Serial analysis of transcription factor binding sites

转录因子结合位点的系列分析

• 批准号: 6916492
• 负责人: ROBYN MEECH
• 金额: $ 18.77万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916492
• 关键词: RNA interference; binding sites; bioinformatics; biotechnology; chromatin immunoprecipitation; cytogenetics; gene expression; high throughput technology; laboratory mouse; microarray technology; molecular cloning; polymerase chain reaction; serial analysis of gene expression; technology /technique development; transcription factor

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.

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阻遏子元件中。标准的分子方法来识别这些元素是费力的，而生物信息学分析不能揭示在特定环境中发挥作用的元素。拟议的研究将开发和验证一个高通量的程序，为特定的背景下识别的转录因子结合位点。该程序结合了染色质免疫沉淀（CHIP）和富集序列的快速系列分析，称为基因组位点系列分析（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（2 lbp）的识别基序（NRSE），可以很容易地通过生物信息学预测。将预测的元件与使用SAGS鉴定的实际细胞结合位点进行比较。将P19细胞中与NRSF结合的100- 300 bp的基因组片段通过CHIP富集，并使用新型限制性内切酶从片段的每个末端切割独特的26 bp标签。这些标签将被连接起来，并按顺序排列。然后提取单个标签的序列并映射到小鼠基因组以鉴定结合位点和相关基因。我们还将在NRSF无效胚胎中使用微阵列分析和在P19细胞中RNAi介导的NRSF表达抑制后确定NRSF的直接调控靶点。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个强大的组合，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差异活性的调控序列。这样的信息在设计用于基因治疗载体的特异性靶向启动子序列中可能是特别有用的。