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

Abstract

ABSTRACTMicrococcal nuclease sequencing (MNase-seq) is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Analysis of MNase-seq data is complex due to variable degrees of sample quality, sequence bias of the endonuclease, and a high sequencing depth requirement. Therefore, we developed an optimized analysis pipeline, publicly available via Nextflow. We show the nucMACC pipeline is consistent and robust and provide recommendations for minimum sequencing depth, MNase titrations, and spike-in use. Besides, the pipeline provides high-resolution nucleosome positions and an automated way of calling non-canonical nucleosomes, un-stable nucleosomes, hyper-accessible nucleosomes, hypo-accessible nucleosomes, and stable canonical nucleosomes. Using the new nucMACC pipeline, we characterized nucleosomal properties, such as stability and accessibility, and explore how these properties change to regulate gene expression. Intriguingly, we find a distinct group of un-stable nucleosomes enriched at the TSS of promoters marked with motif one, an M1BP TF-specific motif. We show that Eukaryotic organisms can modulate nucleosome stability and change the RNA polymerase pausing rate. Modulation of stability is a dynamic process regulating development and response to environmental stimuli and represents a novel mechanism of gene regulation inD. melanogaster.