López-Fuentes Eunice, Hernández-Hernández Grecia, De Las Peñas Alejandro, Castaño Irene
Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, A.C. División de Biología Molecular, San Luis Potosí, Mexico.
Methods Mol Biol. 2022;2542:71-89. doi: 10.1007/978-1-0716-2549-1_5.
Chromatin architecture has an enormous impact on gene regulation, DNA replication, repair, and packaging. Chromatin is organized in a complex hierarchical manner in which distant fragments of DNA can interact with each other through DNA loops. DNA loops can interact between themselves to form topologically associated domains (TADs) that are further organized into functional compartments. In the last two decades, Chromatin Conformation Capture (3C technology) and its high-throughput derivatives allowed detailed analysis of the chromatin architecture. The 3C method is based on ligation of distant fragments brought together by DNA looping. The method analyzes a particular genomic region of interest and quantifies the interactions between a defined fragment with all the surrounding fragments of the region. It consists of four steps: (1) The long-distance interacting chromatin fragments are fixed with formaldehyde in whole cells which are then lysed; (2) the fixed chromatin is digested with a carefully chosen restriction enzymes to separate intervening DNA fragments; (3) the fragments brought into proximity by DNA looping are ligated in conditions favoring intramolecular ligation; and (4) the interactions are quantified by quantitative PCR using the TaqMan technology and unidirectional primers. Herein, we describe the use of this methodology to analyze the chromatin conformation at a subtelomeric locus containing three genes encoding adhesins and several cis-regulatory elements, in the pathogenic yeast Candida glabrata.
染色质结构对基因调控、DNA复制、修复及包装有着巨大影响。染色质以复杂的层级方式组织，其中DNA的远距离片段可通过DNA环相互作用。DNA环之间相互作用形成拓扑相关结构域（TADs），这些结构域进一步组织成功能区室。在过去二十年中，染色质构象捕获技术（3C技术）及其高通量衍生技术使得对染色质结构进行详细分析成为可能。3C方法基于对由DNA环聚集在一起的远距离片段进行连接。该方法分析特定的感兴趣基因组区域，并量化一个定义片段与该区域所有周围片段之间的相互作用。它包括四个步骤：（1）在全细胞中用甲醛固定远距离相互作用的染色质片段，然后裂解细胞；（2）用精心选择的限制性酶消化固定的染色质，以分离中间的DNA片段；（3）在有利于分子内连接的条件下，连接由DNA环拉近的片段；（4）使用TaqMan技术和单向引物通过定量PCR对相互作用进行定量。在此，我们描述了使用这种方法来分析致病性酵母光滑念珠菌中一个包含三个编码黏附素基因和几个顺式调控元件的亚端粒位点的染色质构象。
