This project will lead to an increased understanding of how genomic processes are coordinated. The investigators are studying a DNA sequence called a Matrix Attachment Region (MAR), which can bind to a dynamic structure within a cell's nucleus called the nuclear matrix (or scaffold). MARs are thought to be important for DNA replication, nuclear organization and gene expression. This project, which uses the model plant Arabidopsis thaliana, will address a major question affecting the use of transgenic plants in agriculture. Frequently the expression of a transgenic trait is extremely variable from one transformant to the next, reflecting complete or partial silencing of transgene expression. Studies in both plants and animals have shown that adding MARs to transgenes can increase the average level of expression, probably by reducing the frequency of silencing. However, increased expression does not always occur, and even MAR- containing transgenes are sometimes silenced. This present project seeks to understand how MARs function, and why their effects are not always the same. In particular, the investigators will ask if MAR-flanked transgenes are transcriptionally silenced (not transcribed into RNA), or if the transgenes are actively transcribed, but the RNA is degraded as rapidly as it is produced. Importantly, they will use technology allowing them to compare transgenes with and without MARs at the known genomic positions, and to relate differences in MAR effects to differences in genomic features of each location, such as proximity to native genes or the presence of transposons, repetitive DNA, or modified versions of the histone proteins that package DNA into the chromosome. Direct measurements of transcription of each transgene will also be used to test the hypothesis that MARs act primarily at the transcriptional level.
Broader Impacts. This research will contribute to ongoing efforts to link the biochemistry of gene expression with organizational structures in the nucleus, and to a better understanding of the mechanisms controlling gene silencing and gene expression. A better understanding of these issues, including the molecular mechanisms of MAR effects, will support more sophisticated applications in eukaryotic genetic engineering, including new technologies to reduce transgene silencing. Examples of potential applications include the use of MARs to stabilize disease resistance, as well as pathway engineering for crop improvement, bioenergy applications, or production of pharmaceuticals and chemical feedstocks in non-food species. The project will also provide training and mentoring for a graduate student and a postdoctoral scholar. In addition, funding from this project will allow continuation and expansion of a successful collaboration with two Granville County Middle School teachers and the North Carolina Museum of Life and Science. The project has the potential to reach at least 10 middle schools in the Granville and Durham Counties, with as many as 1,000 middle school students directly benefiting from distribution and implementation of 'Science in a Suitcase' instructional kits (10 teachers, 100 students per middle school engaged in math and science classes for three years). Specific objectives include: engaging 10 middle school math and science teachers each year in a two day instructional workshop; providing follow-up support through the Museum of Life and Science staff; and providing instructional kits at no-cost to teachers who participate in the workshop.
Matrix attachment regions (MARs) are operationally defined as DNA sequences that bind to the nuclear matrix. We, and others, have shown that a tobacco MAR called Rb7 can be used to increase and stabilize transgene expression (termed a "MAR effect") in genetically modified organisms. Unfortunately, little is known about the molecular mechanism(s) involved in this MAR effect. A primary barrier that prevents us from understanding the mechanism is the difficulty encountered when analyzing transgenes without being able to control for major variables such as the genomic integration site or the complexity of integration patterns. To overcome these barriers and control for such variables we used a recombinase-mediated MAR excision system, which allowed us to produce five plant lines, each representing a different transgene (luciferase) insertion site, that either had, or lacked, flanking Rb7 MARs. We found that the Rb7 MARs stabilize luciferase trangene expression and transcriptional activity when the transgene integrates into genomic regions that are more condensed and inaccessible (heterochromatic), but have little effect when the transgene integrates into genomic regions that are not condensed (euchromatic). Our data support the hypothesis whereby the Rb7 MAR can prevent a transgene from being transcriptionally silenced when the transgene has integrated into condensed, inaccessible genomic regions. Our research contributes to ongoing efforts in many laboratories aimed at linking the biochemistry of gene expression (and gene silencing) with genome organization and subnuclear structure. In addition to the basic importance of such knowledge, we have contributed to having a better understanding of these issues, which will support more sophisticated applications in genetic engineering of higher eukaryotes. In plants, for example, one clear application involves the use of MARs to prevent transcriptional silencing of RNA interference (RNAi) inducing constructs that are being used to confer disease resistance and create gene knockdowns. More generally, as genetic engineering efforts expand to include larger numbers of genes and more sensitive modulation of their expression, scientists will need much more sophisticated tools and technologies that will depend on basic information such as that produced from our research.
