Background

Now that genomes are being sequenced at a high rate, investigators wish to understand their regulation. In living cells, genomes are not simply made of DNA, but rather of a large amount of proteins that dynamically interact with, and modulate the function of, DNA. This dynamic structure is called “Chromatin”. Chromatin plays an essential role in gene expression regulation, DNA replication and repair as well as many epigenetic phenomena, therefore impacting on cellular functions including growth, differentiation and development. Mutations affecting chromatin regulation are implicated in many diseases including cancers, neurodegeneration diseases and developmental diseases.


Chromatin is regulated by protein factors that non-covalently modify nucleosome structure using ATP as an energy source, and by factors that catalyze the addition of functional groups to the N-terminal tails of histones. All those chromatin regulators have to be targeted to specific chromosomal regions to achieve their respective function(s). Interestingly, however, chromatin regulators generally do not have DNA-binding domains. They therefore rely on interactions with some DNA-binding proteins to reach their appropriate genomic locations. All those interactions need to be tightly regulated in space and time. The regulation of chromatin has been characterized in great details for a few genes, but its global impact on genomes and their regulation is vastly unknown due to lack of appropriate technologies. Recently, We and others have developed a technology allowing protein-DNA interactions to be monitored across the entire genome, providing an opportunity to study nuclear processes in a global way.


The Technology

DNA microarrays have been used to identify genes whose expression is affected by mutations in transcription factors and chromatin regulators. In these genome-wide expression analysis experiments, the relative expression level between cells expressing either a wild type or a mutated transcriptional regulator is monitored across all the genes of the genome using cDNA hybridization to DNA microarrays. This technology is useful since it can identify all the transcriptional changes associated with a perturbation in a specific regulator. However it has a number of limitations, in particular in defining the direct and physiologically relevant targets of the regulator. First, genome-wide expression analyses cannot distinguish between direct and indirect effects at individual target genes. Second, yeast cells contain multiple chromatin regulators whose partially redundant functions will not be uncovered by a single mutation. Third, the use of deletion mutants to measure gene expression provides a steady-state measurement of cells that have adapted to the mutations. Fourth, conditional alleles often cause partial loss of function, and the analysis is complicated by the loss of viability or cell cycle arrest under non-permissive conditions.


The genome-wide location analysis, we have developed has been used to discover the physiologically relevant and direct targets of many DNA-binding proteins and chromatin regulators. In a genome-wide location analysis experiment (also called ChIP-chip), the genes bound by a chromatin-associated protein are identified by a combination of chromatin immunoprecipitation (ChIP) and DNA microarrays. Since genome-wide location analysis does not involve the use of mutants, results from these experiments are free of the downstream effects described above for expression studies. Since the genome-wide location analysis does not provide information about the functional consequence of the binding observed, a combination of these two genome-wide technologies allows a comprehensive study of nuclear processes. The genome-wide location analysis technique is depicted and described below.

Briefly, cells are fixed with formaldehyde, harvested, and disrupted by sonication. The DNA fragments cross-linked to a protein of interest are enriched by immunoprecipitation with a specific antibody. After reversal of the cross-links, the enriched DNA is amplified and labeled with a fluorescent dye (Cy5) using ligation-mediated polymerase chain reaction (LM-PCR). A sample of DNA that was not enriched by immunoprecipitation is also subjected to LM-PCR in the presence of a different fluorophore (Cy3), and both immunoprecipitation (IP)-enriched and –unenriched pools of labeled DNA are hybridized to a single DNA microarray containing all yeast intergenic and intragenic DNA sequences. The results of three independent location analysis experiments are used to determine the binding sites for a single regulator under a single experimental condition. The IP-enriched/unenriched ratio of fluorescence intensity obtained from the three independent experiments is used with a weighted-average analysis method to calculate the relative binding of the protein of interest to each sequence represented on the array. An error model allows us to assign binding in a probabilistic manner (P value).