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

众所周知，DNA结合因子调控真核生物的转录。因此，了解这些因子如何以及在哪里与启动子DNA相互作用是很有意义的。到目前为止，这些信息主要是从蛋白质提取物和无核小体DNA模板的调查中获得的。未来我们对转录过程的理解将需要新的工具来评估体外结果的有效性，并确定转录复合体在体内如何运作。这项研究的总体目标是开发一种新的方法来表征核转录因子与其在细胞启动子中的结合部位之间的体内相互作用。这一策略依赖于甲醛快速高效地将蛋白质与其DNA结合部位在体内进行交联的能力。固定的染色质然后被机械分离或酶切成更小的片段，并对共价连接的蛋白质和感兴趣的DNA复合体进行免疫浓缩。在65℃下，复合体中的交联物是可逆的，从而产生单独的蛋白质和DNA组分，分别用于蛋白质印迹分析或聚合酶链式反应分析。尽管到目前为止还没有对活细胞进行测试，但在体外对碱性/亮氨酸拉链转录因子和植物染色质的初步结果表明，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索基因表达的分子机制的能力发生重大的范式转变。这项工作可能解决的重要生物学问题包括：1)确定体内转录因子的启动子结合部位，2)分离转录因子调控的新基因，3)分析已知启动子位点上因子的DNA结合活性的变化，以及4)识别多基因因子家族中的哪个成员与活体中的共同靶点结合。这项研究有望为理解基因在细胞中的调控提供新的工具。由于基因是生物体发育、内稳态和防御的基础事件，我们设想，在这里的工作将促进我们对多细胞生物体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.