Light-Dependent composition of transcriptional complexes in plants
Due to their sessile nature, plants have evolved very sophisticated mechanisms to respond and adjust to the changes in environmental conditions. They do so through interconnected signaling pathways that trigger global changes in gene expression. Information about transcriptional regulation in response to different stimuli in plants relies mostly on the identification of the signaling pathways and transcription factors involved in a particular response. Importantly, investigations in yeast and humans indicate that the activity of the RNA Polymerase II (RNAPII) complex itself is subjected to a tight regulation by multiple interacting factors that respond to endogenous and exogenous stimuli. Therefore, environment-dependent changes in the activity of this transcriptional machinery seem a plausible new layer of regulation to fine-tune the transcriptional response to external stimuli in plants. In this proposal, we aim to use two complementary approaches based on quantitative mass spectrometry to isolate protein complexes in plant cells at different phases of the transcription cycle and, more important, in response to a major environmental stimulus, light. The first approach relies on the use of deactivated CRISPR/Cas9 to target proximal promoter regions and isolate proteins complexes involved in early events of light-induced transcription. In the second approach, we will identify RNAPII-associated complexes specific of transcription initiation and late elongation/termination. It relies on the use of antibodies that recognize Ser5P and Ser2P marks in the RNAPII, hallmarks of transcription initiation and termination, respectively, to isolate the region-specific RNAPII-associated complexes. Together, these two approaches will allow us i) to identify region-specific transcription machinery components in plants, both conserved and likely plant-specific ii) to study the dynamic composition of these complexes during the response to light.