Chromation Immunoprecipitation Assay

Chromation Immunoprecipitation Assay1

Background ChromatinDNA and Nucleosome Histones (H2A, H2B, H3, H4)Heterochromatin and Euchromatin

In eukaryotes DNA is found in vivo in complex with proteins and RNA. It is divided between heterochromatin (highly condensed) and euchromatin (less extended). The major components of chromatin are DNA and histone proteins, although many other chromosomal proteins have prominent roles too. The fundamental unit of chromatin is the nucleosome, which consists of 2 copies each of H2A, H2B, H3 and H4 histones, and approximately 147 bp of DNA wrapped almost two times around the octamer.2

The basic unit of chromatin, the nucleosome, consists of DNA that wraps around an octamer of histones. The histone tails protrude from the nucleosome core and are subjected to different post-translational modifications. Nucleosomal DNA can also be methylated by DNA methyltransferase activities. The types of post-translational modifications of histones and the degree of DNA methylation determine a specific chromatin structure. Two morphologically distinct types of chromatin can be distinguished, namely euchromatin and heterochromatin. Euchromatin is associated with gene-rich and transcriptionally active domains of dispersed appearance characterized biochemically by hyperacetylation of histones and hypomethylation of both histones and DNA. In contrast, highly condensed heterochromatin is linked to gene-poor and transcriptionally inactive domains such as centromeres and telomeres. Biochemically, heterochromatin is characterized by hypoacetylation of histones, and hypermethylation of histones and DNA. In addition, non-coding RNAs have been recently recognized as players in chromatin remodeling, primarily involved in heterochromatin formation and transcriptional or post-transcriptional gene silencing.3Chip Assay Immunoprecipitation of a protein of interest from a chromatin preparationHelps determine DNA sequences associated with the proteinChIP has been used for mapping the localization of post-translationally modified histones and histone variants on the genome,

ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it.4and for mapping DNA target sites for TFs and other chromosome-associated proteins

This assay determines whether a certain protein-DNA interaction is present at a given location, condition, and time point.5

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71. Dna-protein cross link and cell lysis (30-40 mins)Cross link ChIP - XChIP Uses formaldehyde to covalently attach proteins to target DNAdimethyl adipimidate, disuccinimidyl suberate, succinimidyl proprionate or succinimidyl succinate can be used with formaldehyde Glycine stops cross linking lysis Lysis buffer fragmentation by sonication 500 to 1000bpBroad range of chromatin associated factors can be analyzed

Native ChIP NChIP No cross- linking Cells are lysed Fragmentation by micrococcal nuclease digestion 150 to 900bp Nucleosome based resolution mono to heptanucleosomes

for proteis tightly associated to chromation- histones and modifications

DNA and proteins are commonly reversibly cross-linked with formaldehyde (which is heat-reversible) to covalently attach proteins to their target DNA sequences. This ensures that protein-DNA complexes are maintained during the ChIP procedure. Formaldehyde cross-links proteins and DNA molecules within ~2 of each other, and thus is suitable for looking at proteins which directly bind DNA. The short cross-linking arm of formaldehyde, however, is not suitable to examining proteins that indirectly associate with DNA, such as those found in larger complexes.

To remedy to this limitation, long-range bifunctional cross-linkers such as dimethyl adipimidate, disuccinimidyl suberate, dithiobis(succinimidyl proprionate) or ethylene glycol bis(succinimidyl succinate) have successfully been used in combination with formaldehyde, to detect proteins on their target genes, which with formaldehyde alone are refractory to detection (22). Because the use of long-range cross-linkers is likely to increase the risk of precipitating irrelevant proteins, they should be restricted to analysis of proteins previously established to be in the DNA-binding complex of interest. Cross linking is time dependent. (2- 30 mins) Excessive cross linking reduces antigen accessibility and sonication efficiency. Glycine quenches formaldehyde and stops the reactionRegardless of the cross-linking method, the chromatin is then fragmented, either by enzymatic (micrococcal nuclease) digestion after cell lysis, or by sonication of whole cells or nuclei, to fragments of 200-1,000 base pairs (ideal) in length (and average of 500 base pairs is considered as optimal) encompassing mono- to heptanucleosomes. This step (sonication) is important because it renders chromatin soluble and dictates resolution of assay. The extent to which one can fine map the location of a protein to the genome depends on extent of DNA fragmentation.

Sonicationefficiency is dependent on the cell line, cell density, extent of cross linking.

82. Immunoprecipitation (8-10 hours) Add Primary Antibody (1-10g per 25g Protein) wash and add protein A/G agarose beads- A and G beads (1:1) in RIPA bufferOvernight incubationWash samples

You can purify and quantify DNA from the sample from previous step

93. Elution and reverse Cross link (4- 10 hours) elute DNA using elution buffer DNA PurificationPCR purification kit - RNAse A and heat at 65o C. Phenol:choloroform Proteinase K and heat. Use phenol:choloroform and ethanol precipitate with glycogen10 ChIP-PCR: Quicker and cost effective

CHiP-chip : DNA purified from IP chromatin is labelled with fluorescent dyes using ligation mediated PCR and hybridize to microarray

ChIP-seq: combine ChIP and direct sequencing

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After cross linking and lysis. Fraction with nuclear pellets is collected and chromatin is sheared. Chromatin sa,mples are incubated with antibodies in ultrasonic bath 15 min 4C. After centrifugation they are mixed with Protein A beads for 45 min 4cMany washes. Add chelex 100 suspension to beads and boil 10 min.Cool add proteinase K. Incubate in shaker 30 min 50C 1400rpm.Boil again. After centrigugatio PCR12

After centrifugation they are mixed with Protein A beads for 45 min 4cMany washes. Add chelex 100 suspension to beads and boil 10 min.Cool add proteinase K. Incubate in shaker 30 min 50C 1400rpm.Boil again. After centrigugatio PCR

13ReferencesCollas, P., & Dahl, J. A. (2008). Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Frontiers in Bioscience: A Journal and Virtual Library, 13, 92943.

2. Nelson, J. D., Denisenko, O., & Bomsztyk, K. (2006). Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat. Protocols, 1(1), 179185. 14