Improved cohesin HiChIP protocol and bioinformatic analysis for robust detection of chromatin loops and stripes. Improved cohesin HiChIP protocol and bioinformatic analysis for robust detection of chromatin loops and stripes

Chromosome Conformation Capture (3 C) methods, including Hi-C (a high-throughput variation of 3 C), detect pairwise interactions between DNA regions, enabling the reconstruction of chromatin architecture in the nucleus. HiChIP is a modification of the Hi-C experiment that includes a chromatin immunoprecipitation (ChIP) step, allowing genome-wide identification of chromatin contacts mediated by a protein of interest. In mammalian cells, cohesin protein complex is one of the major players in the establishment of chromatin loops. We present an improved cohesin HiChIP experimental protocol. Using comprehensive bioinformatic analysis, we show that a dual chromatin fixation method compared to the standard formaldehyde-only method, results in a substantially better signal-to-noise ratio, increased ChIP efficiency and improved detection of chromatin loops and architectural stripes. Additionally, we propose an automated pipeline called nf-HiChIP (https://github.com/SFGLab/hichip-nf-pipeline) for processing HiChIP samples starting from raw sequencing reads data and ending with a set of significant chromatin interactions (loops), which allows efficient and timely analysis of multiple samples in parallel, without requiring additional ChIP-seq experiments. Finally, using advanced approaches for biophysical modelling and stripe calling we generate accurate loop extrusion polymer models for a region of interest and provide a detailed picture of architectural stripes, respectively. Overall design: FA-EGS-HiChIP for SMC1 and standard FA-HiChIP for CTCF performed in the HG00731 lymphoblastoid cell line from the 1000 Genome Project.

染色体构象捕获（Chromosome Conformation Capture，3C）技术，包括Hi-C（一种高通量的3C衍生技术），可检测DNA区域间的成对相互作用，从而实现细胞核内染色质高级结构的重构。HiChIP是对Hi-C实验的改良版本，其引入了染色质免疫共沉淀（chromatin immunoprecipitation，ChIP）步骤，能够实现全基因组范围内目标蛋白介导的染色质相互作用的鉴定。在哺乳动物细胞中，黏连蛋白复合体（cohesin protein complex）是建立染色质环的核心调控因子之一。本研究报道了一种优化的黏连蛋白HiChIP实验方案。通过全面的生物信息学分析，我们证实相较于仅使用甲醛的标准固定方法，双重染色质固定法可显著提升信噪比、增强ChIP效率，并优化染色质环与结构条纹的检测效果。此外，我们开发了一款名为nf-HiChIP的自动化分析流程（https://github.com/SFGLab/hichip-nf-pipeline），可从原始测序读段数据出发处理HiChIP样本，最终输出一系列显著染色质相互作用（环）结果；该流程能够高效并行分析多个样本，且无需额外开展ChIP测序（ChIP-seq）实验。最后，我们借助先进的生物物理建模与条纹调用技术，分别针对目标区域构建了精准的环挤压聚合物模型，并详细解析了染色质结构条纹。实验整体设计：针对千人基因组计划（1000 Genome Project）中的HG00731淋巴母细胞系，分别开展染色体结构维持蛋白1（SMC1）的FA-EGS-HiChIP实验与CCCTC结合因子（CTCF）的标准FA-HiChIP实验。