Chromatin immunoprecipitation ChiP

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... 

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. 

Occupancy of TBP at promoters in vivo was examined by chromatin immunoprecipitation (ChIP) assay (Kuras and Struhl, 1999 Li et al., 1999). The results indicate that the level of TBP occupancy at promoters correlates well with transcription level and is stimulated by activators. Interestingly, TBP occupancy is dependent on Srb4, a subunit of the Pol II holoenzyme. This indicates that TBP recruitment is an important mechanism of transcriptional activation in vivo and is interdependent with holoenzyme recruitment. 

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. 

Key words Chromatin immunoprecipitation (ChIP), RNA polymerase II, Gene expression. Next-... 

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