The Dean lab has recently cloned Arabidopsis (VAN) genes that mediate vernalization, the low temperature-induced promotion of flowering. Their structure and action on expression of their downstream target, FLC, suggest that they function analogously to Drosophila Polycomb-Group genes, thus explaining the epigenetic basis of vernalization. The parallels between Arabidopsis VAN and Drosophila Polycomb-Group gene suggest that the VAN proteins will act in a multi-subunit complex that binds to specific DNA sequences to maintain a transcriptionally silent chromatin state. To further investigate the molecular basis of vernalization, the objectives of the proposed work will be:
1) To characterize the proteins interacting with VRN2. This objective will be achieved through the use of affinity purification of protein complexes, the components of which will be identified using tryptic peptide fingerprinting and MALOI ToF/Q-Tof analysis
2) To test whether VRN2 bind directly to FLC. This will be achieved with gel-shift and BIACORE assays using purified recombinant VRN2 protein and sub clones covering FLC and flanking genomic sequences. If binding is detected actual binding sites will be determined using in vitro footprint analysis. The in vivo relevance of these sites will be confirmed by analysis of site-directed mutants in transgenic plants. If FLC is not a direct target we will use SELEX to define preferred DNA-binding sites to aid future target identification. The project that I will develop during this post-doctoral position should allow me to acquire new skills in the field of protein biochemistry especially the techniques associated with proteomics. Moreover the program involves a genetic approach in order to evaluate the function of the candidate genes. The acquisition of these techniques is of high interest to main the near future in order to secure a permanent position back in France.