Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP)
Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP)
复制标题
DOI：
10.1038/s41596-019-0285-9
复制
发表时间：
2020-02-12
期刊：
影响因子：
14.8
通讯作者：
Stanton, Benjamin Z.
中科院分区：
文献类型：
--
作者：
Gryder, Berkley E.;Khan, Javed;Stanton, Benjamin Z.
为捕捉蛋白质-锚定染色质相互作用而开发的方法(通过成对末端标签测序和HiChIP进行的染色质相互作用分析)已经对基因组的3D折叠原理产生了巨大的洞察力，但由于测序深度的归一化，因此无法准确测量染色质相互作用和接触域组织的全球变化。在此，我们描述了染色质结构绝对定量的协议-HiChIP，这是一项进步，允许确定样品之间蛋白质锚定的染色质相互作用的绝对差异。用我们的方法，将确定的比例的小鼠和人的固定核混合并进行内切酶消化。染色质接触通过生物素-dATP掺入和邻近连接获得，随后是温和剪切、芯片、生物素捕获和配对末端测序。3D接触是从人类基因组中的配对末端标签(PETS)中计算出来的，并被归一化为老鼠基因组中的全部宠物。由于正交归一化允许观察全局变化，该方法将使人们能够更定量地了解转录控制和组织特异性表观遗传记忆的拓扑决定因素。通过我们的方法，我们发现组蛋白脱乙酰酶的快速抑制通过产生许多新的异常接触来扰乱超级增强子的功能。有关数据分析的代码，请访问。该协议报告了执行Aqua-HiChIP的实验和生物信息学细节，从细胞培养到染色质相互作用排名在6天内。
Methods developed to capture protein-anchored chromatin interactions (chromatin interaction analysis by paired-end tag sequencing and HiChIP) have yielded tremendous insights into the 3D folding principles of the genome, but are normalized by sequencing depth and therefore unable to accurately measure global changes in chromatin interactions and contact domain organization. We herein describe the protocol for absolute quantification of chromatin architecture (AQuA)-HiChIP, an advance that allows the absolute differences in protein-anchored chromatin interactions between samples to be determined. With our method, defined ratios of mouse and human fixed nuclei are mixed and subjected to endonuclease digestion. Chromatin contacts are captured by biotin-dATP incorporation and proximity ligation, followed by gentle shearing, ChIP, biotin capture and paired-end sequencing. 3D contacts are counted from paired-end tags (PETs) from the human genome and are normalized to the total PETs from the mouse genome. As orthogonal normalization allows observation of global changes, the approach will enable more quantitative insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory. With our approach, we have discovered that rapid histone deacetylase inhibition disrupts super enhancer function by creating many new aberrant contacts. The code for data analysis is available at . This protocol reports both experimental and bioinformatic details to perform AQuA-HiChIP, going from cell culture to ranking chromatin interactions within 6 d.