Optimized MNase-seq pipeline reveals a novel gene regulation via differential nucleosome stability

Micrococcal nuclease sequencing (MNase-seq) is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease, and the requirement for high sequencing depth. Here, we present the newly developed nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. nucMACC represents the first systematic, and genome-wide approach for detecting unstable ("fragile") nucleosomes. We characterized the regulatory nucleosomal landscape in D. melanogaster and S. cerevisiae. Two functionally distinct sets of promoters were identified, one associated with an unstable nucleosome and the other being nucleosome depleted. Chromatin structure analysis shows that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies. TeaserThe nucMACC pipeline quantifies the local and global functional alterations of chromatin structure.