Our approach to separate non-specific and specific chromatin interactions in our ChIP-3C protocols is to use sonication to fragment chromatin fibers (), which is very different from the 3C protocol that uses restriction digestion to gently fragment chromatin fibers. The use of sonication in ChIP-3C has worked well [Kumar et al., 2007
; Pan et al., 2008
]. Chromatin interactions bound by SATB1 and PML have been found by sonication-based ChIP-3C. RNA interference was used to knock down SATB1 and PML, and this procedure was found to change the chromatin interaction profile as well as the expression of associated genes [Kumar et al., 2007
]. Sonication-based ChIP-3C was also performed on the TFF1 region [Pan et al., 2008
], and could recapitulate the TFF1 chromatin interaction shown to be present previously by restriction enzyme-based ChIP-3C [Carroll et al., 2005
]. Further, sonication-based ChIP-3C was used to show that ERα mediates the TFF1 interactions, by the use of RNA interference to knock down ERα which abrogated the chromatin interaction and associated gene expression [Pan et al., 2008
]. In addition to the demonstrated successes of the sonication-based ChIP-3C protocols, we will further show the benefits of sonication through a theoretical argument and several lines other experimental evidence, including whole genome ChIP sequencing and mapping experiments; and our own unpublished data on sonication-based molecular interaction mapping methods.
We believe that the use of restriction enzymes to gently digest the material could retain non-specific interactions where two chromatin fragments float close to each other in the crowded cellular nucleus. We and others have used sonication to fragment chromatin. While previously underappreciated, sonication is very vigorous, and could possibly break up weak non-specific interactions (). Moreover, chromatin is sonicated to a region of about 200-1000 bp, as opposed to 3-4 kb fragments created by 6-bp restriction enzyme cutters. A much smaller chromatin fragment would be sterically hindered from ligating to non-specific interactions that are not in very close proximity, as compared with a longer chromatin fragment which would be much more flexible. In line with this idea, a review article analyzing all 3C and related methods suggests that 6-bp restriction enzyme cutters do not give such good resolution, and recommends the use of 4-bp cutters for analysis of <10-20 kb loci [Simonis et al., 2007
]. Another benefit of the use of sonication instead of restriction enzyme digestion is that incomplete digestion products can be avoided. Given that incomplete digestions can form 20-30% of a library [Simonis et al., 2007
], this is a large amount of noise that could be eliminated with sonication instead of restriction enzyme digestion.
Further, adding ChIP would enrich the specific protein-bound chromatin interaction complexes and wash away the non-specific chromatin fragments that were already detached from specific chromatin complexes by sonication, hence providing an even purer chromatin DNA pool for chromatin interaction analysis. Therefore, with this composition, sonication-based ChIP-3C protocols should have much less complication by high level non-specific interaction noises than the 3C-like methods.
Therefore, in comparing restriction enzyme digested chromatin, sonicated chromatin, and sonicated plus ChIP-enriched chromatin, we would expect to see different profiles from different detection techniques, with different levels of information. Simply performing reverse cross-linking of the chromatin and sequencing all material would be similar to a ChIP-Seq experiment if ChIP-enriched chromatin were used as the input (). Performing 3C on restriction enzyme digested chromatin would be quite noisy, whereas 3C on sonicated chromatin would be less noisy, and ChIP-3C on sonicated chromatin would be the least noisy and specific for chromatin interactions bound to the protein of interest (). Similar to 3C, 4C on enzyme digested chromatin would be quite noisy, whereas 4C on sonicated chromatin would be less noisy, and ChIP-4C on sonicated chromatin would be the least noisy and specific for chromatin interactions bound to the protein of interest ().
Sequencing from different experimental approaches
3C and 4C from different experimental approaches
Experimental evidence from ChIP-Seq () and our sonication-based 4C data () support the hypothesis that sonication can “shake off” non-specific interactions, giving rise to more specific data. First, ChIP-Seq data show very specific binding peak results. ChIP-Seq methods commonly employ sonication to fragment the chromatin. In ChIP-Seq, formaldehyde cross-linking captures chromatin complexes, followed by sonication, and complexes are all immunoprecipitated in ChIP. Therefore, all reverse cross-linked ChIP DNA should, after mapping and sequencing, be reflected as binding density (). This includes chromatin sequences that are not directly bound to the protein of interest, but which are involved in the complexes. As such, if weak non-specific interactions are present in chromatin complexes, they should be shown as regions of binding density in ChIP-Seq data (most ChIP-Seq protocols use sonication to fragment chromatin). In particular, if it were true that sonicated material shows high levels of non-specific interactions due to flexible polymer dynamics such that nonspecific interactions within 100 kb could occur, then we should see gradually sloping ChIP peaks. However, this is not the case, and most ChIP-Seq peaks, including ERα ChIP-Seq peaks, are very tight and narrow ().
Second, our sonication-based 4C results are also very specific (). In this 4C experiment, we used sonication to fragment the chromatin, which differs from the reported standard 4C protocols. Our 4C data, which is also non-ChIP enriched, shows a steep drop in the number of ligated sequences after 1kb. The number of ligated sequences remains 0 until it reaches the first interacting sequence ~50 kb away, whereupon it rises to several hundreds of sequences, indicating specific chromatin interactions. The lack of sequences between 1 to ~50 kb indicates that we do not detect any interactions despite using an unprecedented number of sequences (0.46 million sequences); hence the observations from restriction enzyme-based 3C, 4C, and 5C that distance-based, non-specific interactions up to 100 kb due to flexible polymer dynamics are present do not appear to be recapitulated when sonication is used.