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。这种信息包含在与特定转录因子结合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。建议的研究将开发和验证一种高通量的程序，用于特定背景的转录因子结合位点的鉴定。该程序结合了染色质免疫沉淀(CHIP)和对丰富序列的快速序列分析，被称为基因组位点序列分析(SAGS)。对于SAG的发展，我们将使用神经限制性沉默因子(NRSF)作为样本，因为它有一个异常长(2 LBP)的识别基序(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SAG确定的实际细胞结合位点进行比较。在P19细胞中与NRSF结合的100-300bp的基因组片段将通过芯片进行浓缩，并将使用一种新型的限制性内切酶从片段的两端切割出独特的26bp标签。这些标签将被连接在一起，并全部排序。然后，将提取单个标签的序列，并将其映射到小鼠基因组，以识别结合位点和相关基因。我们还将在NRSF缺失的胚胎中使用微阵列分析，并在RNAi介导的P19细胞中抑制NRSF的表达后，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球分析，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、药物治疗后或在各种病理条件下具有不同活性的调控序列。这些信息在设计用于基因治疗载体的特定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.