Name: Shannon Mills
Position: Honors in the Major Undergraduate
Contact: esm07c@fsu.edu
Hometown: Crawfordville, FL
Education: B.S. Biological Science (2011), Florida State University

Thesis Research: Characterization of maize transgene-reactivated mutants

Epigenetic regulation of gene expression results from changes in chromatin structure, DNA methylation and/or histone modifications. Such modifications allow for tissue specific expression of genes. The silent b1 genomic transgene (BTG-s) can be used as a tool to study epigenetic gene regulation in maize. A forward genetics screen based on reactivation of the silent BTG was conducted using EMS mutagenized maize. Through this screen several mutants, designated transgene reactivated (tgr), were identified that exhibited phenotypes corresponding to varying levels of transgene reactivation. Published work demonstrates that mutations of genes whose proteins are involved in the RNA-dependent DNA methylation (RdDM) pathway cause transgene reactivation (McGinnis et al., 2006). DNA methylation of promoter regions is frequently correlated with low gene expression levels.

The goal of this work is to characterize tgr mutants and determine if their phenotypes result from mutations in genes involved in the RdDM pathway. To do this, several molecular assays will be applied to compare the tgr mutants to previously existing and identified mutants. Bisulfite conversion will be used to evaluate DNA methylation of 6 different mutants in order to see if there are differences in methylation between the silenced and reactivated states of the transgene. For some experiments, the focus will only be on one mutant, tgr2, to allow for a more in depth analysis. Candidate gene expression for genes encoding proteins believed to be involved in an RdDM pathway will be evaluated in tgr2 using RT-PCR. A decrease in expression levels of one of these genes would suggest that the mutation which results in reactivation is affecting that gene. Previous work shows that a reduction in the number of 24-nt small interfereing RNA (siRNA) in maize correlates to a mutation in a gene that expresses a protein believed to be involved in the RdDM pathway (Nobuta et. al. 2008). SiRNA levels in tgr2 will be quantified using northern blot analysis.

Epigenetic regulation is involved in development and control of cellular functions, as well as in controlling repetitive and/or non-protein coding regions of the genome. Changes in the regulation of certain sequences in humans are correlated with the onset of cancers, diseases, or tumors. Further understanding of the maize tgr mutants will advance our understanding of epigenetic regulation in maize, a genetic model organism and major agricultural crop in the United States. A foundational understanding of maize’s epigenetic regulation processes can then be applied to other plants and humans.