Johnson Kirby D, Bresnick Emery H
Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, 387 Medical Sciences Center,1300 University Avenue, Madison 53706, USA.
Methods. 2002 Jan;26(1):27-36. doi: 10.1016/S1046-2023(02)00005-1.
Analysis of physiological mechanisms that control transcription often requires extrapolation of in vitro measurements into in vivo mechanisms. This extrapolation is complex, as mammalian genes are commonly organized into broad chromosomal domains, and such domains cannot be readily reconstituted in vitro. Thus, the nucleoprotein structure of chromosomes constitutes a considerable impediment to elucidating transcriptional mechanisms. The development of assays to measure protein-DNA interactions and chromatin structure in living cells has greatly facilitated progress in understanding physiological transcriptional mechanisms. Chromatin immunoprecipitation (ChIP) is a powerful approach that allows one to define the interaction of factors with specific chromosomal sites in living cells, thereby providing a snapshot of the native chromatin structure and factors bound to genes in different functional states. ChIP involves treating cells or tissue briefly with formaldehyde to crosslink proteins to DNA. An antibody against a protein suspected of binding a given cis-element is then used to immunoprecipitate chromatin fragments. Polymerase chain reaction analysis of the immunoprecipitate with primers flanking the cis-element reveals whether a specific DNA sequence is recovered in an immune-specific manner and therefore whether the protein contacted the site in living cells. The central focus of this review is the use of ChIP to study transcriptional activation over long distances on chromosomes.
对控制转录的生理机制进行分析通常需要将体外测量结果外推至体内机制。这种外推很复杂，因为哺乳动物基因通常被组织成宽泛的染色体结构域，而此类结构域无法在体外轻易地重建。因此，染色体的核蛋白结构对阐明转录机制构成了相当大的障碍。用于测量活细胞中蛋白质 - DNA相互作用和染色质结构的检测方法的发展极大地推动了对生理转录机制的理解。染色质免疫沉淀（ChIP）是一种强大的方法，它能让人们确定活细胞中因子与特定染色体位点的相互作用，从而提供天然染色质结构以及与处于不同功能状态基因结合的因子的快照。ChIP包括用甲醛短暂处理细胞或组织，使蛋白质与DNA交联。然后使用针对怀疑与给定顺式元件结合的蛋白质的抗体来免疫沉淀染色质片段。用位于顺式元件两侧的引物对免疫沉淀产物进行聚合酶链反应分析，可揭示是否以免疫特异性方式回收了特定的DNA序列，进而确定该蛋白质在活细胞中是否与该位点接触。本综述关注的核心是利用ChIP研究染色体上远距离的转录激活。
