Role of the DDB1 complex in integrating chromatin remodelling and DNA repair during plant development – DDB1 complex

Due to the critical importance of light for photosynthesis, plants have adapted their photosynthetic machinery and their development to use this energy source optimally, as well as to protect themselves from photodamage. They have therefore evolved complex mechanisms to respond to light signals and to control their development. For example, dark-grown seedlings display an elongated phenotype, which is necessary for them to grow up through the soil to reach the light. Upon light exposure, the leaves expand, stem elongation is inhibited, and cell differentiation is initiated. This process, known as photomorphogenesis, is mediated by the coordinated photoregulation of several thousand genes. Genetic studies in Arabidopsis thaliana and tomato have identified mutants affected in photomorphogenesis that have led to the discovery of a series of negative regulators encoded by the COP/DET/FUS loci. Analysis of the cop/fus mutants revealed that the affected gene products are involved in ubiquitin-mediated proteolysis, while DET1 was found to be part of a nuclear complex containing the plant homolog of UV-Damaged DNA Binding protein DDB1, a protein involved in DNA damage repair in mammalian cells. We have demonstrated that the DET1/DDB1 complex interacts with nucleosomes, suggesting that it may play a key role in chromatin remodelling during photomorphogenesis. Starting from these results obtained in the plant system, characterizion of the human ortholog of Arabidopsis DET1 (hDET1) subsequently revealed that it is part of a ubiquitin ligase complex containing DDB1, hCOP1 and CUL4A, which is involved in the control of c-Jun transcription factor accumulation. An emerging picture is therefore that DET1/DDB1 complexes play key roles in a range of regulatory mechanisms such as DNA repair, chromatin remodelling and protein ubiquitylation, and that these mechanisms are interconnected at the molecular level. The current project proposes to explore DDB1 complexes in plants by identifying their components and to explore their multiple roles in chromatin-level effects and DNA repair during photomorphogenesis. The project comprises three main objectives, based on state-of-the-art biochemical, molecular genetic, and cell biological methods: A. To investigate the molecular mechanisms controlling chromatin structure around light-regulated genes during photomorphogenesis, B. To characterize specifically the function of DET1-containing DDB1 complexes in the chromatin level regulation of gene expression during photomorphogenesis, and C. To examine the role of DDB1 complexes in DNA repair. This ambitious research proposal is based on a four year programme that will involve three French laboratories with demonstrated international reputations. The majority of tools, such as transgenic Arabidopsis lines, mutants, protocols, and tiled arrays are already functional in the proponent laboratories, and so the project will provide an excellent platform for the training of students and young researchers. The proposed programme of research will 1. define the changes occurring at the chromatin level around light-regulated genes during photomorphogenesis, 2. clarify the role of the basal chromatin regulating machinery, and in particular DET1-containing DDB1 complexes, during photomorphogenesis, 3. identify the genomic targets of DET1 and DDB1, 4. clarify the role of DDB1 complexes in DNA repair, and 5. explore the possibility that plant histones are targets for ubiquitylation by DDB1 complexes.