1. ABSTRACT
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 invitro-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.