Schultz-Norton Jennifer R, Ziegler Yvonne S, Likhite Varsha S, Yates John R, Nardulli Ann M
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
BMC Mol Biol. 2008 Oct 30;9:97. doi: 10.1186/1471-2199-9-97.
DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor alpha (ERalpha) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERalpha using full-length receptor and endogenously-expressed nuclear proteins.
We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERalpha. Purified ERalpha and HeLa nuclear extracts were combined with oligos containing ERalpha binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERalpha, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERalpha, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERalpha activity and expression of estrogen-responsive genes.
The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.
与DNA结合的转录因子招募一系列影响基因表达的共调节蛋白。我们先前证明DNA作为雌激素受体α(ERα)构象的变构调节剂,改变调节蛋白的募集,并影响雌激素反应性基因表达,并且推断开发一种使用全长受体和内源性表达的核蛋白分离与DNA结合的ERα相关蛋白的方法将是有用的。
我们开发了一种新方法来分离与DNA结合的ERα相关的大型蛋白质复合物。将纯化的ERα和HeLa核提取物与含有ERα结合位点的寡核苷酸混合,并在琼脂糖凝胶上分级分离。分离蛋白质-DNA复合物,并使用质谱分析来鉴定与DNA结合受体相关的蛋白质。我们不是简单地鉴定与ERα相互作用的单个蛋白质,而是鉴定了具有多种酶促和催化活性的相互连接的蛋白质网络,这些蛋白质不仅与ERα相互作用,而且彼此相互作用。对其中一些蛋白质的表征表明,除了它们先前确定的功能外,它们还影响ERα活性和雌激素反应性基因的表达。
我们开发的琼脂糖凝胶分级分离方法将有助于鉴定与DNA结合的转录因子相互作用的蛋白质,并且应该很容易适用于各种培养细胞系、DNA序列和转录因子。