Cloning of Ah receptor bound regulatory DNA

Ah 受体结合调节 DNA 的克隆

• 批准号: 6904566
• 负责人: Gary H. Perdew
• 金额: $ 14.02万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6904566
• 关键词: aromatic hydrocarbon receptor; cell nucleus; chromatin immunoprecipitation; crosslink; gene expression; genetic regulatory element; molecular cloning; nucleic acid sequence; polymerase chain reaction; receptor binding; technology /technique development; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): In an attempt to understand the role of a receptor or transcription factor (TF) in basic cellular functions it is necessary to determine the genes that are regulated by that factor. This is usually determined through the use of methods that assess changes in mRNA levels (e.g. subtraction libraries, DNA microarrays) of specific genes upon activation or overexpression of a TF. However, these methods often do not detect low abundance genes, tend to identify only highly induced genes, or fail to identify novel genes. Chromatin immunoprecipitation cloning is a method that was recently published that clones regulatory DNA sequences bound to a specific TF. With the sequence of the human and mouse genome known, the sequence of the cloned DNA fragments can be used to determine the genes directly regulated by a given TF. This method can be considered an unbiased method to determine genes directly regulated by a TF, because there are two copies of each gene in each cell and identification of a gene is independent of whether it is induced or repressed. However, the current approach is quite laborious and has only yielded a limited number of identified genes. The central hypothesis to be tested is that enhanced chromatin immunoprecipitation cloning is a superior technique to identify genes that are directly regulated by a specific transcription factor. We have added a number of steps to the procedure in order to use PCR to amplify the isolated DNA fragments. This method has been named "PCR Amplified Cloning of Chromatin Immunoprecipitated Products" (PAC-ChIP). Cross-linked nuclei instead of cells will be used to allow the method to be effectively used in tissues. An additional goal of the proposed studies is to identify a large number of target genes directly regulated by the Ah receptor/ARNT complex. Thus, specific aim one proposes to develop the methodology to efficiently clone specific regulatory DNA sequences that are bound by the Ah receptor/ARNT heterodimer. These studies will develop a method to efficiently clone a significant number of regulatory sequences regulated by the Ah receptor from cells or tissue. This method should in the future be utilized in biology for a wide range of mechanistic studies.

描述（由申请人提供）：为了了解受体或转录因子（TF）在基本细胞功能中的作用，有必要确定受该因子调节的基因。这通常是通过使用评估 TF 激活或过度表达时特定基因 mRNA 水平变化（例如消减文库、DNA 微阵列）的方法来确定的。然而，这些方法通常不能检测低丰度基因，往往只能识别高度诱导的基因，或者无法识别新基因。染色质免疫沉淀克隆是最近发表的一种方法，可克隆与特定 TF 结合的调控 DNA 序列。在已知人类和小鼠基因组序列的情况下，克隆的 DNA 片段的序列可用于确定受给定 TF 直接调控的基因。该方法可以被认为是确定由 TF 直接调节的基因的无偏方法，因为每个细胞中每个基因有两个拷贝，并且基因的识别独立于它是被诱导还是被抑制。然而，目前的方法非常费力，并且仅产生了有限数量的已识别基因。要测试的中心假设是，增强染色质免疫沉淀克隆是一种识别受特定转录因子直接调控的基因的优越技术。我们在程序中添加了许多步骤，以便使用 PCR 扩增分离的 DNA 片段。该方法被命名为“染色质免疫沉淀产物的 PCR 扩增克隆”(PAC-ChIP)。将使用交联的细胞核而不是细胞，以使该方法能够在组织中有效使用。拟议研究的另一个目标是鉴定大量由 Ah 受体/ARNT 复合物直接调节的靶基因。因此，具体目标是开发一种方法来有效克隆由Ah受体/ARNT异二聚体结合的特定调节DNA序列。这些研究将开发一种方法，从细胞或组织中有效克隆大量受 Ah 受体调节的调节序列。这种方法将来应该在生物学中用于广泛的机制研究。