense gene transcripts, and other non-coding RNAs. Once we have identified all methylated loci, we will use novel sequence libraries to classify them as transposons, other repeats, or unique sequences. This analysis is likely to reveal functional non-coding elements that are invisible to other methods. Since methylation is critical in many mammalian gene regulation phenomena, the methods developed in this proposal will clearly move the ENCODE project toward its goal of identifying functional elements in the human genome. [unreadable] [unreadable]

The National Plant Genome Initiative (NPGI) was established in 1998 as a coordinated national plant genome research program by the Interagency Working Group on Plant Genomes (IWG-PG). In the 10 years since the program started, the field of plant genomics has made tremendous progress, changing the way that plant research is conducted, attracting a new generation of scientists to the field and contributing a wealth of new information and knowledge with potential application to agriculture. The purpose of this workshop is to discuss the status of plant genome research in the U.S. after 10 years of the NPGI and to consider the challenges and opportunities for the next decade. The workshop will bring together stakeholders with broad range of expertise in high-throughput genomics, plant biology, bioinformatics and databases drawn from the academic, private and international sectors. Workshop outcomes will be captured in the form of a paper to be developed by the workshop participants and circulated to the broader community. The paper will be accessible through http://pbio.salk.edu/pbioe/ and TAIR (http://www.arabidopsis.org).

This project specifically addresses an important aim of the 2010 project - to identify genetically stable loss-of-function mutations in all Arabidopsis genes. Computational analysis of the initial genome sequence of Arabidopsis thaliana in 2000 provided evidence for the existence of approximately 25,500 genes. Intensive efforts during the past six years to experimentally annotate this genome sequence have identified many additional protein-coding and non-coding RNA genes, expanding the gene list to 31,128 genes. Importantly, even the most current version of the genome annotation does not include many hundreds of unannotated non-coding (large and small) RNA genes. The identification of T-DNA/transposon insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the current set of sequence-indexed integration sites reveals that mutations for ~6,000 "new" genes have not yet been identified. This project will use specific gene-directed PCR to identify two insertion mutations for each of these 6,000 genes, thereby allowing completion of the "uni-mutant" set for annotated Arabidopsis genes. In addition, the project will provide for the genotyping of these mutations which segregate for these T-DNA insertions to identify homozygous mutants in all genes that do not lead to lethality or infertility. In addition, large-scale genotyping of the known Salk, SAIL, Wisconsin and GABI-KAT T-DNA insertion mutant lines will be done as part of the effort to obtain homozygous insertion mutants in the 25,000 genes initially identified by the Arabidopsis Genome Initiative. Combined, these efforts will result in the production of a "unimutant" set of homozygous insertion mutations for the core set of plant genes. When completed, this resource will enable the initiation of whole genome phenotypic screens, allowing the identification of genes important for understanding basic plant biology as well those genes whose functions are important to further improvement of food, biomass and energy production.

Broader Impacts. The genomic resources developed by this project will be widely available to a large number of researchers, will provide the basis for a variety of research projects that rely upon whole genome information and will enable genome-wide mutant screens for any visible phenotype of interest. All of the mutant plants will be available to the broader research community as soon as they are produced through the Arabidopsis Biological Resource Center (ABRC). All DNA sequence data (T-DNA insertions and genome sequences) will be deposited in GenBank, and TAIR (http://www.arabidopsis.org). The data will also be made available via the SIGnAL database (http://signal.salk.edu/cgibin/tdnaexpress), allowing browsing/retrieval of these data types, integration with the genome annotation, transcription units and DNA methylation, cDNA/ORF clones, and with all public insertion mutant sequences.

An equally important aspect of the project is the hands-on training in plant genome research that will be provided at a variety of levels, including outreach to minority high school and undergraduate students.

DESCRIPTION (provided by applicant): Transcription factors (TF) have a crucial role in controlling gene expression, and exploring their molecular targets, binding partners and mode of regulation is essential to understand any plant biological process. Arabidopsis offers some unique advantages for the development of large-scale genomic approaches for the study of TFs function such as the ease and low cost to generate large transgenic collections, or the propensity of most gene-regulatory regions to be circumscribed to a short region upstream the transcription start site. The potential of these types of strategies is greatly exemplified in a recent report from our laboratory where, by using a fraction of the full TF collection, a novel clock component was identified. For these reasons, we started gathering all available Arabidopsis TFs from the different ORFeome resources (Salk, Pekin-Yale, REGIA, TIGR and RIKEN) to generate a complete TF collection. However, our findings from resequencing the different clones revealed a large overlap between the collections and several hundred mislabeled or missing ones, resulting in a final coverage close to 75% of all the Arabidopsis transcription factors and regulators. Here, we propose to generate an homogenous gold standard GATEWAYTM compatible collection containing every Arabidopsis TF. The corresponding coding sequences will be cloned in the same vector using the available ORFeomes as the template resource when possible. The remaining 25% missing ones will be generated by following different complementary amplification protocols and subsequently cloned in the same vector backbone. In addition, we propose to create and distribute to the community, nine application-ready genomic collections containing each TF in fusion with different tags for a multitude of applications. These nine collections will allow (1) overexpression screens in plants of wild type as well as EAR or VP64 translational fusions, (2) protein-protein interaction screens, (3) protein-DNA interaction screens, (4) subcellular localization and, (5) bacterial recombinant protein expression. In addition, in collaboration with Dr. Joe Ecker at the Salk Institute, we propose to test and compare the efficiency of different protein epitope-tags suitable to perform ChIP-seq experiment. A collection of TF tagged with the selected epitope will be generated. Finally, we propose to devise a simple protocol to perform yeast one-hybrid screens with full TF collections at a reasonable cost. We strongly believe these resources have the potential to greatly enhance research in Arabidopsis and other crop species. As the knowledge gap is being filled, the study of transcriptional networks in Arabidopsis will ultimately help us understand the biochemical complexity of multicellular organisms and positively impact the biomedical research community.

The completion of the first plant genome sequence from the model plant Arabidopsis thaliana in December 2000 sequence ushered in a new era in plant biology. This coordinated international project drew in scientists from 25 laboratories in the U.S., Europe and Japan to develop a high quality sequence resource that is freely accessible to all. The subsequent expansion of genome sequencing efforts to other model and crop plants, functional genomics efforts and development of new tools and technologies has moved the whole field forward in understanding the structure and function of plants. As the first decade of genome-enabled plant biology draws to a close, it is time to develop a new vision for plant biology for 2011-2020 that builds on these tremendous advances. A small workshop will bring together scientists from around the world working on a range of plant systems and processes to develop a vision document that will outline a forward-looking, bold, science-driven roadmap for the plant sciences at an international scale. The draft document will be available for comment at http://pbio.sal.edu/pbioe/ and the final version submitted for publication in December 2009.

Plant biologists need many completely sequenced and functionally annotated genomes within each species in order to fully exploit the power of evolution to understand how an organism functions and adapts to its environment. Researchers interested in natural variation in Arabidopsis propose to generate genomic DNA sequences from over 1000 inbred strains, driving technology developments in both hardware for the DNA sequencing itself and in software development to make sense of the DNA sequence data. The goal of this research project is to record the genetic variation in the entire genome of many strains of the reference plant Arabidopsis thaliana. We will develop and apply cutting edge DNA sequencing approaches using the reference plant A. thaliana to address questions of fundamental importance about plant evolution and gene function. The complete genome sequences for 200 accessions, produced as a result of this project, will provide the first complete view of haplotype structure for Arabidopsis thaliana and will allow future studies of epigenetic variation among different individuals in a population or within a species, a potential source of phenotypic diversity. The patterns of sequence and structural variation will reveal important insights into the dynamics of genome change and pinpoint potentially functionally important sources of genetic and epigenetic variation. Moreover, these data will enable subsequent mechanistic studies through experimental manipulation of Arabidopsis strains.

The 1001 Arabidopsis Genomes Project (http://1001genomes.org) will provide detailed genotyping data of wild strains that will complement the efforts of individual investigators to phenotype these same accessions for thousands of traits of interest. For example, this research has the potential for rapid advancement toward the mechanisms by which plants adapt to various climates, utilize soil nutrients and resist pathogen infection. The knowledgebase produced from the 1,001 Arabidopsis Genomes Project will yield direct and measurable outcomes for deployment of similar traits in economically important crops for a changing global environment.

Broader Impacts of the Proposed Research
The impact of this project will be in two broad areas. First, the completion of the planned research will result in important new resources for the plant biology community: large-scale information on genetic variation among closely related genotypes. The very limited availability of whole genome sequence variation information has negatively impacted a variety of research endeavors such as the understanding of adaptive evolution or the development of association mapping. All of the DNA sequence data will be made freely and easily accessible to the research community. The long-term impact of these enabling tools and technologies on agriculture and forestry is expected to be profound, providing fundamental knowledge for the construction of new plant varieties with superior agronomic traits. An equally important aspect of this program is training, which will be provided at a variety of levels, including outreach to high school and undergraduate students as well as postdoctoral mentoring.

Intellectual merit. The response of organisms to their environment is the consequence of biological processes that turn genes on and off. These regulatory processes involve the concerted activity of many genes and proteins. Hence, genes and proteins are not independent units, rather their actions are deeply interconnected, influencing each other in their activity and regulation. Experimental approaches that combine different types of data from different types of experiments can be a very powerful way to gain new insights into the gene networks that control these processes. Plant science is now at the crossroads of being able to harvest all of this information for all the genes in a genome. However, very little effort has been made to undertake the more daunting task of assigning biological meaning or functions of all these genes and gene networks on a global scale. During the past decade, vast quantities of genomic resources have been generated, including many genome sequences, knockout collections of plants with mutations in most genes, large clone collections that allow facile manipulation of the expression of any gene, along with various types of genome maps that tell us when genes are turned on and off. By integrating all of these different kinds of data and using all of the resources developed over the past decade, we now aim to create a new type of map called a ?plant growth network?. The goal of this research is to apply a ?systems biology approach? using the tools of genomic science (genome sequences, protein; DNA maps, gene expression maps, gene knockout mutation collections, and other datasets) to understand how plant hormone responsive gene networks control the growth of plants. The long-term impact of this research on agriculture and forestry is expected to be profound. This fundamental information will enable engineering of new crop varieties with superior agronomic traits and will dramatically promote the ability to engineer plants to adapt to a rapidly changing environment.

Broader impacts. The broader impacts of this project will be in two areas. First, the completion of this research will result in important new results for the plant biology community ? it will produce a set of large-scale transcriptional regulatory network information for responses which are linked to all of the major growth-regulating plant hormones. These growth modules are currently very limited in availability thus this knowledge-base will have a large impact on a variety of research studies in both basic plant science and agriculture. All of the results of the research will rapidly be made accessible to the research community. An equally important broad impact area of this research program is student training, which will be provided at a variety of levels, including outreach to high school and undergraduate students as well as postdoctoral mentoring.

Intellectual merit: Plant biologists need many completely sequenced and functionally annotated genomes within each species to fully exploit the power of evolution and to understand how an organism functions and adapts to its environment. This project will contribute to an international effort aimed at generating genomic DNA sequences from over 1,000 inbred strains of Arabidopsis thaliana, while driving technology developments in both the hardware for the DNA sequencing itself and in software development to make sense of the sequence data. Specifically, this project will generate genome, transcriptome, and methylome sequences. The genome sequences will provide an unparalleled view of haplotype structure and structural variation for Arabidopsis thaliana. The transcriptome sequence data will assist in genome annotation and identification of naturally occurring splice isoforms. Lastly, the DNA methylome sequencing data will permit identification of epiallelic variation on a population-wide level. These three sequencing data sets (genetic, transcriptional and DNA methylomes) will dovetail nicely with each other to provide a comprehensive set of variants within the Arabidopsis thaliana population. Combining genetic, epigenetic and transcriptional variation from different individuals in a population or within a species will provide a potential source for analyzing phenotypic diversity via quantitative trait loci mapping or genome-wide association studies. Moreover, these data will enable subsequent mechanistic studies through experimental manipulation of Arabidopsis thaliana strains and will complement the efforts of individual investigators who are using the same accessions to catalog phenotypes for thousands of traits.

Broader impacts: The impact of this project will be in two broad areas. First, the completion of the planned research will result in important new resources for the plant biology community: large-scale information on genetic, epigenetic and transcriptional variation within a species. All of the DNA, cytosine methylation and RNA sequence data will be made freely and easily accessible to the research community. The long-term impact of these enabling tools and technologies on agriculture and forestry is expected to be profound, providing fundamental knowledge for the construction of new plant varieties with superior agronomic traits. An equally important aspect of this program is training, which will be provided at a variety of levels, including outreach to high school, undergraduate and graduate students as well as postdoctoral mentoring.

DESCRIPTION (provided by applicant): Epigenetic regulation of gene transcription, specifically when related to changes in DNA-methylation patterns (methylome), is a plausible mechanism underlying long-term environmental contributions to neuropsychiatric disorders. For example, pharmacological or environmentally-induced methylome alterations may lead to the silencing or aberrant activation of genes involved in the postnatal maturational process of brain circuitry, leading to functional and behavioral alterations appearing when the system reaches maturity. We and others have shown that activation of oxidative stress mechanisms during the period of maturation of brain inhibitory neurons leads to permanent neurochemical changes and schizophrenia-like behavior when animals reach adulthood. However, the mechanisms by which oxidative stress leads to disruption of the brain maturational process are unknown. Oxidative stress and inflammatory mediators are known to lead to epigenetic alterations in cancer, and activation of such mechanisms may have profound consequence during critical periods of brain maturation. Our preliminary findings suggest that activation of oxidative stress mechanisms during early life may produce epigenomic modifications, due to methylome changes, that affect neurodevelopment and thus may underlie the origins of the schizophrenia syndrome and possibly other mental disorders. We will test this hypothesis during postnatal development of frontal cortex of mice subjected to two developmental manipulations, known to lead to schizophrenia-like behavioral and neurochemical alterations in early adulthood. Three specific aims will be developed: Aim 1 will use MethylC-Seq to produce genome-wide, single-base resolution maps of methylated cytosines (methylome) during mouse postnatal brain-development at the tissue and brain cell-type levels, and determine the consequences of methylation changes at the transcriptional level by RNA-Seq (transcriptome). Aim 2 will determine the methylome and transcriptome changes induced by two non-overlapping neurodevelopmental models of schizophrenia in the two major neuronal populations in frontal cortex, and will produce transcriptome data for all inhibitory subtypes at two developmental time points. Aim 3 will determine whether treatment-induced methylome and transcriptome changes can be observed in peripheral blood cells (neutrophils). Health Impact: The proposed studies will produce a complete map of the mouse frontal cortex methylome, at the tissue and cell-type level, during the period of postnatal development until adulthood. Moreover, it will delineate the methylome changes and transcriptional consequences produced by two developmental manipulations that lead to schizophrenia-like behavior in adulthood, at the neuronal and peripheral tissue level. By making the data publically available, it will serve as a standard reference for methylome and transcriptome databases that can be consulted in relation to neuropsychiatric disorders with known and unknown developmental origins.

Intellectual Merit: This project aims to develop genome-wide experimental tools and technologies for analyzing gene regulation and function in Arabidopsis. Computational analysis of the initial genome sequence of Arabidopsis thaliana, completed in the year 2000, provided evidence for the existence of approximately 25,500 protein-coding genes. Intensive efforts during the past ten years to experimentally annotate this genome sequence have now identified 31,128 genes, which include additional protein-coding genes as well as non-coding RNA genes. The identification of T-DNA insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the available set of TDNA mutants reveals that ~24,000 additional mutant alleles are needed to create a comprehensive homozygous mutant collection. This research specifically addresses the following goals: to identify two genetically stable loss-of-function mutations in all Arabidopsis genes and to complete the goal of isolating two homozygous alleles for every gene in the genome. llumina paired-end deep sequencing will be used to identify mutations in these "missing" genes, thereby allowing completion of the "unimutant" for the annotated set of Arabidopsis genes. Further development of the T-DNA-Seq method for large scale capture and sequencing of ~ one million T-DNA insertion sites will allow identification of the inventory of essential genes as well as provide insights into the mechanism of T-DNA integration and associated gene silencing events.

Broader Impacts: The genomic resources developed by this project will be widely available to a large number of researchers and will provide the basis for a variety of research projects that rely upon whole genome information. Completion of the proposed research will provide a new important resource for the plant biology community, enabling a variety of genome-wide mutant screens for any visible phenotype of interest. An important feature of this research is that all of the mutant plants/populations will be available to the research community as soon as they are produced. The beneficiaries of this program will be the entire plant biology community, providing essential reagents necessary to elucidate the functions of the Arabidopsis genes. Additionally, the new technology developed to rapidly and inexpensively index insertion mutants by next generation sequencing in any plant or animal will have applications far beyond Arabidopsis research. The long-term impact of these enabling tools and technologies on agriculture is expected to be profound, providing fundamental knowledge for the construction of new plant varieties with superior agronomic traits. An equally important aspect of our research program is the hands-on training in plant genome research that will be provided at a variety of levels, including outreach to minority high school and undergraduate students.

This INSPIRE award is partially funded by the Genetic Mechanisms and Cellular Dynamics and Function Programs in the Division of Molecular and Cellular Biosciences, by the Plant Genome Research Program in the Division of Integrative Organismal Systems, and by the Division of Emerging Frontiers in the Directorate for Biological Sciences, and also by the Biotechnology, Biochemical, and Biomass Engineering Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems in the Directorate for Engineering.

Epigenetic modifications to the DNA and protein components of chromatin are important determinants of gene expression, and dynamic epigenetic changes are thought to underlie the ability of organisms to rapidly respond to environmental perturbations. Much of what is understood about such regulation has been obtained through so-called ensemble approaches, which determine epigenetic modifications from cells isolated from mixed tissue types; thus, the data represent an average of epigenetic and gene expression profiles of multiple cells. Recent technical advances have made it possible to obtain DNA sequence information and gene expression profiles for single cells, but it is not yet possible to obtain epigenetic information from single cells. The ability to monitor dynamic epigenetic profiles in single cells would revolutionize the field of epigenetics by enabling hitherto impossible studies of stochasticity, stability, and heritability of epigenetic responses. This project seeks to develop and implement an innovative pipeline for controlled hormone and stress perturbation of plant cells, followed by single-cell analysis of genome-wide DNA methylation profiles. An integrated micro- and nano-fluidic device will be built to enable single-cell isolation from specific cell types and subsequent chromatin sorting. Then DNA methylation information will be extracted from the isolated cells via new kinetic analysis of real-time sequencing data produced by ultrasensitive FRET-based single molecule sequencing. If successful, this new technology will enable analysis of epigenomic profiles of single cells, thus making it possible to address fundamental questions about epigenetic regulation in a revolutionary new way, not only in plants, but also in mammalian and microbial cells.

In addition to the scientific impact, the project will have broad educational impacts by offering opportunities for cross-disciplinary training at the interfaces of molecular biology and nano-engineering. Postdoctoral and graduate students will be involved directly in carrying out the research, and the technology developed in the project will be incorporated into hands-on laboratory training modules for both graduate and undergraduate students.

? DESCRIPTION (provided by applicant): Understanding the exact cell-type composition in brain regions is fundamental to integrating physiological, behavioral and neurochemical data to systematically understand the brain structure and function. At present, although the major categories of cell-types present in brain have been defined, the different subtypes within these categories, as well as their location and connectivity are far from understood. DNA methylation (mC) is a stable covalent modification that persists in post-mitotic cells throughout their lifetim, defining their cellular identity. It was recently demonstrated that mC patterns in brain are highly dynamic throughout development, and that there are clear differences between the major types of cells i.e., neurons and glia, in the rodent and human cortex. These analyses have been taken a step further and have produced data at the cell-type level that shows that each neuronal type carry specific mC signatures in their genomes that define the population they belong to. These results now open the possibility of producing a catalog of cell-types in brain defined by methylome signatures. This proposal will utilize this cell-type-specific base-resolution methylome data to produce complete maps of cell-types in the rodent brain in situ, and by this means develop a systematic inventory and census of cell types in the brain based on an integrated view of their molecular identity. Based on preliminary results showing clear-cut differences between the methylomes of specific neuronal types, we propose to use cutting edge technology to discover and test specific differentially methylated regions that define, at the molecular level, cell-populations in the frontal cortex of mice. The results obtained will be made publically available, and will serve as foundation to produce a complete genomic census of cell-types in a brain region that can be scaled to the whole brain. If successful, the approach could be ultimately tested in the primate brain. This proposal is thus responsive to RFA MH-14-215 BRAIN Initiative: Transformative Approaches for Cell-Type Classification in the Brain (U01).

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a highly heritable, but genetically complex, neurodevelopmental disorder. Increasing evidence points to an interaction between genetic vulnerability and environmental risk factors in the generation of ASD. Human genetics and animal models suggest that alterations in synapse formation and maintenance may be fundamental to the etiology of ASD, and recent human exome-profiling studies suggest transcription and chromatin remodeling functions may be affected. Epigenetic regulation of gene transcription, including through developmentally dynamic and cell type-specific patterns, is a plausible mechanism mediating long-term environmental contributions to ASD. Environmental impacts on the overall configuration of DNA methylation (methylome) may lead to aberrant silencing or activation of genes involved brain circuitry maturation, with subsequent functional and behavioral consequences. Recently, the first analysis of whole- genome single-base resolution methylome maps of developing frontal cortex in mice and humans revealed extensive methylome reconfiguration during development from fetal to young adult. Importantly, a specific form of cytosine methylation, in non-CG sites, accumulated preferentially in neurons, coinciding with the period of synaptogenesis in both species. These results point to a potential role of the neuronal methylome in healthy development of neural circuits that could be particularly vulnerable to pathological disruption. Leveraging on these data, as well as on preliminary data showing dynamic changes in CG methylation patters during the transition between embryonic and early life in mouse brain, this proposal will test the hypothesis that alteration of specific forms of DNA methylation are involved in the origins of ASD. To test this hypothesis, Aim 1 will produce single-base resolution methylome maps in the two major neuronal populations in frontal cortex i.e. excitatory and inhibitory neurons, in a well-established model of ASD, the maternal immune activation (MIA) model. Additionally, to test for additive effects of environmental exposures in a compromised gestation, Aim 2 will expose the MIA animals to the environmental toxin PBDE. Finally, Aim 3 will analyze the methylome changes produced by MIA in animals that are deficient for the autism risk gene Shank3 to test for gene x environment interactions. The transcriptional consequences of methylome changes will be assessed by RNA- Seq for each neuronal population at each time-point during development, and long term behavioral consequences will be assessed through a battery of behavioral tests relevant to ASD. New, sophisticated computational analysis procedures will be used to integrate diverse and large-scale empirical data sets to provide a powerful and stringent test of our hypotheses.

Project Summary/Abstract The epigenome is the ensemble of chemical modifications of DNA and chromatin that modulates genomic activities such as the transcription of messenger RNA, which further serves as the template of protein syntheses. The epigenome plays instrumental roles in gene regulation during human development and in healthy as well as disease conditions. Although the genome is nearly identical in each cell in the human body, epigenomes are highly dynamic across tissues and between cell types composing the tissues/organs. The current knowledge of the epigenome is largely built on analyses of human tissues without distinguishing the potential distinct epigenomes of individual cells. The heterogeneity of certain biological processes, such as the reprogramming of somatic cells to the highly medically valuable induced pluripotent stem cells (iPSCs), also requires the capability of analyzing the epigenome and transcriptome of single cells. A powerful approach for studying epigenomic regulation is to profile multiple components of the epigenome (e.g. DNA methylation or histone modifications) and the transcriptome from the same sample. Such multi-dimensional analysis of single cells is highly challenging since most current methods are uni-dimensional. High-throughput sequencing based methods will be developed by the project to analyze multiple epigenomic components, including the DNA methylome and chromatin accessibility, and the transcriptome of single human cells. These methods will be first developed using cultured human cells and later adapted to primary human tissues. As a model for assay development, the methods will be applied to study single cell epigenomic diversity during the somatic cell reprogramming process that generates iPSCs. If successfully developed, these methods will greatly facilitate epigenomic studies of diverse cell types in complex human tissues and in heterogeneous diseases such as cancer.

This project seeks to develop a facile method to mine plant-microbe interactions for key molecular targets of microbial factors that influence plant performance. Plants and microbes traffic molecules to each other during interactions that range from symbiotic to pathogenic. Plant biologists are beginning to appreciate the continuum of these interactions at the molecular level. Most current experimental paradigms center on pathogenic or symbiotic microbes in mono-association with a specific plant. These systems are highly developed and feature various molecular and genetic tool kits for research. However, many important plant-microbe interactions, especially those causing devastating diseases, are experimentally under-developed. Here a method will be developed that can serve as a general tool to understand protein-protein interactions in any plant-microbe interaction. The project is expected to produce original insights into the dynamic and intimate interactions between plants and their microbiota. The method could be subsequently widely deployed as an integral part of functional study of plant microbiota, thereby broadly impacting plant science research. The project will provide interdisciplinary training for undergraduates, doctoral and post-doctoral as well as outreach to high school and undergraduate students.

The method to be developed can serve as a general tool to understand protein-protein interactions in any plant-microbe interaction. If successful, the project will provide an unprecedented source of plant microbe protein interactome information that will be easily accessed and utilized by the research community. The data will be made publicly available via a curated PPIN database. The method is a high-throughput yeast two-hybrid (Y2H) system named ProCREate which exploits Cre-lox recombination to enable proteins to induce reporter gene expression of Cre Recombinase and subsequent Cre-recombination of plasmids containing mutant loxP sequences. The irreversible double mutant loxP linkage of each protein's corresponding coding sequence allows the identification of protein interactions using Illumina paired-end sequencing and bioinformatics analysis.

Abstract Understanding the exact cell-type composition in brain regions is a fundamental step when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although major categories of cell- types present in cortex have been defined through a handful of specific markers, the different subtypes within these categories, as well as their location and connectivity are far from understood. DNA methylation (mC) is a stable covalent modification that persists in post-mitotic cells throughout their lifetime, defining their cellular identity. Methylation patterns are cell type specific, differentiating the major types of cells i.e., neurons and glia, in the rodent and human cortex, as well as differentiating neuronal types in mouse cortex. However, little is known about what identifies the populations of neurons belonging specific categories within a brain region e.g. pyramidal neurons in different cortical layers or areas of frontal cortex. This project proposes to develop the technology necessary to address the challenge of producing single-neuron mC maps in a cortical layer-specific manner using human postmortem frontal-cortex tissue. Expansion of this methodology will ultimately lead to a thorough classification of all neuronal types in the human brain.

Abstract Understanding the exact cell-type composition in the different regions of the mouse brain is a fundamental step when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although major categories of cell-types present in the mouse brain have been defined through a handful of specific markers, the different subtypes within these categories, as well as their location and connectivity are far from understood. Epigenomic signatures such as DNA methylation (mC) and open chromatin are stable modifications that persist in post-mitotic cells throughout their lifetime, defining their cellular identity. Open chromatin as well as methylation patterns are cell type specific, differentiating the major types of cells i.e., neurons and glia, in the rodent and human cortex, as well as differentiating neuronal types in mouse brain. Research Segment 1 of this center proposes to produce a catalog of methylation and open chromatin patterns at the single-cell level throughout the entire mouse brain. Analysis of the combined data will permit the discovery of cell-type specific regulatory regions that will allow the production of transgenic mouse lines and viral tools that will become available to the community.

Abstract Understanding the exact cell-type composition in the different regions of the mouse brain is a fundamental step when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although major categories of cell-types present in the mouse brain have been defined through a handful of specific markers, the different subtypes within these categories, as well as their location and connectivity are far from understood. Epigenomic signatures such as DNA methylation (mC) and open chromatin are stable modifications that persist in post-mitotic cells throughout their lifetime, defining their cellular identity. Open chromatin as well as methylation patterns are cell type specific, differentiating the major types of cells i.e., neurons and glia, in the rodent and human cortex, as well as differentiating neuronal types in mouse brain. Research Segment 1 of this center proposes to produce a catalog of methylation and open chromatin patterns at the single-cell level throughout the entire mouse brain. Analysis of the combined data will permit the discovery of cell-type specific regulatory regions that will allow the production of transgenic mouse lines and viral tools that will become available to the community.

Project Description/Abstract - Administrative Core The Administrative Core will coordinate all scientific, administrative and external reporting activities of the CEMBA research program. The central objective of the Core is to facilitate reaching the milestones of discovery laid out in the CEMBA Research Segments, Data Core and Overall program research strategies. The Center Directors and the Scientific Leadership Committee will be the ultimate decision-making body for all CEMBA administrative and scientific policies. The Core will support a Program Manager as key personnel for overseeing all Center activities, and an administrative assistant for help in meeting planning and the generation of manuscripts and progress reports. The Core will coordinate bi-weekly CEMBA research team meetings, monthly leadership committee conference calls and the annual CEMBA retreat. The administrative core will also serve as the central conduit for interaction between CEMBA and the overall BICC Network. These activities include oversight of data sharing between the CEMBA-DC and the BCDC, participation of CEMBA investigators in BICCN conference calls and working groups, attendance of team members at BICCN in-person meetings, and the development of reports, websites and other outreach activities as an integral part of the BICCN.

Abstract Cytosine methylation and histone modification are epigenomic marks with effects on transposable elements (TE), transcription of genes and heterochromatin formation. While occurring mainly in a CG-dinucleotide context, DNA methylation in brain cells contains nearly an equal amount of non-CG methylation (mCH). mCH accumulates in neurons and correlates with transcriptional repression at a period coinciding with synaptogenesis and neuronal maturation. Embryonic CG-methylation patterns also change dramatically during the period between birth and the second postnatal week. The DNA methyltransferase Dnmt3a is highly expressed in brain during this period. Preliminary data in this application suggests that this enzyme is responsible for the accumulation of mC in neurons during the perinatal period. A conditional knockout mouse was created, in which deletion of Dnmt3a in pyramidal neurons occurs during the late embryonic period (~E15, driven by Neurod6-Cre). Contrary to results showing a shortened lifespan in animals with earlier embryonic deletion (driven by Nestin-Cre), or lack of phenotype when the deletion occurs past the second postnatal week (driven by CamK2a-Cre), NeuroD6-driven Dnmt3a-KO (pyrDnmt3a-KO) animals show no postnatal mC accumulation, have significantly altered gene expression, and develop pronounced changes in behavior without changes in lifespan. These results support the hypothesis that mC accumulation and patterning in neurons requires precise regulation of Dnmt3a activity during neuronal development. Based on these findings, it is proposed that mC accumulation during the perinatal period may be essential for the spatial and temporal gene regulation required for proper synapse development and circuit formation. This hypothesis will be tested by delineating the dynamics of Dnmt3a-dependent mC accumulation during brain development, by characterizing the disruptions in methylation patterns, transcriptional dysregulation and histone modifications in animals carrying a deletion of Dnmt3a in pyramidal and inhibitory neurons from cortex and hippocampus (Aim 1). To understand the mechanisms of activation of Dnmt3a during postnatal cortical development, this proposal will identify its binding-partners during the developmental transition between the first and second postnatal week in neurons using mass spectrometry of Dnmt3a immunocomplexes. It will also assess the requirement of these binding partners for Dnmt3a function by transcriptional knockdown experiments in cultured cells and animals using a viral deliver system (Aim2). Finally, this proposal will characterize the effects of Dnmt3a deletion on neuron development and synaptogenesis (Aim3).

  Project Summary/Abstract  Chromatin organization and epigenomic modifications such as cytosine methylation play instrumental roles  in gene regulation during human development and in healthy and disease states. Although the genome is  nearly identical in each cell in the human body, chromatin conformation and cytosine DNA methylation are  highly dynamic across cell types and during development. Previous studies showed that chromatin looping  can be regulated by cytosine methylation. However, our current state of knowledge of the interactions  between chromatin organization and DNA methylation is built on analyzing cultured cells and bulk tissues.  The interaction between cell-­type specific chromatin looping and methylation in heterogeneous tissues  remain largely unexplored. This project will develop single-­cell multi-­omic methods to jointly analyze  chromatin conformation and cytosine methylation from the same cell. The methods will lead to high quality  cell-­type specific chromatin conformation maps of human tissue, and the analysis of the relationship  between chromatin conformation and cytosine methylation. If successful, the proposed methods will greatly  facilitate the study of gene regulation in complex human tissues and in heterogeneous diseases such as  cancer.    

Abstract  Understanding the exact cell-­type composition in the different regions of the human brain is a fundamental step  when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although  major categories of cell-­types present in the human brain have been defined through a handful of specific  markers, the different subtypes within these categories as well as their location are far from understood.  Cytosine DNA methylation (mC) is a stable epigenomic signature that persists in post-­mitotic cells throughout  their lifetime, defining their cellular identity. Open chromatin marks gene regulatory elements that control cell  type-­specific gene expression patterns. Single cell DNA methylation and open chromatin profiles have been  successfully used to identify de novo distinct cell types in heterogeneous tissues including the brain.  This U01  aims to produce a first version of an epigenomic cell atlas at the single-­cell level across the human brain.   Multi-­modal integration between epigenomic and transcriptomic signatures will allow the identification of new  cell types and unique cell-­type markers that will become available to the community. Epigenomic profiling of  human brain cells permits the discovery of cell type-­specific regulatory regions, which will facilitate the  functional analysis of genetic variants associated with psychiatric and neurological disorders.  

ABSTRACT The mammalian brain is composed of 50% neurons and 50% non-neuronal glial cells, including astrocytes (20%) which regulate synaptic transmission and provide metabolic support for neurons, oligodendrocytes (25%) that speed up neuronal action potential conduction, and microglia (5-15%) which are tissue-resident macrophages with important roles in homeostasis and protection from infection. The ability to genetically target subsets of glial cells within a circuit, e.g. astrocytes in a given layer of the cortex, or astrocytes in the cortex and not in the thalamus, is lacking. Further, the ability to specifically target glial cells at early stages of development, without targeting other cells including neurons, is a challenge due to the promoters used often being expressed embryonically by radial glia that give rise to both neurons and glial cells. Therefore, there is a pressing need to identify new genetic points of entry to specific subsets of glia within a circuit at specific times, in order to allow cell-type specific manipulation to interrogate the role of glia in circuit function. To achieve this, a systematic classification of glial cell types and subtypes in the adult and developing brain is required. This project will approach this within the visual system of the mouse, using an innovative, recently developed single- cell multi-omics method to profile transcriptome and methylome from the same cell, called single-nucleus methylCytosine and Transcriptome sequencing (snmCT-seq). Applying snmCT-seq to study glial cell diversity will lead to a high-resolution classification of glial cell types across developmental stages and major regions of the mouse brain. Aim 1 will use snmCT-seq to profile glial cells from the adult (4 month) mouse visual system - retina (input region), dLGN (thalamic relay) and visual cortex (target area). This will identify cell-type and region-type specific regulatory elements that can be used to genetically access glial cells. In Aim 2 this project will analyze the same brain regions as a timecourse across late embryonic and early postnatal development, to identify stage-specific regulatory elements that can be used to target glial cells in early development. Aim 3 will test candidate regulatory elements for the ability to drive transgene expression in specific sub-sets of glial cells at specific developmental timepoints, using viral delivery strategies, to generate new genetic access to glial cell types. The outcomes of this work will provide an inventory of glial sub-types within a defined circuit, identify stage- and region-specific markers of glial cells, and identify regulatory elements that can target subtypes of glia. The viral tools developed in Aim 3 will enable researchers to target and manipulate glial cells in a circuit- specific manner, to interrogate the contribution of glial cells to neuronal circuit function. The use of viruses will allow use in multiple mammalian species, expanding the utility of this resource.

PROJECT SUMMARY  Brain  cells  exhibit  profound  molecular  and  cellular  changes  during  aging.  Epigenomic  marks  such  as  DNA  methylation are associated  with age  in  multiple human  tissues,  suggesting an  alteration  of  transcriptional  regulation during aging.  However,  age-­associated  epigenomic  signatures  have  not  been determined  with  cell-­ type  specificity  in  the  brain.  Single-­cell  epigenomic  strategies,  such  as  single-­cell  DNA  methylation  and  open  chromatin  profiling  assays,  are  powerful  strategies  for  de  novo  identification  of  cell-­type  specific  epigenome  landscapes in heterogeneous tissues. At the same time, these strategies uniquely allow the identification of cell-­ type specific regulatory elements that control gene expression patterns in complex tissues. The proposed project  will  complement  and  build  upon  current  NIH-­supported  BRAIN  Initiative  efforts  to  produce  an  epigenomic  cell  atlas  of  the  aging  mouse brain  at  the  single-­cell  level.   Single-­cell  DNA  methylome  and  chromatin accessibility  data will be generated to allow identification of cell types and cell-­type specific regulatory elements in the brains  of  middle-­aged  (9  month  old)  and  aged  (18  month  old)  mice,  and  in  brains  of  aged  mice  subject  to  caloric  restriction.  Aging-­associated  epigenomic  signatures  will  be  identified  through  comparison  to  single-­cell  epigenomic data generated from young mice generated by a BICCN U19 Center for Epigenomics of the Mouse  Brain Atlas  (CEMBA).  Through  generation of a  comprehensive epigenome-­based brain  cell  reference atlas  of  the aging mouse brain, the proposed research will provide invaluable resources for the aging-­research field.    

PROJECT SUMMARY ? Core B: Epigenomics The epigenetic modifications found in chromatin (DNA methylation and post-translational modifications of histones) are involved in gene regulation during development and differentiation. Core B will generate epigenomics data from massive parallel, multi-omic sequencing from human and mouse and xenopus developing neural tube. The Core will identify gene regulatory elements and epigenomic variants having the potential to cause or influence phenotypes. The Program PI and Director of Core B have worked together extensively in the past with great success producing significant mouse neural tube epigenomic data. Dr. Ecker has worked broadly in the area of genomics and epigenomics and in the development methodologies employing multi-modal ?multi-omics? techniques for generation multiple types of NGS datasets from single cells. The data generated from Core B, as well as imported from Project I, II and III, will be delivered to Core C for extraction of results which will be delivered to each of the Projects for further validation. These goals will be accomplished by developing the key pipelines of Core B that involve data production, and analysis: 1] Bulk whole genome bisulfite sequencing (WGBS) Pipeline. The Ecker lab published the first human methylome and thus has significant expertise in the workflow for data production. Our data workflow was established, and standard operating procedures developed and adopted, by the ENCODE project and will take advantage of the same features of our WGBS workflow that has made it successful for that effort. 2] Single cell methylome sequencing (snmC-seq) pipeline. The methods for production and analysis of single cell methylome data snmC-seq2 and more recently snmC-seq3, were established and standard operating procedures develop for the NIH BRAIN initiative. 3] Multi-omics analysis combining two different NGS datasets measured from single cells. These include snMethyl-3C-seq, snPaired-seq and snMethyl-HiC. The Epigenomics Core and the Bioinformatics Core will work together to perform analysis of all epigenomic data produced by Core B, will work with existing pipelines or create new pipelines as need demands, and will share data the Projects I, II and III. We anticipate that 25,000 single cell methylomes/yr will be generated, along with detailed analyses. Data processing quantification of genome wide unmethylated and methylated cytosine base calls are generated using an algorithm that we previously developed called Methylpy. Clustering of snmC-seq2 data for cell type classification will utilize both mCG and mCH patterns to effectively classify both neuronal and non-neuronal cell types in the developing neural tube. These analyses will result in a prioritized list of candidate marker features (genes and predicted regulatory elements) that will be provided to the project PIs for further examination and validation.