COCA-seq: genome-wide mapping of O-GlcNAc-associated open chromatin [RNA-Seq]

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. Overall design: 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.

O-连接β-N-乙酰葡糖胺修饰（O-GlcNAcylation）是一种广泛存在的可逆翻译后修饰，可通过复杂机制调控非组蛋白，进而影响可及染色质环境中的基因转录调控网络组织。这类以无组蛋白区域为特征的核质环境，通过动态的染色质可及性，从根本上支持O-GlcNAc介导的基因调控。然而，当前技术仍难以解析调控复杂转录调控网络的O-GlcNAc与开放染色质的互作关系，现有技术缺乏探究这种时空互作的分辨率。
本研究报道了一种通用策略，可在全基因组范围内系统且化学选择性地分析与O-GlcNAc修饰相关的染色质可及性（COCA-seq，化学选择性O-GlcNAc-染色质可及性测序）。通过低通量与高通量层面的全面验证，我们证实了COCA-seq在O-GlcNAc化学选择性与开放染色质特异性两方面的双重保真度。我们利用该技术探究了乳腺癌的阿霉素耐药性，深入分析了这一复杂生物学过程中涉及的关键调控基因与转录因子，如NRF1与XBP1。
通过将批量RNA测序（bulk RNA-seq）与COCA-seq整合分析，我们获得了多组学视角，阐明了药物外排、应激稳态等相关生物学过程与通路，进而揭示了O-GlcNAc相关开放染色质调控肿瘤耐药性的潜在机制。COCA-seq作为一款通用且多用途的工具，可应用于多种生物学场景，有望揭示全基因组范围内O-GlcNAc相关开放染色质区域的分布特征，并解析其背后糖基化修饰的生物学意义。
实验设计概述：我们采用化学选择性代谢标记策略结合脱氧核糖核酸酶I（DNase I）处理，来分析与O-GlcNAc修饰非组蛋白结合的开放染色质区域。简要而言，将人乳腺癌细胞（MCF-7）及其阿霉素耐药株（MCF-7/DOX）用200 μM 1,6-Pr2GalNAz进行代谢标记。随后对O-GlcNAz修饰的染色质进行交联，以5 U/mL脱氧核糖核酸酶I进行酶切，并通过生物正交化学进行富集。将与O-GlcNAc修饰蛋白结合的基因组DNA片段去交联、进行片段筛选（100~750 bp）后，进行下一代测序，即化学选择性O-GlcNAc-染色质可及性测序（COCA-seq）。同时，我们开展了RNA-seq以分析MCF-7与MCF-7/DOX细胞间的差异基因表达。COCA-seq与RNA-seq均设置了2次生物学重复。