Rodríguez-Gil Alfonso, Riedlinger Tabea, Ritter Olesja, Saul Vera V, Schmitz M Lienhard
Department of Oncohematology and Genetics. Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío;
Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Member of the German Center for Lung Research, Justus-Liebig-University.
J Vis Exp. 2018 Apr 2(134):57272. doi: 10.3791/57272.
Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.
对细胞外信号作出适当的基因表达，即组织和谱系特异性基因转录，严重依赖于高度明确的染色质组织状态。细胞核的动态结构由多种机制控制，并塑造转录输出程序。因此，以一种可靠的方式确定基因座特异性染色质可及性很重要，这种方式最好不依赖于抗体，因为抗体可能是实验变异性的潜在混杂来源。染色质可及性可以通过多种方法测量，包括甲醛辅助调控元件分离（FAIRE）分析，该分析能够在相对少量的细胞中确定一般染色质可及性。在这里，我们描述了一种FAIRE方案，该方案能够简单、可靠且快速地鉴定蛋白质占据率低的基因组区域。在该方法中，使用甲醛作为交联剂将DNA与染色质蛋白共价结合，然后将其剪切成小片段。随后通过酚：氯仿萃取富集游离DNA。与代表总DNA的对照样品相比，通过定量聚合酶链反应（qPCR）或DNA测序（DNA-seq）确定游离DNA的比例。染色质结构较松散的区域在游离DNA样品中富集，从而能够鉴定染色质压缩程度较低的基因组区域。
