Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements
Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements
复制标题
使用串联的转录因子反应元件串联阵列对激活的转录因子进行全蛋白质组分析。
DOI：
10.1073/pnas.1217657110
复制
发表时间：
2013-04-23
影响因子：
11.1
通讯作者：
Qin, Jun
中科院分区：
文献类型：
--
作者：
Ding, Chen;Chan, Doug W.;Qin, Jun
转录因子(TF)是一类与特定DNA序列或转录因子反应元件(TFRE)结合的蛋白质家族，在许多细胞过程中发挥调节作用。由于转铁蛋白丰度低，在蛋白质组水平上直接定量测定转铁蛋白仍然是一个挑战。在这项研究中，我们报告了一种亲和试剂的发展，它允许在蛋白质组水平上鉴定内源性转录因子。亲和试剂是由合成的DNA组成的，该DNA含有大多数TF家族的共识TFRE(CatTFRE)的串联阵列。通过使用catTFRE从细胞中富集转录因子，我们能够从单个细胞系中鉴定出多达400个转录因子，从11种细胞类型中总共鉴定出878个转录因子，覆盖了基因组中编码DNA结合因子的基因产物的50%以上。我们进一步证明，通过使用基于无标记MS的定量方法，CatTFRE下拉可以定量地测量响应于外源刺激的TF的DNA结合活性的蛋白质组范围的变化。将CatTFRE应用于药物效应的评价，我们描述了Tf激活的全景，为阐明药物作用的分子机制提供了候选。我们预计catTFRE亲和策略将在生物医学研究中得到广泛应用。
Transcription factors (TFs) are families of proteins that bind to specific DNA sequences, or TF response elements (TFREs), and function as regulators of many cellular processes. Because of the low abundance of TFs, direct quantitative measurement of TFs on a proteome scale remains a challenge. In this study, we report the development of an affinity reagent that permits identification of endogenous TFs at the proteome scale. The affinity reagent is composed of a synthetic DNA containing a concatenated tandem array of the consensus TFREs (catTFRE) for the majority of TF families. By using catTFRE to enrich TFs from cells, we were able to identify as many as 400 TFs from a single cell line and a total of 878 TFs from 11 cell types, covering more than 50% of the gene products that code for the DNA-binding TFs in the genome. We further demonstrated that catTFRE pull-downs could quantitatively measure proteome-wide changes in DNA binding activity of TFs in response to exogenous stimulation by using a label-free MS-based quantification approach. Applying catTFRE on the evaluation of drug effects, we described a panoramic view of TF activations and provided candidates for the elucidation of molecular mechanisms of drug actions. We anticipate that the catTFRE affinity strategy will find widespread applications in biomedical research.