ACCESSIBILITY OF GENOMIC REGULATORY ELEMENTS IN MACROPHAGES

The packing of eukaryotic genomes into chromatin plays a fundamental role in controlling DNA accessibility, important for different processes such as transcription, DNA replication and repair. Faithful transcriptional control in eukaryotic cells relies on the precise interplay between regulatory elements in the DNA, nucleosomes and transcription factors (TFs). The aim of this study was to analyze: 1) the nucleosome organization and chromatin accessibility at regulatory elements in differentiated cell types (notably murine macrophages) and 2) the role of the macrophage master regulator Pu.1, ATP-chromatin remodelers and H2A.Z histone variant in regulating chromatin accessibility. We generated high-resolution genome-wide nucleosome maps (by Micrococcal Nuclease digestion) centered on TSS-distal Pu.1 binding sites. We found regularly spaced nucleosome arrays with a nucleosome-depleted region centered on Pu.1 binding peaks. On the contrary, high nucleosome occupancy overlapping regions bound by Pu.1 in macrophages was detected in cells depleted of, or not expressing Pu.1 or in in vitro-reconstituted chromatin. Our findings suggest that Pu.1 actively maintains nucleosome depletion at regulatory regions. We then focused on the role of chromatin remodelers highly expressed in macrophages in regulating nucleosome landscape. In particular, we found that Brg1 strongly co-localizes with Pu.1 at macrophage regulatory regions, suggesting its active role in organizing chromatin accessibility at regulatory elements. We then investigated the genomic localization of histone variant H2A.Z. We found that it is highly associated with regulatory regions and Pu.1-bound sites in macrophages and its binding to macrophage genomic regions is affected by inflammatory stimulus. Finally we generated genome-wide DNase-seq, FAIRE-seq and ATAC-seq maps to study DNA accessibility and its changes after inflammatory stimulus.