COCA-seq: genome-wide mapping of O-GlcNAc-associated open chromatin

O-GlcNAcylation, a prevalent reversible post-translational modification, intricately alters non-histone proteins, influencing the organization of gene transcriptional regulation within the accessible chromatin environment. This nucleoplasmic landscape, characterized by histone-free regions, fundamentally enables O-GlcNAc-mediated modulation through dynamic accessibility. However, unraveling the O-GlcNAc-open chromatin interplay that governs sophisticated transcriptional regulatory networks remains constrained by current techniques, which lack the resolution to probe this spatiotemporal crosstalk. Here, we reported a general strategy to systematically and chemoselectively profile O-GlcNAc-associated chromatin accessibility on a genome-wide scale (COCA-seq). Through comprehensive validation across low- and high-throughput levels, we demonstrated COCA-seq's dual fidelity in both O-GlcNAc chemoselectivity and open chromatin specificity. We employed it to delve into doxorubicin resistance for breast cancer, scrutinizing pivotal regulatory genes and transcription factors implicated in this complex biological event, such as NRF1 and XBP1. By integrating bulk RNA-seq with COCA-seq, we offered a multiomics perspective, shedding light on related biological processes and pathways like drug efflux and stress homeostasis, thereby uncovering potential mechanisms by which O-GlcNAc-associated open chromatin orchestrates tumor drug resistance. COCA-seq emerges as a general and versatile tool across various biological contexts, poised to reveal the landscape of O-GlcNAc-associated open chromatin regions across the genome and decipher the significance of glycosylation behind it. We employed a chemoselective metabolic labeling strategy coupled with DNase I treatment to profile open chromatin regions bound by O-GlcNAc-modified non-histone proteins. Briefly, human breast cancer cells (MCF-7) and their doxorubicin-resistant counterparts (MCF-7/DOX) were metabolically labeled with 200 μM 1,6-Pr2GalNAz. The O-GlcNAz-modified chromatin was crosslinked, digested with 5 U/mL DNase I, and enriched via bioorthogonal chemistry. Genomic DNA fragments associated with O-GlcNAc-modified proteins were then de-crosslinked, size-selected (100-750 bp), and processed for next-generation sequencing (Chemoselectively O-GlcNAc-Chromatin Accessibility Sequencing, COCA-seq). In parallel, we performed RNA-seq to analyze differential gene expression between MCF-7 and MCF-7/DOX cells. Two biological replicates of COCA-seq and RNA-seq were both performed.