Cloning of Ah receptor bound regulatory DNA

描述(由申请人提供)：为了试图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控的基因。这通常是通过使用一些方法来确定的，这些方法评估了在转录因子激活或过度表达时特定基因的mRNA水平的变化(例如，消减文库、DNA微阵列)。然而，这些方法往往没有检测到低丰度基因，往往只识别高度诱导的基因，或者无法识别新的基因。染色质免疫沉淀克隆是最近发表的一种克隆与特定转铁蛋白结合的调控DNA序列的方法。在人类和小鼠基因组序列已知的情况下，克隆的DNA片段的序列可以用于确定由给定的转铁蛋白直接调控的基因。这种方法可以被认为是确定由转铁蛋白直接调控的基因的一种无偏见的方法，因为每个细胞中每个基因都有两个拷贝，并且基因的识别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。需要检验的中心假设是，增强型染色质免疫沉淀克隆是识别由特定转录因子直接调控的基因的一种优越技术。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“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来允许该方法在组织中有效地使用。拟议研究的另一个目标是确定大量直接受ah受体/Arnt复合体调控的靶基因。因此，一个特定的目标是开发一种方法来有效地克隆与ah受体/Arnt异源二聚体结合的特定调控DNA序列。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受ah受体调控的调控序列。这种方法将来应该在生物学中用于广泛的机械学研究。

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.