Cloning of Ah receptor bound regulatory DNA

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，有必要确定由该因子调节的基因。这通常是通过使用评估特定基因在TF激活或过表达时mRNA水平变化的方法来确定的（例如减法文库，DNA微阵列）。然而，这些方法往往不能检测到低丰度的基因，往往只能识别高度诱导的基因，或者不能识别新的基因。染色质免疫沉淀克隆是最近发表的一种克隆与特定TF结合的调控DNA序列的方法。随着人类和小鼠基因组序列的已知，克隆DNA片段的序列可用于确定特定TF直接调控的基因。这种方法可以被认为是确定TF直接调控基因的无偏方法，因为每个细胞中每个基因都有两个拷贝，并且基因的鉴定与它是被诱导还是被抑制无关。然而，目前的方法相当费力，只产生了有限数量的已识别基因。要验证的中心假设是，增强染色质免疫沉淀克隆是一种识别由特定转录因子直接调节的基因的优越技术。为了使用PCR扩增分离的DNA片段，我们在程序中增加了一些步骤。该方法被命名为“PCR扩增克隆染色质免疫沉淀产物”（PAC-ChIP）。将使用交联核代替细胞，从而使该方法有效地应用于组织中。拟议研究的另一个目标是确定由Ah受体/ARNT复合体直接调节的大量靶基因。因此，具体的目标之一提出了发展的方法，以有效地克隆特异性调控DNA序列结合的Ah受体/ARNT异源二聚体。这些研究将开发一种方法，有效地从细胞或组织中克隆大量由Ah受体调节的调控序列。这种方法将来应该在生物学中广泛应用于机理研究。

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.