Department of Genetics, University of Georgia, Athens, Georgia, USA.
Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Dev Dyn. 2022 Apr;251(4):729-742. doi: 10.1002/dvdy.430. Epub 2021 Oct 23.
Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis.
To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences.
Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis.
细胞命运的决定受序列特异性转录因子与动态染色质景观之间的相互作用所调控。斑马鱼为探究驱动这些细胞命运选择的机制提供了一个强有力的系统，尤其是在早期胚胎发生的背景下。然而，与常规染色质分析方法相关的技术挑战，已经减缓了我们对在斑马鱼胚胎发生过程中染色质变化、转录因子结合和细胞分化之间的确切关系的理解。
为了克服这些挑战，我们对靶向切割和释放（CUT&RUN）和 CUT&Tag 染色质分析方法进行了改编，使其可应用于斑马鱼，并将这些方法应用于从整个中胚层期胚胎分离的组织中，以生成 H3K4me3、H3K27me3、H3K9me3、RNA 聚合酶 II 和组蛋白变体 H2A.Z 的高分辨率富集图谱。使用这些数据，我们确定了一小部分基因，这些基因可能在斑马鱼和小鼠原肠胚形成过程中都处于双重调控状态，为胚胎发育过程中 H2A.Z 景观的演变提供了证据，并证明了 CUT&RUN 检测 H3K9me3 在重复序列上富集的有效性。
我们的结果表明，将 CUT&RUN 和 CUT&Tag 方法与斑马鱼系统的优势相结合，在脊椎动物胚胎发生的背景下定义新兴染色质景观，具有强大的功能。
