Plants belonging to the legume family are able to interact symbiotically with nitrogen fixing bacteria named rhizobia, allowing these plants to grow in agricultural systems with low nitrogen inputs and thus to be important actors of sustainable agriculture. This symbiotic interaction leads to the formation of a new organ on the roots of the host plants, called the root nodule, inside which atmospheric nitrogen is fixed for the benefit of the plant. Nodule development is specifically controlled by the nuclear factor Y (NF-Y) transcription factor (TF) NF-YA1 that belongs to the CCAAT-box binding factor family. The heterotrimeric NF-Y complex is composed of the DNA-binding subunit NF-YA associated with two histone-like subunits NF-YB and NF-YC. Interestingly NF-Y is both a sequence specific transcription factor, binding CCAAT boxes, but also shows nucleosome-like properties, promoting chromatin accessibility for other master TFs. In animals, NF-Y is a central regulator of cell cycle progression and each NF-Y subunit is encoded by a single gene while in plants, small multigene families have specialized expression patterns and functions. Indeed, NF-YA1 is rapidly, strongly and specifically expressed during nodule development, and KO mutant lines only form few small nodules that lack a functional meristem. The aim of this project is to thoroughly understand the mode of action of NF-YA transcription factors in nodule development, a specific organogenesis. In WP I, we will determine using ChIP-Seq the binding sites for this TF during nodule development. IN addition, comparative transcriptome analysis using RNA-Seq in WT, mutant and overexpression backgrounds will be performed. The combination of these approaches will identify direct target genes of NF-Y complexes. In WP II, the epigenetic impact of NF-YA1 as a chromatin modifying TF will be investigated. In both WT and mutant backgrounds, we will perform ChIP-seq, using antibodies against active and repressive histone modifications and determine chromatin accessibility with a genome-wide ATAC-Seq approach with a particular focus on the potential target genes identified in WPI. The role of NF-YA1 on nuclear organization will be then investigated using chromatin conformation capture (3C) approaches. Then, the crosstalks between hormonal regulatory pathways, essential for nodulation and associated to chromatin modifications, and the epigenetic impact of NF-YA1 will be investigated. In WP III we will identify and characterize proteins interacting with NF-YA1 within multiprotein complexes to regulate gene expression and chromatin structure. This will be achieved by tandem affinity purification coupled to mass spectrometry (TAP/MS). In WP IV, we will determine whether NF-YA1 interacts with long non-coding RNAs (lncRNAs) in order to modulate genome topology and regulate target gene expression. For this purpose, lncRNAs interacting with NF-YA1 will first be identified using RNA-immunoprecipitation (RIP-Seq) assays and their role in target gene expression and chromatin structure will then be characterized. WP V will be dedicated to data analysis and integration using JBROWSE to allow a comprehensive picture of “omic” datasets and of the mode of action of NF-YA1 during nodulation. Integration of these data will serve to select a set of NF-YA1 targets and partners (proteins and lncRNAs) for functional studies in WP6. This project will associate research teams from the LIPM in Toulouse and IPS2 in Paris-Saclay with complementary skills in transcription factor analysis, chromatin-related techniques, epigenetics and hormone physiology. Data generated in the NODCCAAT project could help defining new strategies to promote better nodule development in legume crops during adverse conditions and to transfer nodule development into non-nitrogen-fixing crops thereby contributing to the ongoing worldwide effort to increase biological nitrogen fixation in agriculture.