Simultaneous Integration of Combinatorial Histone Marks by the Atypical PHD-finger Protein EDM2 in Arabidopsis Thaliana

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While the role of DNA in life processes is generally limited to the storage of genetic information, proteins have many structural roles and control nearly all biochemical processes in cells. Eukaryotic cells contain certain types of proteins, termed histones, which physically associate with DNA. Originally histones were found to be critical for the organized compaction of DNA that enables the DNA to fit into the nuclei of eukaryotic cells and to be faithfully transmitted (after duplication) to each of both daughter cells after cell divisions. During the past decade, a new type of biological code critical for the proper function of genes has emerged. This so-called histone or epigenetic code is manifested in certain chemical modifications of DNA and DNA-associated histone proteins. In contrast to the DNA code, which provides instructions for the synthesis of proteins, the histone code defines the activity states of genes and determines to what extent their genetic information is utilized. Numerous types of chemical modifications that can mark histone proteins at defined positions within their amino acid sequences have been identified. In some cases defined histone marks have been associated with enhanced or suppressed activity of genes. For example, the addition of an acetyl-tag to the 4th amino acid (a lysine) of the histone type H3 is often found in highly active genes, while methyl-tags at amino acid 9 (also a lysine) of H3 has been associated with suppressed genetic activity. It is also well established that certain proteins can ?read? this histone code and bind to these defined marks. When recruited to defined DNA regions by docking to their cognate histone marks, such histone binding proteins can alter activity levels of their respective target genes. Intellectual Merit-This project focuses on the characterization of a new type of histone binding protein in Arabidopsis plants. The EDM2 protein, which was originally identified as a critical regulator of plant immune responses, was found by the PI?s laboratory to specifically bind to the histone H3 protein bearing certain combinations of three different chemical marks. Responsible for this unusual property is a part of EDM2 related to the known-PHD finger domain. While conventional PHD-finger domains have been found previously to dock to either single or double histone marks, it is remarkable that the atypical PHD-finger domain of EDM2 appears to be unable to bind to single or double marks, but is only able to ?read? triple histone modifications. This novel mode of context-dependent histone binding behavior points to the existence of an additional, so far unknown, layer of the histone code, which is based on the combination of defined single marks. The PI proposes to perform a detailed analysis of histone-binding characteristics of the EDM2-type PHD finger domain. Besides biochemical experiments to understand how this type of ?histone reader? module distinguishes between different combinations of histone marks, studies will also be performed to identify which plant genes bear such ?higher-level of histone code words? that can be read by EDM2. If successful, this study will substantially advance our basic understanding of epigenetics, which is the biological discipline focused on the function of histone marks and chemical modifications of DNA. Broader Impacts-In addition to its scientific significance, this project will have broader societal impacts. An important component of the proposed project is an involvement of undergraduate students, mainly from minority groups. The project will be linked to classes on plant biochemistry, biotechnology and molecular biology taught by the PI and the ongoing NSF-REU Plant Cell Biology program within the Center for Plant Cell Biology at UC-Riverside. In addition, the proposed project will provide a strong platform for training of a postdoctoral scholar and a graduate student and prepare them for careers as senior researchers in academia or industry. The goals of the America COMPETES Act of 2007 that emphasize the importance of comprehensive professional training and awareness of the responsible and ethical conduct of research are a top priority of this project. By regulating an immune receptor gene, EDM2 has an important role in mediating resistance of plants against diseases. Therefore, the significance of this study goes beyond its impact on basic epigenetics and will likely benefit society by allowing new solutions in crop disease protection.