Zheng Qianwen, Wu Xiaotong, Li Xin, Mo Xianming, Xiang Bo, Chen Jing
Department of Pediatric Surgery and Laboratory of Pediatric Surgery, West China Hospital/West China School of Medicine, Sichuan University, Chengdu, China.
Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
Front Cell Dev Biol. 2024 Dec 4;12:1475912. doi: 10.3389/fcell.2024.1475912. eCollection 2024.
Histone modification signatures mark sites of transcriptional regulatory elements and regions of gene activation and repression. These sites vary among cell types and undergo dynamic changes during development and in diseases. Oocytes produce numerous maternal factors essential for early embryonic development, which are significantly influenced by epigenetic modifications. The profiling of epigenetic modifications during oogenesis remains uniquely challenging due to the presence of numerous tightly wrapped granulosa cells. Here, we successfully established a low-input CUT&Tag (Cleavage Under Targets and Tagmentation) method tailored for zebrafish stage I oocytes. This advanced technique enables high-resolution profiling of histone modifications and DNA-binding proteins, critical for understanding chromatin dynamics in developing oocytes. In this study, we detailed the workflow for this technique, including the isolation of pure stage I oocytes without somatic cells, library construction and quality monitoring. Our results demonstrate the method's efficacy by identifying distinct histone modification patterns and analyzing differentially expressed genes in oocytes with and without granulosa cells. We also successfully profiled divergent histone modifications in oocytes derived from wild-type and mutants. These advancements overcome technical challenges in epigenetic research on zebrafish oocytes and establish a solid foundation for exploring the epigenetic regulatory mechanisms of maternal contribution.
组蛋白修饰特征标记转录调控元件的位点以及基因激活和抑制区域。这些位点在不同细胞类型中有所不同，并在发育过程和疾病中经历动态变化。卵母细胞产生许多对早期胚胎发育至关重要的母体因子，这些因子受到表观遗传修饰的显著影响。由于存在大量紧密包裹的颗粒细胞，卵母细胞发生过程中表观遗传修饰的分析仍然具有独特的挑战性。在此，我们成功建立了一种针对斑马鱼I期卵母细胞量身定制的低输入CUT&Tag（靶向切割与转座）方法。这项先进技术能够对组蛋白修饰和DNA结合蛋白进行高分辨率分析，这对于理解发育中卵母细胞的染色质动态至关重要。在本研究中，我们详细介绍了该技术的工作流程，包括不含体细胞的纯I期卵母细胞的分离、文库构建和质量监测。我们的结果通过识别不同的组蛋白修饰模式以及分析有颗粒细胞和无颗粒细胞的卵母细胞中差异表达的基因，证明了该方法的有效性。我们还成功分析了野生型和突变体来源的卵母细胞中不同的组蛋白修饰。这些进展克服了斑马鱼卵母细胞表观遗传研究中的技术挑战，并为探索母体贡献的表观遗传调控机制奠定了坚实基础。
