Serial analysis of transcription factor binding sites

描述（由申请人提供）：生物学中的一个中心问题是定义在特定细胞环境中控制基因表达的信息。这些信息包含在结合特定转录因子的增强子和抑制子元件中。识别这些元素的标准分子方法是费力的，而生物信息学分析不能揭示在特定环境中起作用的元素。拟议的研究将开发和验证一种高通量程序，用于特定上下文的转录因子结合位点鉴定。该方法将染色质免疫沉淀（CHIP）与富集序列的快速序列分析相结合，称为基因组位点序列分析（SAGS）。对于SAGS的开发，我们将使用神经限制性沉默因子（NRSF）作为范例，因为它具有异常长的（2 lbp）识别基序（NRSE），可以很容易地通过生物信息学预测。预测的元件将与使用SAGS识别的实际细胞结合位点进行比较。p19细胞中被NRSF结合的100-300bp基因组片段将通过CHIP富集，并使用一种新的限制性内切酶从片段的每一端切割出独特的26bp标签。这些标签将被串联起来，并按顺序排列。然后提取单个标签的序列并将其映射到小鼠基因组中，以确定结合位点和相关基因。我们还将利用微阵列分析NRSF缺失胚胎和rnai介导的P19细胞中NRSF表达抑制后，确定NRSF的直接调控靶点。总之，这些研究将提供转录因子结合位点的全局分析以及这种结合与基因表达的相关性。SAGS和微阵列分析有望成为一种强大的组合，用于识别在发育过程中、药物治疗后或各种病理条件下具有差异活性的调控序列。这些信息在设计基因治疗载体的特异性靶向启动子序列时可能特别有用。

DESCRIPTION (provided by applicant): A central problem in biology is defining the information that controls gene expression in particular cellular contexts. This information is contained within enhancer and repressor elements that bind to specific transcription factors. Standard molecular approaches for identifying these elements are laborious, while bioinformatic analyses cannot reveal elements that function in particular contexts. The proposed studies will develop and validate a high throughput procedure for context specific identification of transcription factor binding sites. The procedure combines Chromatin Immunoprecipitation (CHIP) with rapid serial analysis of enriched sequences and is called Serial Analysis of Genomic Sites (SAGS). For development of SAGS we will use the Neural Restrictive Silencer Factor (NRSF) as an exemplar because it has an unusually long (2 lbp) recognition motif(NRSE) that can be easily predicted by bioinformatics. Predicted elements will be compared to actual cellular binding sites that are identified using SAGS. 100-300bp genomic fragments that are bound by NRSF in P 19 cells will be enriched by CHIP, and a novel restriction enzyme will be used to cleave unique 26bp tags from each end of the fragments. These tags will be concatenated, and sequenced en masse. Sequences of individual tags will then be extracted and mapped to the mouse genome to identify binding sites and associated genes. We will also identify direct regulatory targets of NRSF using microarray analysis in NRSF null embryos and after RNAi-mediated inhibition of NRSF expression in P19 cells. Together these studies will provide global analysis of transcription factor binding sites and correlation of this binding with gene expression. SAGS and microarray analysis promises to be a powerful combination for identifying regulatory sequences that are differentially active during development, after treatment with drugs or in a variety of pathological conditions. Such information may be particularly useful in the design of specifically targeted promoter sequences for gene therapy vectors.