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Binding Sights in Chromatin by X-ChIP.

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Mapping Protein/DNA Interactions by Cross-Linking [Internet]. Paris: Institut national de la santé et de la recherche médicale; 2001.

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Immunoprecipitation using in vivo, formaldehyde-fixed chromatin technique (X-ChIP) has been providing a powerful tool to unravel basic mechanisms in gene expression. Wide-spread application of this method for the analysis of many chromosomal proteins has allowed substantial progress in the current understanding of gene regulation and chromosome domain structure. Major breakthroughs have been achieved, for example, in the sequence of events that follow mitosis and lead to resetting of transcriptional competence by chromatin remodelling complexes (1) and the replication machinery (2). Other important achievements in gene regulation were the dynamics of recruitment of TBP and associated factors at promoters (3) and changes in histone modification (4). Further, the structure and role of heterochromatin at telomeres, centromeres, and developmentally regulated complex loci in the epigenetic control of gene silencing (5–9) have been thoroughly investigated with this method (see also Ref. 10). The method relies, in the first place, on the availability of high-quality antibodies. Affinity-purified and monoclonal antibodies should be tested for their efficiency in immunoprecipitation and immunoistochemistry. The latter may be highly indicative of the ability of the antibody to recognize a given epitope in formaldehyde-fixed preparations. The efficiency of immunoprecipitation is normally quite low (10-50 micrograms). Assuming that a given factor may be associated with roughly 0.1% of the genome, to get enough material to be analyzed by PCR or ligation and cloning (0.5-5 ng of DNA), each chromatin immunoprecipitation has to contain the equivalent of 25-50 micrograms of DNA. Thus, tissue culture cells, yeast, and other homogeneous cell suspensions are routinely used. Drosophila and mouse embryos have been also successfully used. More detailed comments and technical tips on the procedure in various systems are discussed in Ref. 10. Indeed, by "visualizing" regulatory proteins in vivo at high resolution, new functional aspects of regulatory proteins in the context of living interphase and mitotic chromosomes may be anticipated. Nevertheless, the occupancy of specific genomic sites by a given protein does not always allow easy extrapolation of the functional relevance of those binding sites. Formaldehyde may freeze transient interactions occurring in vivo, which reflect regulated binding of factors acting on the same site. Thus, the presence of factors at specific DNA sequences may simply reflect the chance that a protein has to access a particular site. The relevance of "binding" of a protein for gene function has then to be evaluated by other means. To this concern, appropriate functional tests should be considered before embarking on X-ChIP analysis. Because immunopurified chromatin contains all binding sites in the genome for a given factor, the exciting possibility exists that this method will contribute to a genome-wide identification of target sites of transcription factors (11).Finally, the X-ChIP assay is a powerful tool to take high resolution "pictures" of proteins in the context of chromosome structures. By fixing, we take a snapshot of a largely dynamic situation. Thus, like the single frames in a movie, one has to wait until the end before writing about it.

Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM)

PMID:
21413365
[PubMed]
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