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. ? ? ?
