Bill Thompson

DNA replication is a highly choreographed process that integrates many aspects of genome structure and function, including transcriptional activity, chromatin structure, epigenetic states, and 3-D structure. However, almost all of our knowledge about DNA replication in higher eukaryotes comes from studies in metazoans. Evolutionary variation in DNA replication programs and their genetic control have not been studied in any plant system. The project will provide summer research and professional development experiences to undergraduates from underrepresented groups. The students will be actively recruited through established connections with HBCUs. The project will support the continuation of an annual summer workshop for under-represented highschool students, exposing them to maize genetics and modern plant research techniques. It will also support the Maize-10-Maze demonstration project during Year 3, and the production of a book combining the artistry and science of maize mutants to illustrate the genetic potential of important crops to the general public.

Intellectual Merit In spite of continued strong interest in epigenetic aspects of genome function, we do not yet know very much about the process by which plant and animal cells transmit epigenetic marks through multiple rounds of DNA replication and cell division. Because cell-to-cell inheritance is crucial to the function of epigenetic marks, understanding their biology requires an intimate knowledge of DNA replication as well as tools to study events occurring in S phase. In the proposed project, we will use the tools and knowledge of DNA replication that we developed in our previous PGRP project to characterize epigenome dynamics during S phase in two important model systems, Arabidopsis and maize. We will use combinations of flow cytometry and in vivo labeling to dissect multiple stages within S phase of cells in suspension culture and in planta. Using a genome-wide approach and making extensive use of deep sequencing technology, we will then investigate questions such as, Are post-translational modifications made immediately after replication of a given stretch of DNA? Are there differences in the timing of different types of modifications, or of modification events in different parts of the genome? To what extent does siRNA contribute to heterochromatin replication and inheritance? Do matrix attachment sites define domains with similar replication and modification kinetics? Do the attachment sites change during S phase? In addition, we will carry out experiments with mutants and artificial micro RNA knockdowns to explore the functional consequences of inhibiting selected modification pathways. Broader Impacts Knowledge of epigenome dynamics and the underlying mechanisms of epigenetic inheritance will enhance our understanding of fundamental processes in development and evolution, and will have practical impacts on plant tissue culture and micropropagation, plant breeding, and biotechnology. The effort will bring together investigators with expertise in biochemistry, molecular biology, genetics, genomics, and bioinformatics, and support a productive collaboration between two major research institutions. An excellent training environment for graduate and postdoctoral students will be provided, and selected undergraduates will be given the opportunity to participate in various aspects of the research. We plan two principal outreach efforts, one at each institution. At NCSU, we will continue and expand our successful collaboration with two Granville County Middle School teachers and the North Carolina Museum of Life and Science. We will update the ?Science in a Suitcase? unit on Genetics that we created in our present project, and continue to hold workshops for teaching training. At CSHL, we will inaugurate a program in epigenetics at the Dolan DNA Learning Center. This program will target advanced high school and faculty at two year and agricultural colleges. It will seek to update faculty and provide resources for teaching about epigenetics, and will provide a combination of web materials and podcasts as well as resources for experiments on imprinting and epigenetic inheritance. We will also host a regional workshop at a location to be determined, perhaps in conjuction with a plant biology professional meeting. The program at CSHL is expected to involve staff and PIs from NCSU as well as CSHL personnel.

Matrix Attachment Regions, or MARs, are thought to play an important role in chromatin organization in eukaryotic nuclei, and in many instances they have large positive effects on expression of transgenes. However, the mechanism(s) by which these effects are exerted remain obscure. We believe the difficulty reflects at least two major complicating factors. First, MAR effects on transcriptional silencing may be confounded by post-transcriptional gene silencing (PTGS), and the relative importance of these two types of silencing can vary with species, developmental stage, transgene copy number, and other factors. In order to reduce this confounding, we propose to measure transcription per se in addition to reporter gene expression, and to carry out parallel experiments in wild type and PTGS-defective backgrounds. Secondly, most MAR studies compare populations of individuals in which transgenes are integrated in a variety of genomic positions, configurations and copy numbers. This heterogeneity leads to high noise levels, and obscures locus-specific and general effects. To address this problem, we propose to use site-specific recombination (SSR) to excise MAR sequences from test constructs integrated into characterized sites with known genomic features. We have shown that both flanking MARs can be excised from a transgene in planta, allowing us to create isogenic lines carrying a single copy transgene with and without MARs. This approach will permit precise, locus-by-locus comparisons of MAR effects at defined locations in the Arabidopsis genome. Interpretation will be greatly facilitated by the ability to relate each insertion to its context in a sequenced genome, and the wealth of cytogenetic and epigenetic information available for this model system. To obtain a mechanistic insight into MAR effects on transgene expression, we propose the following specific aims: 1. Create and characterize isogenic transgenic lines with and without MAR elements 2. Evaluate MAR effects in WT and PTGS-defective backgrounds 3. Characterize MAR effects at the level of transcription 4. Describe MAR effects on chromatin state and DNA methylation Pairs of isogenic lines will be evaluated by reporter gene expression, transcript abundance, and an RNA polymerase occupancy assay to assess transcriptional activity. Transgene loci and nearby genomic sequences will be examined for chromatin conformation, DNA methylation, and histone modifications. In addition, we will evaluate characterized isogenic loci in a genetic background deficient in post-transcriptional silencing. Broader Impacts: Our results will provide a basis for thinking about the biological effects of MARs in transgenes, and the function of natural MARs in normal genomic contexts. In addition, the knowledge gained will contribute to future genetic engineering technologies. The project will be undertaken in an excellent, ethnically diverse training environment, and will provide training opportunities at undergraduate, graduate, and postdoctoral levels. Outreach efforts will include interactions with middle school students and teachers through established programs at NCSU.

We propose to take advantage of the unexpected opportunity to collaborate with the successful outreach effort initiated through Dr. Ralph Dean?s PGRP award at NCSU. Our activity within the collaborative effort will focus on middle schools in a rural/low wealth county with a substantial minority population. To maximize synergy, we propose to work with one or more schools that feed into the high school(s) served by our partner program, directed by Dr. Dean. We will introduce students to PGRP research processes through hands on activities and face-to-face interaction with North Carolina State and researchers and teachers. The goal of our program is to introduce students to our PGRP associated research, as it pertains to the NC Standard Course of Study, through hands on activities and face-to-face interaction with NCSU researchers. Teachers from the selected middle schools will spend 6 weeks during June/July of 2006 participating in our PGRP research working with researchers to translate their summer experiences into lesson plans and related instructional materials. The curriculum developed will translate our PGRP research into new modules of middle school curriculum aligned to the North Carolina Standard Course of Study. At strategic points in the 2006-2007 school year, the senior research personnel will make presentations to the middle school classes to discuss and demonstrate key concepts. Taken together, these proposed activities will create opportunities for teachers to participate in our research program, as well as offering a well coordinated program of hands-on, face-to-face interaction between research scientists and middle school students.

We propose to purchase an ABI SOLiDTM sequencing platform with ancillary equipment for sample preparation and array-based targeted genomic capture. The equipment brings deep-read sequencing technology to NCSU, which is needed for continued competitiveness and research productivity. It will be housed with other core genomic equipment in NCSU's Genome Science Lab (GSL). The new equipment will serve a diverse user community and will be an essential tool in research requiring i) full genome and targeted resequencing, ii) SNP detection, genotyping, genetic mapping, iii) small RNA discovery, and iv) transcriptional profiling. Research projects directly benefiting from the acquisition of the ABI SOLiDTM focus on understanding the relationship between genetic variation and phenotypic diversity and range from understanding how life persists in extreme environments to determining the mechanisms by which adaptive variation in butterfly wing patterns arise. The acquisition of the ABI SOLiDTM will be coupled with the development of targeted genomic capture methodologies and the establishment of core bioinformatics support for the GSL community. This combination of state-of-the-science equipment, technical expertise, and informatic support, makes the GSL unique among core genomic facilities, facilitates technology transfer among our diverse user community, and provides novel training experiences for our undergraduate and graduate students and postdoctoral researchers.