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.
PI: Vitaly Citovsky, State University of New York, Stony Brook, NY 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. The following major sets of data were obtained: 1. Involvement of KDM1C histone demethylase-OLD1 otubain-like histone deubiquitinase complexes in repression of plant genes. Covalent modifications of histones, such as acetylation, methylation and ubiquitination, are central for regulation of gene expression. While KDM1-containing repressor complexes have been implicated in histone demethylation, methylation and deacetylation, whether or not they can also mediate histone deubiquitination remains unknown. We identified an Arabidopsis otubain-like deubiquitinase OLD1 which directly interacts with the Arabidopsis KDM1C in planta, and showed that both KDM1C and OLD1 are involved in transcriptional repression of the same target genes via histone deubiquitination and demethylation. We also showed that OLD1 binds plant chromatin and has enzymatic histone debubiquitinase activity. Thus, we suggested that, during gene repression, lysine demethylases can directly interact and function in a protein complex with histone deubiquitinases. 2. Characterization of the role of histone acetylation in regulation of root elongation. Transcriptional repression by histone modification represents a universal mechanism that underlies critical biological processes, such as neurogenesis and hematopoietic differentiation, in animals. In plants, however, the extent to which these regulatory pathways are involved in development and morphogenesis is not well defined. SWP1/LDL1 is a component of a plant corepressor complex involved in regulation of flower timing. We report that SWP1 also plays a role in regulation of root elongation by repressing a root-specific gene LRP1 via histone deacetylation. Thus, SWP1 likely functions as a regulator of developmental events both in the shoot and the root meristem. These research activities were integrated into the teaching of science at junior educational levels, including numerous undergraduates (most of whom are minorities) who worked on the project and learned scientific thinking and modern bioexperimentation.
