ChIPing Away at Global Transcriptional Regulation
The regulation of gene expression impacts all aspects of cell biology and biochemistry. As we gain a greater understanding of the mechanisms involved in this process, we also begin to unveil its complexities. The delicate balancing act played out by the m
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Introduction Our understanding of gene expression has come a long way from early definitions of how a gene is transcribed and translated into a functional protein e.g. (1). For example, the current number of transcription factors known to this point has grown from the few that encompassed the basic transcriptional machinery to thousands. Many of these regulatory proteins play very specific roles and can have far reaching implications on the fate of the cell. Furthermore, the initial process of gene transcription can no longer be viewed as an isolated event. A multitude of regulatory events may occur that can influence not only whether or not a gene is even transcribed but also quantity and stability e.g., DNA methylation (2).
Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_11, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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Jackson, Paris, and Takahashi
Even the expression of one gene can have huge implications on the expression of others. How genes are transcribed can be as basic or complex as the number of genes in the genome. Until recently, much of this work had been carried out on a per gene basis. However, two advances that have occurred in the past decade changed the way we now view genomics and have pushed our knowledge of transcriptional regulation to new levels. The first was the completion of the human genome sequencing projects (3, 4). This has been pivotal in providing the necessary road map of all the genes including all of the intergenic regions. The second has been the advent of microarray technologies (5). By combining the information learned from sequencing the genome and applying it to microarrays, we are now capable of investigating gene expression at the genome wide scale. The question of what sequence to lay down on an array in order to profile transcriptional regulation of a gene has always been a difficult question to answer. However, the completion of both the mouse and human genomes have allowed for the generation of promoter arrays. These, in combination with CpG island arrays, have been used in a variety of genome-wide analyses of protein–DNA interactions including histones, transcription factors, and the machinery for both transcription and replication (6, 7). More recently, interest has been growing in the scientific community surrounding DNA modifications such as methylation acquired during one’s lifetime. Once again, by using antibodies raised against the methylated form of cytosine (5-methylcytosine), the same promoter arrays can be used to profile the extent and location of DNA methylation (8, 9). In the following protocol, we provide a robust method which we have used extensively with both spotted CpG island arrays developed in-house and Agilent CpG island arrays. However, the current protocol is versatile enough that it can be used in conjunction with a number of commercially available platforms. For example, with minor modifications, it can also be applied to p
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