STAGE and FAIRE for Regulatory Element Identification

本提案的总体目标是开发和联合收割机两种全新的实验基因组技术，用于识别非编码DNA中的功能调节元件。第一种是全基因组方法，用于识别任何测序基因组中的蛋白质结合位点。它是基于DNA的高通量序列标签分析，DNA在染色质后被回收，|免疫沉淀（ChIP）。我们将这种方法称为STAGE，即基因组富集的序列标签分析。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局限性。然而，STAGE用于此目的的实用性受到ChIP技术的限制，该技术必须一次一种蛋白质进行，并且受到其他限制，例如针对感兴趣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调控元件的程序，而不需要抗体或亲和标签（FAIRE，用于甲醛辅助分离调控元件）。FAIRE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，从而使调控元件的高通量表征成为可能。因此FAIRE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C。elegans和human人。为了证明FAIRE在酵母之外的实用性，需要像STAGE这样的无偏检测方法。相反，FAIRE是ChIP独立的，因此可用于交叉验证基于ChIP的STAGE结果。我们两种独立的新技术之间的这种自然协同作用使每一种技术都更加强大，并有可能加速任何测序基因组中蛋白质结合位点和调控元件的鉴定。

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.