The long-term goal of this research is to enable scientists to manipulate the identity of specific tissues in plants to increase their agricultural potential. For example, this research may identify a factor that can be inhibited to enable leaf tissue to begin producing root proteins, and thereby form roots. Unshackling the constraints imposed by a fixed tissue identity could have a substantial practical impact by enabling modification of plants to enhance their utility for food, fiber, and fuel. All organisms contain the blueprint of how tissues are formed and what proteins are produced in genes in a highly organized dynamic complex of proteins and nucleic acids referred to as chromatin. Factors play a critical role in regulating production of tissue-specific genes that determine the developmental identity of specific tissues such as leaf and root. How genes are expressed in different tissues is of particular interest in plants because changes in gene expression during development are extremely malleable in this kingdom. Although some of the factors have been identified that control tissue-specific genes, very little is known about the regulatory mechanisms and machinery that enable genes to transition from one state or the other. The protein will study one of those factors to decipher the mechanism by which it can control gene regulation in plants. The proposed research is also aligned with the education mission of NSF. The Principal Investigator (PI) strongly supports undergraduate research and has considerable record of engaging undergraduates in research projects that provide them with a transformative scientific experience. These types of research experiences often play a critical role in prompting students to pursue a Science, Technology, Engineering and Math (STEM) career. In addition, graduate students will undertake the bulk of the proposed research, which will provide them with a solid foundation in science associated with research involving genomics, biochemistry, and genetics. The PI has an extensive experience in teaching undergraduate and graduate students and strongly believes in motivating topics in the classroom based on current efforts in the lab. Lessons learned by working with students on the proposed research will provide the bases by which the PI will continue to transform the educational experience of students as head advisor in the Department of Biochemistry.
The fundamental organizational subunit of chromatin is the nucleosome, which consists of DNA wrapped around an octamer of histone proteins. Factors that control chromatin structure, referred to as chromatin remodelers, play a critical role in regulating expression of tissue-specific genes that determine the developmental identity of specific tissues such as leaf and root. The protein that will be studied in this work, the chromatin remodeler PICKLE (PKL), is thought to contribute to both regulation of tissue-specific genes by serving as a component of a chromatin-based switch that enables both negative and positive regulation of these genes. Thus deciphering the mechanism of PKL action at these loci will illuminate for the first time how the machinery of plants enables fluid determination of gene expression and developmental identity. The proposed experiments will elucidate a fundamental chromatin-based pathway that restricts developmental identity in plants and thus, are likely to identify novel targets by which developmental identity can be manipulated in recalcitrant species. This proposal will examine the role of the chromatin remodeling protein PKL in determination of developmental identity in Arabidopsis thaliana, a plant that is widely used for laboratory research. PKL alters the structure of nucleosomes and is necessary for proper expression of genes that specify developmental identity. Many of these tissue-specific genes are regulated by the repressive epigenetic mark H3K27me3 (trimethylation of lysine 27 of histone H3 ? a specific modification of a histone protein), which is used to prevent expression of tissue-specific genes in inappropriate tissues (for example to keep root genes off in leaf tissue). PKL is necessary for both repression and activation of H3K27me3-regulated genes, indicating that it acts as both a repressor and an activator of gene expression. This proposal will address the hypothesis that PKL is a component of a chromatin-based switch that enables both negative and positive regulation of developmental genes. This proposal will characterize how PKL contributes to chromatin structure and expression from genes that are regulated by H3K27me3. Specifically, the project will analyze the chromatin of the entire genome to distinguish between different models of PKL action. It will examine how PKL activity is regulated by identifying the modification state of nucleosomes that interact with PKL as well as test the hypothesis that nucleosomes remodeled by PKL are a better substrate for the complex that promotes the H3K27me3 modification. Genetic and biochemical screens will be used to identify new factors and pathways that enable PKL-dependent processes.
