SGER: Development of a Novel Approach for the In Situ Analysis of Transcription Factor-DNA Complexes

9817820 Arias DNA结合因子在真核生物中调节转录已得到充分证实。因此，了解这些因子如何以及在何处与启动子DNA相互作用是有意义的。迄今为止，这些信息主要是从蛋白质提取物和无核小体DNA模板的研究中获得的。我们对转录过程的理解的未来进展将需要新的工具来评估体外结果的有效性，并确定转录复合物如何在体内运作。本研究的总体目标是开发一种新的方法，用于表征核转录因子与细胞启动子中的结合位点之间的体内相互作用。该策略依赖于甲醛在体内快速有效地将蛋白质与其DNA结合位点交联的能力。然后将固定的染色质机械分离或酶切成较小的片段，并对共价连接的蛋白质和感兴趣的DNA复合物进行免疫富集。复合物中的交联在65 ℃下是可逆的，从而分别产生用于Western印迹或PCR分析的单个蛋白质和DNA组分。虽然未经测试的活细胞，初步结果与基本/亮氨酸拉链转录因子和植物染色质在体外表明，所提出的方法在技术上是可行的。这项工作的成功结果将导致我们探测基因表达的分子机制的能力的重大范式转变。这项工作可能解决的重要生物学问题包括：1）鉴定转录因子的体内启动子结合位点，2）分离受转录因子调节的新基因，3）分析已知启动子位点处因子的DNA结合活性的变化，以及4）鉴定多基因因子家族中的哪一个成员与体内的共同靶位点结合。这项研究有望为理解基因在细胞中是如何调控的提供新的工具。由于基因是生物体发育、体内平衡和防御中基本事件的基础，我们设想这里的工作将促进我们对多细胞生物如何协调这些过程的理解。

9817820AriasIt is well established that DNA-binding factors modulate transcription in eukaryotes. It is thus of interest to know how and where these factors interact with promoter DNA. To date, such information has largely been obtained from investigations with protein extracts and nucleosome-free DNA templates. Future advances in our understanding of transcriptional processes will require new tools for evaluating the validity of in vitro results and determining how transcription complexes operate in vivo. The overall goal of this research is to develop a novel method for characterizing in vivo interactions between nuclear transcription factors and their binding sites in cellular promoters. The strategy relies on the ability of formaldehyde to rapidly and efficiently cross-link proteins with their DNA-binding sites in vivo. Fixed chromatin is then isolated mechanically or enzymatically-cleaved into smaller fragments, and immuno-enriched for the covalently-linked protein and DNA complex of interest. Cross-links in complexes are reversible at 65C, thus yielding individual protein and DNA components for Western blot or PCR analysis, respectively. Although untested as yet with living cells, preliminary results with a basic/leucine-zipper transcription factor and plant chromatin in vitro suggest that the proposed methodology is technically feasible. A successful outcome to this work will result in a significant paradigm shift in our ability to probe the molecular mechanisms of gene expression. Important biological questions that may be addressed by this work include: 1) identifying in vivo promoter-binding sites for a transcription factor, 2) isolating new genes that are regulated by a transcription factor, 3) analyzing changes in the DNA-binding activity of a factor at a known promoter site, and 4) identifying which member of a multi-gene family of factors binds to a common target site in vivo.This research is expected to provide new tools for understanding how genes are regulated in cells. As genes underlie fundamental events in an organism's development, homeostasis, and defense, we envision that work here will advance our understanding of how multicellular organisms orchestrate these processes.