STAGE and FAIRE for Regulatory Element Identification

这项提案的总体目标是开发和结合两项全新的实验性基因组技术，以确定非编码DMA中的功能调节元件。第一种是一种全基因组范围的方法，用于在任何已测序的基因组中识别蛋白质结合位点。它基于对染色质|免疫沉淀(CHIP)后恢复的DMA的高通量序列标签分析。我们称之为这个方法阶段，用于基因组浓缩的序列标签分析。STAGE可以克服目前正在为人类和其他大型基因组开发的启动子/基因间微阵列的许多限制。然而，STAGE的实用性受到芯片技术的限制，该技术一次只能处理一种蛋白质，并受到其他限制，如针对目标蛋白质的抗体的质量和可用性。因此，我们建议开发的第二种方法是从总染色质中生化纯化和鉴定所有潜在的调控元件，而不需要抗体或亲和标签(FIRE，用于甲醛辅助分离调控元件)。FIRE通过利用所有活性调控元件共有的染色质特征(即核小体清除)，而不是使用特定的蛋白质来分离它们，从而使高通量表征调控元件成为可能。因此，FIRE可以克服染色质的局限性 免疫沉淀。我们将开发这种方法用于复杂的基因组，如线虫和人类。为了证明FIRE在酵母菌以外的用途，需要一种像STAGE这样的无偏检测方法。相反，FIRE是独立于ChlP的，因此可用于交叉验证基于ChlP的阶段结果。我们两项独立的创新技术之间的这种天然协同使每一项技术都更加强大，并有可能加速识别任何已测序基因组中的蛋白质结合位点和调控元件。

The overall objective of this proposal is to develop and combine two entirely new experimental genomic technologies for identifying functional regulatory elements in non-coding DMA. The first is a genome-wide method for identifying protein-binding loci in any sequenced genome. It is based on high-throughput sequence-tag analysis of DMA that is recovered after Chromatin |mmunoPrecipitation (ChIP). We term this method STAGE, for Sequence Tag Analysis of Genomic Enrichment. STAGE can overcome many of the limitations of promoter/intergenic microarrays that are currently under development for the human and other large genomes. However, the utility of STAGE for this purpose is constrained by the ChIP technique, which must be performed one protein at a time and suffers from other limitations such as the quality and availability of antibodies to proteins of interest. Therefore, the second method we propose to develop is a procedure for biochemically purifying and identifying all potential regulatory elements from total chromatin without the requirement for antibodies or affinity tags (FAIRE, for Formaldehyde-Assisted isolation of Regulatory Elements). FAIRE makes high-throughput characterization of regulatory elements possible by taking advantage of a chromatin feature common to all active regulatory elements (namely, nucleosome clearance) instead of using specific proteins to isolate them. Thus FAIRE can overcome the limitations of chromatin immunoprecipitation. We will develop this method for use in complex genomes such as C. elegans and human. In order to demonstrate the utility of FAIRE beyond yeast, an unbiased detection method like STAGE is required. Conversely, FAIRE is ChlP-independent, and therefore can be used to cross-validate ChlP-based STAGE results. This natural synergy between our two independent novel techniques makes each one more powerful and has the potential to accelerate the identification of protein binding sites and regulatory elements in any sequenced genome.