The precise spatial and temporal control of gene expression is crucial for mediating cellular and developmental processes. A global understanding of transcriptional regulation will require the identification of cis-acting regulatory elements, the factors that bind them, and the regulatory cascades in which these factors function. With the recent determination of the genome sequence of humans and many other organisms it is now possible to identify many genes; however, the factors, DNA sequences and regulatory pathways that control their expression remain poorly understood. Below we discuss the use of chromatin immunoprecipitation and DNA micro-arrays to globally identify targets of transcription factors and use this information to construct regulatory networks.

A variety of indirect approaches have been used to identify targets of transcription factors. Analysis of genes with common expression patterns has led to the identification of shared motifs (e.g. 1); however the factor that binds these motifs is not usually apparent. The monitoring of gene expression patterns with and without a transcription factor of interest using expression microarrays or differential display can identify candidate targets (2). Although comprehensive, this approach identifies alterations in gene expression, which may be due to secondary or downstream signaling events. Similarly, genetic screens for identifying targets are neither direct nor comprehensive. Methods involving in vitro DNA binding selections do not account for the complexities of cooperative binding or cofactor regulation of transcription and therefore do not accurately mimic in vivo binding. Hence, a need exists for a direct method to identify all of the DNA targets of a transcription factor of interest in a single experimental system.

In the last few years a new microarray (chip) technology has allowed the use of chromatin immunoprecipitation (ChIP) (3, 4) to identify in vivo targets of transcription factors on a genome-wide scale; this procedure has been coined 'ChIP-chip'. ChlP-chip has gained increasing popularity as a means of identifying transcription factor targets on a global scale. The experimental approach is built on the premise of reversibly crosslinking proteins to DNA so that the DNA attached to a protein of interest can then be isolated by purifying the protein and subsequently used to probe a microarray composed of regulatory regions containing putative transcription factor binding sites (Plate 5).

This approach was first invented for yeast (5, 6) and has now been successfully performed for a multitude of different transcription factors in many organisms (see Table 13.1). Several important considerations are critical to the ChlP-chip approach including experimental design, array selection, and data analysis. Here we will focus on experimental considerations and then report several studies employing ChlP-chip, which are meant to highlight the various microarray platforms suitable for this type of analysis.

0 0

Post a comment