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

Abstract

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 report 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 demonstrate COCA-seq’s dual fidelity in both O-GlcNAc chemoselectivity and open chromatin specificity. We employ it to delve into doxorubicin resistance for breast cancer, scrutinizing pivotal regulatory genes and transcription factors implicated in this complex biological event. By integrating bulk RNA-seq with COCA-seq, we offer 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.

1. Introduction

Chromatin, an integral component exclusive to the nucleus of eukaryotic cells, comprises a complicated assembly of nucleic acids and histones and exists in two distinct states: closed and open. Closed chromatin ensconces DNA around histones, forming nucleosomes with minimal inter-nucleosome distances, impeding both transcription and replication processes [1]. In contrast, open chromatin, characterized by its accessibility, inclining itself to transcription factors (TFs), RNA polymerase II, etc., thus facilitating gene expression [2]. Open chromatin regions (OCRs) represent segments of chromatin in an accessible state, which contribute to protein-DNA interactions [3]. It has been reported that OCRs contain a large number of TF binding sites and exhibit dynamic histone modifications alongside diminished DNA methylation level which is crucial for modulating gene expression, introducing phenotypic alterations and steering cellular behaviors like differentiation and development [1,2,4].

O-GlcNAcylation, an essential post-translational modification (PTM) that attaches O-linked β-N-acetylglucosamine (O-GlcNAc) moieties to serine or threonine residues of proteins, is widespread across nuclear and cytoplasmic proteins, most notably non-histone proteins (NHPs) like TFs [5]. The critical regulatory roles of O-GlcNAc modification on chromatin-bound proteins and its influence on various facets of transcriptional regulation have been underscored in previous studies, encompassing the stability, transcriptional activity, interaction with chromatin, nuclear localization of NHPs [6]. Given the significant role of NHPs in gene expression regulation, deciphering the distribution of O-GlcNAc-modified NHPs within OCRs assumes paramount importance, offers insights into O-GlcNAc-associated chromatin accessibility and delineates lineage-specific transcriptional regulatory principles. High-throughput sequencing technologies such as DNase-seq and ATAC-seq have been extensively utilized to profile chromatin accessibility, enabling exploration of OCRs on a genome-wide scale across diverse biological processes [7]. However, there is still an absence of approaches that allow genome-wide enrichment of OCRs bound by O-GlcNAc-modified NHPs in the nucleus and accordingly investigation of their connections.