Chromatin ChIP

The chromatin immuno-precipitation assay (ChIP) is a procedure that is employed to determine which genes are... 

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

Chromatin fractionation approaches including ChIP assays have provided evidence for and against uH2A and uH2B being associated with transcriptionally active chromatin [270,281-285]. Most evidence supports uH2A being associated... 

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... 

The basic principle behind the ChIP method is relatively simple It is based on the selective enrichment of a chromatin fraction containing a specific antigen (e.g. transcription factors, DNA binding proteins, modified histones, etc.) by an immunoprecipitation step. Specific (important, see below ) antibodies that recognize a protein of interest or the modified form of a protein can be used to determine the relative abundance of it within DNA regions. 

Before the immunopreciptation step the chromatin has to be fragmented in order to make interactions accessible and to increase the resolution of the ChIP. Two different methods are commonly used fragmentation via sonication of the chromatin, or an enzymatic digestion with micrococcal nuclease. 

The combination of chromatin immunoprecipitation with DNA microarrays allows the genome-wide analysis of the distribution of an antigen. The immunoprecipitated DNA is quantitatively amplified, labeled and used to probe DNA microarrays. In principle ChIP-on-chip methods can be divided into two basic groups, depending on the content of the microarrays which are used (i) microarrays/promoter tiling arrays and (ii) genome tiling arrays. 

For re-ChIP (or double ChIP), two sequential rounds of immunoprecipitations are performed using two different antibodies. Re-ChIP allows one to determine whether two antigens occur together on the same chromatin fragment or on different fragments and can be used to assemble proteins in complexes [23]. 

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

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