Arabidopsis; Retinoblastoma-related protein; stem cells; meristem; cell cycle; embryo-to-seedling transition; interacting protein
Shu Huan, Nakamura Miyuki, Siretskiy Alexey, Borghi Lorenzo, Moraes Izabel, Wildhaber Thomas, Gruissem Wilhelm, Hennig Lars (2014), Arabidopsis replacement histone variant H3.3 occupies promoters of regulated genes, in Genome Biology
, 15, R62.
Kuwabara Asuka, Gruissem Wilhelm (2014), Arabidopsis Retinoblastoma-related and Polycomb group proteins: cooperation during plant cell differentiation and development, in Journal of Experimental Botany
, 65(10), 2667-2676.
Derkacheva Maria, Steinbach Yvonne, Wildhaber Thomas, Mozgova Iva, Mahrez Walid, Nanni Paolo, Bischof Sylvain, Gruissem Wilhelm, Hennig Lars (2013), Arabidopsis MSI1 connects LHP1 to PRC2 complexes, in EMBO Journal
, 32, 2073-2085.
Shu Huan, Gruissem Wilhelm, Hennig Lars (2013), Measuring Arabidopsis chromatin accessibility using DNase I-PCR and DNase I-ChIP assays, in Plant Physiology
, 162(4), 1794-1801.
Shu Huan, Wildhaber Thomas, Siretskiy Alexey, Gruissem Wilhelm, Hennig Lars (2012), Distinct modes of DNA accessibility in plant chromatin, in Nature Communications
, 3, 1281.
Gutzat Ruben Borghi Lorenzo Gruissem Wilhelm (2012), Emerging roles of RETINOBLASTOMA-RELATED proteins in evolution and plant development, in Trends in Plant Science
, 17, 139-148.
Gutzat Ruben, Borghi Lorenzo, Gruissem Wilhelm (2012), Emerging roles of RETINOBLASTOMA-RELATED proteins in evolution and plant development., in Trends in plant science
, 17(3), 139-48.
Gutzat R Borghi L Fütterer J Bischof S Laizet Y Hennig L Feil R Lunn J and Gruissem W (2011), RETINOBLASTOMA-RELATED protein controls the transition to autotrophic development, in Development
, 138, 2977-2986.
Background and working hypothesis. The Retinoblastoma gene (RB) continues to be of intense medical interest and the focus of considerable research efforts because of its role in tumor formation. The currently best-understood function of pRB is the regulation of the E2F/DP family of transcription factors during the transition from G1 to S. pRB also interacts with a number of other proteins and transcription factors, but the biological significance of these interactions remains largely unknown. More recently pRB has emerged as a key regulator of development and differentiation processes, although the underlying molecular and biological mechanisms are not well understood, partially because of functional redundancies of the pRB pathway in mammalian cells. We discovered that the pRB pathway is conserved in plants and that the pRB-related plant homolog RETINOBLASTOMA-RELATED (RBR) is essential during gametophyte development. Using triplex analysis and conditional loss-of-function mutants in Arabidopsis, we have established that RBR is haploid insufficient and required for maintenance and differentiation of stem and pluripotent cell populations. In addition, we discovered that RBR controls the critical transition of the heterotrophic embryo into the photosynthetically active autotrophic seedling by targeting PRC2-mediated H3K27me3-dependent inactivation of late embryo-specific genes. Similar to pRB, RBR interacts with a large number of proteins, several of which are novel and involved in metabolic pathways. We therefore hypothesize that RBR has a novel function, which connects metabolic status and cell cycle control with specific developmental processes.Specific aims. We propose to dissect the RBR pathway in Arabidopsis to understand how RBR connects physiological and developmental status with transcriptional responses and differentiation programs. Specifically, we expect to identify (1) novel genetic networks in which RBR functions, (2) the mechanism by which RBR controls the embryo-to-seedling transition, and (3) the functional consequences of RBR interactions with novel stress- and metabolism-related proteins.Experimental design. Our work will take advantage of genetic and biochemical tools, conditional mutants and functional genomics approaches, and genome-wide profiling technologies. Using conditional expression of RBR-RNAi and RBRcs mutants in combination with epistatic interactions and transcriptome analysis we will establish the genetic and transcriptional network that is targeted by RBR. This work will be complemented by assignment of transcription programs to RBR-dependent regulators. Genome-wide ChIP-Chip in combination with transcriptome analysis will identify genes that become inactivated by RBR/PRC2-mediated H3K27me3 chromatin modification and promoters that are directly targeted by RBR. Mutants in specific metabolic pathways will be transformed with RBRi constructs to explore how RBR may connect metabolic functions to cell cycle regulation and development. We will validate selected RBR-interacting proteins using yeast 2-hybrid or split-YFP systems to expand the regulatory network in which RBR functions and open new research directions for the function of RBR outside of cell cycle control, both for the animal and plant fields as well as tumor biology.Expected value. Since RBR is encoded by a single gene in Arabidopsis and most of plant development is post-embryonic, Arabidopsis RBR represents a unique and genetically powerful system to advance our understanding of the pRB/RBR pathway and its regulatory function in cell cycle control, cell differentiation and important developmental transitions. Our proposed project will provide new insights that may facilitate clinically relevant research and new agricultural applications, especially for the critical embryo-to-seedling transition.