Project

Discipline

Arabidopsis; Retinoblastoma-related protein; stem cells; meristem; cell cycle; embryo-to-seedling transition; interacting protein

Lead
Lay summary
Lead The Retinoblastoma gene (RB) continues to be of intense medical interest because of its role in tumor formation. This project investigates the RETINOBLASTOMA-RELATED (RBR) pathway in Arabidopsis to understand how the RBR protein connects physiological and developmental regulation with transcriptional responses and differentiation programs. Background The currently best-understood function of the RB protein is the regulation of the E2F/DP family of transcription factors during entry into the cell cycle. RB also interacts with a number of other regulatory proteins and transcription factors, but the biological significance of these interactions remains largely unknown. More recently RB has emerged as a key regulator of differentiation processes, although the underlying molecular and biological mechanisms are not well understood. The RB pathway is conserved in plants and the RB-related plant homolog RBR is essential during reproduction. RBR is also required for maintenance and differentiation of stem and pluripotent cell populations. During seedling establishment, which is a critical transition in plant development, RBR cooperates with the Polycomb repressive complex (PRC2) to permanently inactivate late embryo-specific genes.  Scientific Goal The project uses genetic and functional genomics approaches to understand how RBR controls plant stem cell maintenance and connects cell cycle regulation to differentiation processes and developmental transitions. This will identify novel genetic networks in which RBR functions, the mechanism by which RBR controls the embryo-to-seedling transition, and the functional consequences of RBR interactions with novel stress- and metabolism-related proteins.  Significance RBR is encoded by a single gene in Arabidopsis. Since most of plant development is post-embryonic, RBR represents a unique and genetically powerful system to advance our understanding of the RB/RBR pathway and its regulatory function in cell cycle control, cell differentiation and important developmental transitions. The project will provide new insights that may facilitate clinically relevant research and new agricultural applications, especially for the critical plant embryo-to-seedling transition.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Communication	Title	Media	Place	Year

Number	Title	Start	Funding scheme

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.