The fundamental element of chromatin is the nucleosome, which consists of 150pb of DNA wrapped around the histone core. Well-spaced nucleosomes are present in the body gene, while intergenic regions are characterized by fuzzy nucleosomes, with the presence of nucleosome-free DNA portions, which are also called accessible chromatin regions (ACRs). These short DNA sequences, placed in the non-coding genome, host regulatory elements for gene expression, and the lack of nucleosomes ensures access to the DNA of TFs and the transcription machinery. Their prediction can be challenging because they are small DNA sequences disperse in long intergenic -region, but the combination of information about chromatin accessibility, TFs binding profile and DNA sequence will facilitate their identification and it will help the scientific community to predict the expression pattern of specific genes and produce valuable tools. To characterize the chromatin architecture of the regulatory elements of genes responding to LRFR, we used ATAC-seq, which is a technique that uses the enzyme Tn5, a modified version of bacterial transposase, to fragment DNA and insert adaptors used by Illumina sequencing. Tn5 preferentially binds nucleosome-free DNA and hence it allows the isolation and sequencing of ACRs. The limiting step for this technique is the preparation of high-quality nuclei, which we achieved using a purification system based on biotin-streptavidin which is called INTACT. For INTACT, we needed Arabidopsis transgenic lines expressing a nuclear protein tagged with a peptide recognized by BirA, which is a bacterial protein driving the biotinylation of this peptide. Hence, during the first period of the project, I worked on the selection of these transgenic lines and the optimization of the INTACT protocol. The transgenic lines were in two genetic backgrounds: wild type and plants defective for LRFR-mediated auxin biosynthesis, this allowed to test whether the increase of auxin levels affects gene expression through the remodelling of chromatin accessibility. Both genetic backgrounds were analysed in HRFR (control condition), and after 1h or 25h of LRFR, to detect the short and long-term effects of LRFR. In this way, we produced a nice atlas of the ACRs for the LRFR-related genes, which can be used by other members of the host lab and, once the data will be published, by the scientific community interested in studying the expression pattern of specific genes in Arabidopsis at the early developmental stage of seedlings. Moreover, differential analyses revealed the regions showing an increase or decrease of accessibility after 25h of LRFR treatment, leading us to the discovery of new molecular mechanisms.
The results of this project were presented at seminars organized by the CIG and DBMV departments of our university (UNIL), at the Plant Biology Europe conference (June-July 2021) and the International Plant Photobiology Symposium (July 2021). Moreover, the writing of the paper describing the results is undergoing. Moreover, together with Martina Legris, another MSCA fellow of our lab, I participated in the following outreach activities for families and schools: “Science is Wonderful!” organized by the European Commission in Brussels, Belgium on 25-26 September 2019 and “Les Mystères de l'UNIL”, an event organized by the University of Lausanne in 2021, showing the amazing movements of plants mediated by light and experiments that boys and girls could easily reproduce at home to answer basic questions of plant biology.
