Understanding the mode of action of a pioneer transcription factor during symbiotic nodule development – PIOSYM

In eukaryotic nuclei, genomic DNA is compacted via chromatin in which DNA is wrapped around histone tetramers called nucleosomes. Most transcription factors (TFs) are unable to bind to their target sequences in compact chromatin regions or in the presence of nucleosomes. However, a group of TFs called “pioneer” TFs possesses the special ability to bind to inactive chromatin regions thereby modifying the accessibility of the bound regions either directly or via the recruitment of chromatin remodelers. These modifications of the chromosome landscape by pioneer TFs then allow other specific TFs to bind. Consequently pioneer TFs are important determinants of cell fate in many embryonic cell types in animal systems. In plants, pioneer TFs are barely described. Leguminous plants such as the model legume Medicago truncatula are able to interact with nitrogen fixing bacteria named rhizobia. This symbiotic interaction leads to the formation on the roots of the host plant of a new organ called the nodule, inside which atmospheric nitrogen is fixed for the benefit of the plant. The TF NF-YA1 belonging to the CCAAT-box binding factor family of TFs specifically controls nodule development. The objective of this project is to demonstrate that NF-YA1 functions as a pioneer TF regulating the root to nodule developmental transition and to understand its mode of action. The project will be organized in 3 Work packages (WP) aiming to demonstrate different aspects of pioneer TF activity. In WP1, we will assess the ability of NF-YA1 to regulate chromatin accessibility, DNA methylation patterns and 3D chromatin looping organization, characteristic of pioneer TFs. We will a) use a technique called ATAC-seq (Assay for Transposase-Accessible Chromatin followed by sequencing) to asses nucleosome occupancy in both WT and nf-ya1-1 mutant nodules; b) estimate DNA methylation, genome wide, by using BS-seq (Bisulfite conversion and sequencing) again in both WT and nf-ya1-1 mutant nodules; and c) apply chromosome conformation capture approaches to estimate the role of NF-YA1 on chromatin topology in nodules. In WP2, we will identify and characterise potential chromatin modifiers that interact with NF-YA1. We will first perform IP/MS (immunoprecipitation followed by mass spectrometry) assays and/or alternatively we will establish turbo ID-based proximity-dependent labeling techniques to search for transient or weaker protein-TF interactions. As DELLA proteins were previously shown to interact with NF-YA1 complexes, the implication of this interaction on chromatin accessibility will also be tested, as well as the cooperation of NF-YA1 with another type of proposed pioneer TF linked to cytokinin signaling. Selected chromatin modifiers interactions will be confirmed in planta through BiFC (bimolecular fluorescence complementation) as well as FRET/FLIM (Fluorescence energy transfer/ Fluorescent lifetime imaging) assays. WP3 is devoted to the identification and characterization of long noncoding RNAs (lncRNAs) potentially regulating NF-YA1. A combination of open and targeted approaches will also be used to a) isolate lncRNA molecules interacting with NF-YA1 using RIP experiments (RNA Immunoprecipitation) on isolated nodules; and b) selected NF-YA1 and co-regulated lncRNAs interactions will be confirmed using TriFC (tri molecular fluorescence complementation). These results will demonstrate the impact of NF-YA1 in the reconfiguration of the epigenetic landscape during the root-nodule developmental transition. Overall, this project should allow us to better understand a general and innovative mode of action of a plant pioneer transcription factor.