Chromatin-Modifying Co-Repressor Complexes In Plants

Transcriptional regulation of gene expression represents one of the central requirements for many cellular events and for the correct development of the entire organism. Traditionally, transcriptional activation has received the most attention in studies of gene regulation whereas the fundamental role of transcriptional repression has gained recognition only recently. However, while the molecular mechanisms of transcriptional gene silencing in animal systems are being intensively studied, our understanding of these processes in plants is very sparse and, because plants likely utilize unique strategies to establish and maintain chromatin state, only limited use can be made of information available on epigenetic modifications in non-plant systems. To help fill this gap, a comprehensive study of Arabidopsis histone lysine demethylase (LSD1)-like co-repressor complexes was initiated. Three major lines of data were developed: (i) Three components of an LSD1-like co-repressor complex in Arabidopsis were identified: LSD1-like AtSWP1 and AtSWP2 proteins, and a direct interactor of AtSWP1 (but not of AtSWP2), AtCZS, a C2H2 zinc finger histone methyltransferase (HMT). (ii) AtSWP1, AtSWP2, and AtCZS were shown to block expression of a reporter gene in planta. (iii) Using reverse genetics, AtSWP1, AtSWP2, and AtCZS knockout mutants were shown to produce almost identical phenotypes (phenocopies) and chromatin modifications, consistent with the idea that these gene products may function in a single co-repressor complex. These findings will be used as a foundation to seek two specific goals: I. Biochemical characterization of purified recombinant AtSWP1/2, and AtCZS. AtSWP1/2 will be examined for their predicted biochemical activities: histone demethylase, polyamine oxidase, and FAD binding. AtCZS will be analyzed for its function as a HMT. The identity of histone modifications by AtSWP1/2 and/or AtCZS, i.e., specific core histone methylation marks, will be determined. II. Identification and initial characterization of a protein interaction network for activity of the AtSWP/AtCZS co-repressor complexes. Based on our preliminary data, AtSWP1/2 and AtCZS are not the only components of the co-repressor complex. Additional members of this complex will be identified by TAP-tag purification. Protein-protein interactions between these components will be characterized in planta by bimolecular fluorescence complementation (BiFC). The working hypothesis of this research, regards the plant LSD1-like co-repressor complex as a combination of plant-specific and general functional components and architecture. Thus, this project most likely will uncover novel aspects of chromatin-modifying co-repressor complexes that are not possible to address in other model systems. The proposed research activities will be integrated into the teaching of science at junior educational levels. Specifically, the laboratory will train, in addition to postdocs and graduate students, undergraduates and high school students (most of whom are minorities) who will learn scientific thinking and modern bioexperimentation.