Histone modifications ChIP

ChIP-Seq of histone modifications correlated with DNAse Seq of open chromatin in CD4 cells [8, 29]. 

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. 

Histone modifications and how they are affected by toxicants can be investigated by chromatin immunoprecipitation (ChIP). Generally, there are two basic ChIP procedures (a) native ChIP (nChIP) that is performed on native digested chromatin and (b) cross-linked ChIP (xChIP), that includes a reversible chemical cross-link step to stabilize weaker DNA protein binding. The latter can be problematic as the epitope of the antibody may be blocked or destroyed by the cross-link. However, xChIP is more convenient, as samples can be collected and frozen and then precipitated all at the same time, whereas for nChIP the samples need to be precipitated directly. This is a major issue especially when analyzing time courses of PTM events. 

An efficient chromatin immimoprecipi-tation (Chip) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3(6) 1018-1025.doi 10.1038/nprot.2008.66... 

The association between a histone tail modification and a particular functional state of chromatin, came with the demonstration that transcriptionally active chromatin fractions were enriched in acetylated histones, firstly by biochemical co-fractionationation ([8,9] and references therein) and then by Chromatin ImmunoPrecipitation, ChIP [10]. Subsequently, regions of transcriptionally silent constitutive and facultative heterochromatin, were shown, by immunofluorescence microscopy, to be under-acetylated [11,12]. This supported the idea that acetylation of the histone tails, with the associated loss of positive charge and reduction in DNA-binding constant, somehow caused chromatin to become more open (or less condensed ) and thereby more conducive to transcription. While this is likely to be an important contributory factor, it has now become clear that the... 

As we have previously outlined, histones are small nuclear proteins that contribute to pack the DNA within nucleosomes (7,33). Histones are subject to a number of covalent post-translational modifications that collectively influence chromatin remodeling and gene expression. The question now is how to build genome-wide histone-maps. The answer came recently thanks to the combination of ChIP followed by either microarrays (ChIP-chip) or sequencing methods (ChIP-Seq) (31). 

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