Epigenetic Influences on Chromatin-Interacting Proteins and Transcriptional Responses to Stress in Maize

The cis-regulatory elements encoded by the genome and the epigenetic regulation that influences their function are necessary for defining organismal phenotype. However, a gap exists in our understanding of the role that epigenetic regulation plays in the interaction between cis-elements and the trans-acting factors that bind them. The focus of this work is on understanding these relationships by dissecting them in maize (Zea mays). In this dissertation, I describe my efforts to 1) characterize the target genes of RNA-directed DNA-methylation (RdDM), 2) to determine the comprehensive landscape of chromatin-interacting proteins of the b1 tandem repeat paramutation locus (b1TR), and 3) to characterize the physiological and transcriptional responses to severe drought stress in mutant plants that are defective in epigenetic silencing. To characterize the target genes of RdDM, I developed a chromatin immunoprecipitation (ChIP) protocol which enables recovery and analysis of chromatin associated with an epitope-tagged version of MOP1 expressed in mutant plants lacking RdDM components. These tools will be used to determine the genome-wide binding sites of the RNA Polymerase IV:MOP1 transcriptional complex by future members of our lab. To identify the chromatin-interacting proteins of the b1TR, I developed a single-locus immunoprecipitation proteomics method to capture and identify proteins cross-linked to an integrated b1TR transgene that is sufficient for transcriptional silencing. Characterization of the putative b1TR-interacting proteins revealed a potential overlap between the RNA-interference (RNAi) and RdDM pathways in transgene-mediated transcriptional silencing of the B-I allele. I also identified components of the RNA Polymerase II (Pol II) transcriptional complex and RNA-processing proteins, highlighting the involvement of Pol II transcription in b1TR regulation. I explored the role RdDM in stress response by exposing mop1-1 mutant plants which are defective in 24-nt small interfering RNA biogenesis to severe drought. I then analyzed their physiological and transcriptional responses to drought and learned that loss of Mop1 is associated with vegetative and reproductive defects which may be related to the misregulation of thousands of genes involved in stress response. Broadly, this work increases our understanding of gene regulation by identifying proteins that interact with a well-characterized epigenetically regulated cis-element and provides data that can contribute to global efforts for improving food security in a changing climate.