Analyzing Chromatin Structure and Transcription Factor Binding in Yeast

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatinin vivo.The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cellsin vivoto determine the binding of factors tociselements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeastPHO8gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of atransactivator with an altered or perturbed chromatin organization at a specific locus.     

Transcription Factor Binding to Replicated DNA

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运用染色质免疫沉淀技术筛选AP-2α的靶基因

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.染色质免疫沉淀技术(chromatin immunoprecipitation assay,简称CHIP)是目前唯一研究体内DNA与蛋白质相互作用的方法.对与ChIP有关的实验条件进行了优化,获得了较优的实验条件,并运用ChIP实验筛选出了转录因子activator protein-2 alpha (AP-2a)的未知靶基因,对于进一步研究AP-2a的功能和调控网络打下了基础.