PI: Henry W. Bass (Florida State University)
Co-PI(s): Jonathan H. Dennis (Florida State University), Karen M. McGinnis (Florida State University) and Oghenekome U. Onokpise (Florida Agricultural and Mechanical University)
Understanding how genomes are organized functionally is a major challenge in biology. Structurally, the genomes of eukaryotes, such as plants, animals or fungi, are organized into chromatin, which is a complex of DNA and associated proteins. The basic subunit of chromatin is the nucleosome, comprising ~150 base pairs of DNA wrapped around eight histone proteins. It is known that nucleosome positions in the genome vary with changes in gene expression levels. However, the specific relationship between genome structure and gene expression has not been clearly defined. The overall goal of this project is to characterize the chromatin structure of the maize genome in relation to function, specifically with respect to nucleosome occupancy and gene expression. The research uses computational, biochemical, genetic, and microscopic methods to accomplish three specific objectives: (1)DNA microarrays will be developed for genome-wide studies of chromatin structure. In the process new predictive tools will be adapted to the maize genome from technology developed for study of the human genome, (2) genome changes will be investigated in a mutant called mop1, which is thought to regulate chromatin structure through small RNAs and by silencing potentially deleterious genome changes; and (3) chromatin structure of normal plants and mop1 mutants will be visualized using 3D molecular cytology. The project will deliver plots that map sites of nucleosome occupancy in the maize genome and new tools for investigation of chromatin structure. In addition, custom microarrays will be available for use by the broader plant genomics community.
All data will be released to GenBank and will be available on the project website (www.maizenucleosome.org) and on public databases including www.maizegdb.org, www.gramene.org, and www.maizesequence.org. Direct involvement of diverse students in field and laboratory activities will provide training opportunities and exposure to plant genetics and genomic research. The project will continue a unique outreach activity for the public through a Maize-10-Maze chromosome-map field: each of the ten maize chromosomes is displayed as a single row to scale and with mutants in appropriate map positions. This life-size view of the maize chromosomes provides a tangible understanding of genome organization and engages participants in discussion about the importance of genomics research to meet societal challenges.
Project Investigators and co-Investigators compose a team of research professors from Florida State University (HW Bass, JH Dennis, and KM McGinnis) and Florida Agricultural and Mechanical University (OU Onokpise). The overall goal of the project was develop new ways to examine the genetic material of maize, primarily at the level of genome organization and dynamics. The genome of maize is typical of those from plant and animal species in that it is organized into chromatin, which is made up of DNA and various chromosomal proteins. The linear sequence of DNA bases along the chromosome fibers provides information about the location and structure of individual genes, but understanding how they are packaged, accessed, regulated, and expressed remains a major challenge in biology. One structural unit of chromatin that is known as the nucleosome is made up of about 150 base pairs of DNA wrapped around an octamer of eight histone proteins. These nucleosome particles number in the millions per individual cell. Nucleosomes not only help pack the DNA into a small volume, but they also can change in both their location along the DNA and their biochemical properties, which can affect the expression of the nearby genes. Over the lifetime of an organism, different genes must be turned on and off to respond to environmental changes, to mount defense responses, and to differentiate organs and tissues such as fruits or cereal grains. Understanding how plant species modulate their genome structure at the level of chromatin provided the primary rationale and intellectual merit of the project, further summarized at www.maizenucleosome.org. Scientific Findings: Using a combination of computational, biochemical, and genomic approaches, the project achieved several major goals including the first genome-wide nucleosome occupancy prediction for any plant species using a computer program first trained on human nucleosome occupancy data. These results (called "Nucleosome Occupancy Likelihood/NOL" plots) were released to the public via the maize genetics database clearinghouse, www.MaizeGDB.org, and a new genome browser available at www.GENOMAIZE.org (Fincher et al., 2013, Plant Physiology). The primary chromatin mapping scheme developed by this project is summarized in Figure 1. Using a 400 gene microarray-based experiment, the role of Mop1 (a gene implicated in genetic and epigenetic silencing) was examined at two levels, whole-genome and single-gene. Findings suggested a role for MOP1 in the regulation of higher-order chromatin organization, in concert with other regulatory pathways (Labonne et al., 2013 Epigenetics). The most recent outcome with major implications for future discovery research came from analysis of genetic regions that showed variation in chromatin structure from one experiment to the next. A major comparative study revealed that these variable-signal regions may actually represent key regulatory sites. Furthermore, these sites can be mapped using the "degree of digestion" as a controlled experimental parameter. Such assays not only explained the source of signal variation, but also identified areas referred to as fragile nucleosomes or hypersensitive regions, as summarized in Figure 2. Degree-of-digestion mapping developed by this project is expected to provide researchers with a new method for linking chromatin-level genome response to gene expression and traits in maize or other plant species. Broader Impact, Training, and Public Outreach: Over the course of the project, findings were disseminated via multiple public venues, including more than 14 meeting posters, 2 conference talks, 2 invited seminars, 3 published journal articles, and 3 published book chapters. For interactions with the public, the project partnered with the FAMU FACE program (about 20 high school students) each summer. The FACE (Forestry and Conservation Education) summer program is a three-week summer program with the objective of exposing minorities to the scientific disciplines of forestry and natural-resource conservation, including the genetics of plants and related disciplines. In the summer of 2012, the project organized a large public outreach event, the Maize-10-Maze, with over 250 visitors over two days. As part of the FACE summer laboratory at FSU, students isolated genomic DNA from tissues harvested in the field. In the field, the students learned to make crosses, became familiar with about 70 featured mutants, hung informative placards along the chromosome rows, and hosted the visitors Friday and Saturday, June 15-16, 2012. Field day events are summarized online at www.maizenucleosome.org/outreach and shown in part in Figure 3.
