Epigenetics and post-translational control of gene expression at the chomatin level – CHROMAPKA

We analyze protein samples extracted from subcellular fractions of A. thaliana whole plants, in order to determine their amino acid sequences and covalent modifications, mostly phosphorylations that can affect serine, threonine and tyrosine residues. To identify proteins and their modifications that may vary upon stimulation of the plants, we carry out proteomic analyses which consist of the characterization of proteins digested into peptides using a specific enzyme, by the coupling between liquid chromatography and tandem mass spectrometry. Nowadays, to obtain the identity of a phosphorylated peptide, i.e. to determine both its amino acid sequence and the precise site of phosphorylation(s), still remains challenging. Yet it is of prime importance to identify correctly these sequences to be able to reconnect a dynamic phosphorylation to a protein kinase family, and to be able to decipher the phosphorylation cascades triggered within the cell following the external stimulus. Toward that goal, we have developed a bioinformatic tool providing a more reliable identification of phosphopeptides from mass spectra. The originality of this program, named FragMixer, lies in the fact that it can combine the information present in spectrum pairs acquired on each peptide, which allows increasing the probability of correct identification of phosphorylated sequences.

We have been able to analyze samples of nuclear proteins collected from plants submitted to a simulation of pathogen attack for 0, 5, 15, 30 or 45 min. The estimation of their relative abundance between the samples allowed verifying that the vast majority of them did not show significant variations. However, a few yielded a temporal abundance profile that might correspond to a movement of relocalization from/to the nucleus, and ideally from/to the chromatin environment. We need to compare the results obtained on two utterly independent biological preparations. Then, if some proteins show a coherent temporal profile between the two kinetics studies, we will consider validation assays, such as tagging with GFP those proteins that are assumed to relocalize within cells.
Dynamic phosphorylations of proteins may have a strong impact on their activity, stability, subcellular localization, etc. To better identify phosphorylation sites within protein sequences, we have developed a bioinformatics tool which is made available to the scientific community: FragMixer (http://proteomics.fr/FragMixer/). This program allows handling classical mass spectrometry data, which consist of the characterization of each peptide by a given fragmentation mode. It also allows combining the information present in two spectra obtained by two complementary fragmentation modes applied to each peptide, considering that the two spectra are likely to lead to higher confidence identifications.

Our project requires optimally designed proteomic analyses. First, we need to reliably identify phosphorylated sequences, to be able to link interesting substrates to their respective kinases. Second, we want to detect the subcellular relocalization of proteins: a well-thought experimental design and an adequate treatment of quantitative data are critical to extract statistically valid information. We have already developed analytical approaches to answer these questions, which we wish to render publicly available, so that other researches may implement them in other research contexts. In terms of biological knowledge, we hope to highlight or precise mechanisms of regulation in Arabidopsis by identifying key roles of certain proteins (histone desacetylases, etc) in the modulation of gene expression. We thus wish to contribute in a buzzing research area, as evidenced by the huge Encode project on the human genome.

We have started the project by establishing dedicated analytical methods to better characterize phosphopeptide samples. Both mass spectrometry acquisition methods and data interpretation were optimized. These progresses allowing a more robust identification of phosphopeptides have led to a first publication in the Journal of Proteome Research, which was accepted end of October 2012. The developed program, named FragMixer, is made available to the community of ‘proteomists’ at the following address: proteomics.fr/FragMixer/.

The objective of the presented project is to contribute, by proteomics and phosphoproteomics approaches, to the understanding of the molecular mechanisms occurring in time and space at the chromatin level to control gene expression. We wish to reconstitute, as in a molecular movie, which proteins shuttle to or from the chromatin and which get dynamically post-translationally modified to orchestrate a stimulus-triggered re-programming of gene expression. To address that issue, we have chosen a model system in which substantial knowledge of a particular signalling cascade has been acquired, with the clear identification of the protein components recruited from the plasma membrane receptor throughout the cytosol. We have selected the plant Arabidopsis thaliana, studied in the particular situation of a pathogen attack. When Arabidopsis is challenged by pathogens, its innate immune system detects pathogen-associated molecular patterns (PAMPs), which are molecules derived from abundant pathogen structures (e.g. cell wall constituents, etc). Specific signal transduction pathways are then activated to promote the modulation of defined sets of genes and eventually to develop adapted responses. More specifically, a three-tiered cascade of protein kinases known as the mitogen-activated protein kinase (MAPK) module is involved in the adequate response to pathogen aggression. After detection of PAMPs such as flagellin by the plasma membrane receptor FLS2, two main cascade branches involve the successive recruitment of MAP3Kinases, which phosphorylate MAP2Kinases, which finally activate by phosphorylation MAPK3, 4 and 6. Ultimately, MAPKs are expected to activate protein factors responsible for the increased or decreased expression of specific sets of genes, with the goal to counteract the pathogen assault. Some protein targets of MAPKs such as the transcription factors VIP1 and WRKY33 have been identified. However, most parts of the protein machinery orchestrating gene regulation in response to flagellin (flg22) have not yet been identified and no information on its temporal recruitment has been acquired; we propose to contribute to the further assembly of this complex dynamic protein puzzle. Arabidopsis thaliana will thus be used as a model system to understand links between signal transduction, chromatin dynamics and gene regulation.