Mechanism of chromatin accessibility, 3D chromosome organization, and their functions in gene regulation

Abstract of Parent Award: (R35 GM139654 “Mechanism of chromatin accessibility, 3D chromosome organization, and their functions in gene regulation”) Tightly controlled gene expression is essential for the fitness and development of all living organisms, with mis- regulation often leading to disease. Chromatin accessibility is a key factor of gene regulation in eukaryotic cells, where nucleosome positioning can either promote or inhibit transcription. Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) and Micrococcal Nuclease with sequencing (MNase-seq) are two wild- used high-throughput methods for evaluating chromatin accessibility. Although both techniques rely on similar principles and theoretically should yield comparable open chromatin regions, our preliminary studies of published ATAC-seq and MNase-seq data reveal substantial discrepancies. Specifically, out of the 4,800+ nucleosome depleted regions (NDRs) identified by MNase-seq in the Saccharomyces cerevisiae genome, less than half are detected as accessible by ATAC-seq. Given the popularity of these methods, a robust comparison is needed to improve the interpretability of their results. To address this, we will perform MNase-seq and ATAC-seq on matched samples and conduct a detailed bioinformatic comparison, investigating potential biases related to sequence composition, NDR size, and associated factors (Aim 1). Furthermore, to enhance ATAC-seq's sensitivity to small accessible regions, we propose a modified version termed single-hit (sh)ATAC-seq (Aim 2). By sequencing single instead of double-tagmented fragments, this approach will significantly improve the detection of short open regions. Together, we expect the research proposed here to result in more robust measurements and interpretations of chromatin accessibility in future studies.