In Vivo Study of Nuclear-Localized Protein-Protein Interactions in Plant Cells

Recent results suggest that key events controlling light regulated gene expression in plants are import of the phytochrome photoreceptors into the nucleus followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COPl and the C0P9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness. Genetic analyses in Arabidopsis and tomato have revealed that the nuelear protein DETl also plays a key role in the repression of photomorphogenesis. However, up until now the mechanisms underlying its function have remained obscure. In this thesis through a series of in vitro experiments, persuasive evidence is provided that DETl binds to non-acetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, FRET imaging with GFP variants has been used to prove this interaction within the nucleus of living plant cells. DETl is therefore a novel chromatin interacting protein which binds to nucleosomes via interaction with unmodified H2B tails. Given the dramatic photomorphogenic phenotypes of detl mutants, this result infers that chromatin remodelling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis. An important part of this thesis was devoted to the establishment of a reliable FRET-based wide-field microscopy system for the detection of the interactions between GFP- (Green Fluorescent Protein) tagged proteins in living cells. Limits of some of the available techniques are shown together with many data proving that the established system is indeed able to reliably detect protein-protein interactions. Moreover, given the emerging importance of ehromatin remodelling in plants, the FRET-based microscopy system has been used with a histone-based eonstruet in an attempt to reveal chromatin dynamics in living cells. Data are presented about changes of chromatin structures, as detected by FRET measurements, occurring during the cell cycle of living BY-2 cells.