Chromatin immunoprecipitation sequencing

Finally, the deacetylation of histones in methylated gene regions may be selective with respect to the histone affected. Chromatin immunoprecipitation analysis of the histone acetylation status of proviral sequences targeted to defined genome loci in MEL cells showed that the in vitro methylated proviral sequences that were transcriptionally silent in vivo, were hypoacetylated only with respect to histone H3 in contrast, the acetylation status of histone H4 was comparable on the methylated and unmethylated provirus [78]. Moreover, the H3 acetylation status correlated closely with the location of methylated CpGs along the proviral sequence. 

ChIP-on-chip (61) is a technique where DNA arrays (chip) can be used in combination with chromatin immunoprecipitation techniques (ChIP) to study interactions between proteins and DNA sequences. The most common application of this type of arrays is the study of transcription factors (62), but they are also used for the analysis of dynamical transcriptional mechanisms (63), or the study of replication-related proteins such as ORC or histones (64). ChIP-on-chip microarray is based on the isolation of DNA sequences bound to particular proteins by immunoprecipitation (ChIP). Then, isolated DNA... 

Hoffman, B. G. and S. J. Jones. 2009. Genome-wide identification of DNA-protein interactions using chromatin immunoprecipitation coupled with flow cell sequencing. /. Endocrinol. 201 1-13. 

Kaufmann, K., J. M. Muino, M. Osteras, L. Farinelli, P. Krajewski, and G. G. Angenent. 2010. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat. Protoc. 5 457-72. 

If the immunoprecipitation vas performed against a chromatin binding factor vith an as yet unknown binding motif, enriched sequences can be used to retrieve the binding motif. In the same way, previously known binding motifs can be modified. 

This approach, also known as ChIP-on-chip, has been the most used technique to establish histone-maps. Briefly, as we have been detailing, the chromatin fragments are incubated in the presence of an antibody that recognizes a specific histone modification (e.g., methylation on histone 3 or acetylation on histone 4, etc.) (Figure 4). Next, the protein-DNA complex is immunoprecipitated. After reversal of the cross-link, ChIP-enriched DNA and control DNA are amplified by PCR and labeled with fluorescent dyes (Cy3 and Cy5). Finally, the samples are hybridized onto a specific microarray. The ratio of the Cy5 to Cy3 intensities measured for each DNA sequence in the array is a measure of the amount of a specific histone bound to the DNA. 

Step El DNA in the immunoprecipitated chromatin fragments is released by reversing the cross-link and then is quantitated using a sensitive PCR method. The method can be used to analyze the in vivo association of any protein with a specific sequence of DNA by using an antibody against the protein of interest in step B- [See S. E. Rundlett et al., 1998, Nature 392 831]... 

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