ChIP Protocol to Rapidly Characterize DNA-Protein Interactions
V. Palhan, J. Robert, S. Bagga and E. Mueller
Sigma-Aldrich, St. Louis, MO, 63103
In the study of DNA-protein interactions, chromatin immunoprecipitation (ChIP) has emerged as a powerful technique for discovery and characterization of protein-mediated transcriptional regulation. The method is outlined in Figure 1 and relies on the ability of a cross-linking reagent, most typically formaldehyde, to capture the often transient interactions of specific proteins with genomic material. Once thus stabilized, cross-linked chromatin is sheared and then immunoprecipitated using a protein specific antibody. The resulting pellet is enriched in DNA (or RNA) to which the binding protein is cross-linked, and after reversal of the cross-linking the sample contains DNA that can be characterized by a variety of methods.
Figure 1: Schematic of chromatin immunoprecipitation
Early ChIP practitioners developed the method to probe histone interactions with chromatin on transcriptional activity [Dedon et al 1991] as well as the role of the polycomb repressor complex in Drosophila [Orlando and Paro 1993]. In the following decade there has been considerable refinement in ChIP methodology. While this technique still requires optimization for lysis, chromatin shearing and antibody selection and concentration [Orlando, 2000], ChIP has changed significantly in the DNA characterization and detection. ChIPed DNA was first analyzed using Southern analysis and then by PCR techniques, but characterization was later expanded to include several methods to enable microarray analysis [e.g. O’Geen et al, 2006] and high throughput sequencing [Robertson et al, 2007]. These changes have opened the field to wholesale genomic mapping of specific DNA-protein interactions [Rosenfield et al, 2009] and for both abundant and rarer interactions [Pillai et al, 2009 ].
The effect of histones on a gene specific translation is now slowly being teased out of a myriad of ChIP data [Munshi et al, 2009; Strahl and Allis, 2000]. Once this is complete, remaining work will be needed to ascertain which growth conditions drive these histone modifications, and the resultant regulatory changes. ChIP, as currently practiced, is a rather tedious method for this type of discovery. Most of the current published protocols and the commercial kits for ChIP all require up to three days to complete fixation, lysis, shearing and immunoprecipitation. However, the long binding times are only necessary for pulling down rare DNA binding proteins.
A response to this dichotomy has been the development of a ChIP kit based on a flat well plate, with an abbreviated protocol that allows completion of multiple (up to 100) assays in less than a workday. The system is meant for rapid ChIP identification of histone, polymerase and other fairly abundant protein-DNA interactions. The format and protocol allow the researcher to test the effects of growth conditions, time and other variables on these transcriptionally relevant protein-DNA interactions.
Figure 2: Imprint ChIP kit
The simple protocol, outlined in figure 2, was developed for ease of use and speed. The trade-off is that the method cedes the sensitivity needed to pull down rare transcription factors. The method starts with crosslinked cells, and the treated chromatin is isolated after lysis and shearing. Upon addition of an appropriate antibody, immunoprecipitation is facilitated using a stripwell plate coated with protein G. After washing away unbound chromatin, the crosslinks are reversed and the enriched DNA is purified and ready for analysis. The total procedure can be completed in a normal workday, and the kit includes all the necessary reagents for purification as well as a set of antibodies and PCR primers for a positive control. One caveat lies in the fact that this method was conceived for ChIP experiments that had already been well worked out, and so there are a limited number of extra reagents for optimization of crosslinking or lysis.
Information gleaned from chromatin modification can greatly enhance our understanding and treatment of development and related diseases (such as cancer, and autoimmune disorders). Using ChIP technology it is possible to identify histone modification and the translational faction associated with the particular gene. These can then be used to address the regulatory network interactions.
The utility of this streamlined ChIP is shown in the following experiment. A split population of SW480 cells were grown for several days, one set grown with the addition of 5mM butyrate for 20-24 hours. The separate populations were trypsanized, collected by centrifugation, washed in serum free buffer and then cross linked using 1% formaldehyde. After quenching the reaction with glycine, cells were lysed, sonicated and the isolated chromatin frozen for later use. Sonication was completed using a Diagenode bioruptor for 15 minutes of 30 sec sonication "on" and 30 sec "off" at maximum setting. An equivalent of 250,000 cells worth of chromatin was processed using the Imprint CHP1 kit protocol with antibodies for H3K9ac (2µg/well, Sigma H9286), RNA polymerase (2µg/well, Sigma R1530), and H3K27me3 (2µg/well, Millipore 07-449). The enriched DNA was treated to reverse crosslinking, purified, and then quantified using PCR primers for the DNA loci KRT17, MYT1 and SOCS2 (see appendix for primer sequences).
The results, presented in the 2008 Spring meeting of the American Association of Cancer Research, are shown in Figure 3. The relative abundance of the ChIPed DNA is plotted as Butyrate treated (Ct)/ Untreated/normal (Ct); that is the relative differences in IP DNA are normalized.
Figure 3: Butyrate reverses PRC-mediated repression
The data is completely consistent with our current understanding of histone modification. Butyrate is a histone deacetylase inhibitor, and thus would be expected to increase the amount of acetylation as evidenced by an increase in DNA bound to histone 3, acetylated at lysine 9. Conversely, butyrate should have no effect on RNA polII binding, and very little effect is seen with antibody-mediated enrichment by ?-polII. Finally, one would expect methylation of histones to be reduced, since the first step of this process, namely deacetylation, has been inhibited [van der Vlag and Otte, 1999]. In all probed loci the amount the H2K37me3 representation is reduced when compared to the untreated cells. This experiment shows the utility of the kit in that multiple ChIP experiments were able to be carried out in a single day with good results.
A similar result can be seen in a kinetic experiment showing the effects of SAHA on H3K27me3 in DU145 cells. SAHA inhibits the polycomb repressor complex, and works much more specifically but much like butyrate in the example above. In this experiment cells were grown and then induced with 10uM suberoylanilide hydroxamic acid (Vorinostat, SAHA). Cells were collected at 0, 2, 5, 24 and 48 hours, crosslinked with 1% formaldehyde and then frozen. Chromatin was worked up as above and subjected to ChIP using ?-H3K27me3 using either the CHP1 kit [250,000 cell equivalent] or a Staph A protocol [750,000 cell equivalent, Farnham Lab protocols ]; the enriched and purifed DNA was analyzed for the MYT1 locus using qPCR. Figure 4 shows the results; each method showed the expected loss of H3K27me3 loss over time.
Figure 4: Reduction in PRC repression on MYT1 post SAHA treatment chIP with H3K27me3 AB, DU 145 cells.
As mentioned above, the Imprint ChIP kit does sacrifice sensitivity for speed. This is mainly due to the limited binding potential of the plate setup, which is obviously of a fixed capacity while bead-based systems can be scaled. The effect of this finite binding can be seen in the following experiment (Figure 5), in which the signal (H3K9ac) is compared to noise (IgG) from non-specific binding.
Figure 5: Signal to noise with increasing input.
The data shows proportionally greater signal (decreased Cts) as the number of cells are increased, as expected. However, at between 50,000 – 100,000 cells the background noise increases with excess non-specific material available for binding, and the signal to noise drops to unacceptable levels. The point at which noise increases varies with target, but does occur eventually for all studied antibody complexes.
Finally, while this plate mediated ChIP gives rapid results for abundant protein-DNA species, it does not work for rare events. The reason for this failure lies in the fact that not enough of the desired DNA-protein can be bound to give sufficient signal before non-specific interactions swamp out capture. This non-result can also be obtained if too many cells or too much antibody is used per well; Figure 6 shows results comparing ChIP results for the fairly rare EZH2 target (a methylase in the polycomb repressor complex) using either StaphA (2x106 cells) or the Imprint kit (2.4x105 cells).
Figurte 6: Comparison of CHP1 Vs. Staph.A. for a rare target like EZH2; Targetting MYT1 gene post IP
While the StaphA ChIP shows the expected six Ct (64x) enrichment over input sample, the CHP1 kit shows a marginal two Ct increase (4x). The reason for this poor signal is that not enough EZH2-DNA can be captured in the Imprint wells in order to give a reasonable signal. By increasing input almost ten-fold, possible only with the scaleable StaphA protocol, one can clearly delineate enrichment through the crosslinking and immunoprecipitation.
The data from the various experiments above demonstrates the strengths and weaknesses of the Imprint Chromatin Immunoprecipitation kit offered by Sigma (CHP1). The method gives researchers a means to rapidly characterize highly abundant DNA-protein interactions, providing a relatively easy way to perform kinetic or conditional analyses for polymerase- or modified histone-DNA interactions. Post-translational modifications to histone proteins have been found to regulate the packaging of genomic DNA into chromatin and thus the activity of specific genes [Pillali et al, 2009]. These interactions are now thought to play important roles in development and pathogenesis. Many of the advances in understanding of these mechanisms are attributed to the successful development of chromatin immunoprecipitation (ChIP) for in vivo detection of histone modifications as well as chromatin binding regulatory proteins, and the technique is a powerful tool for analyzing histone modifications as well as proteins that bind directly or indirectly to DNA. Sigma's ChIP was conceived as an easy-to-use tool for iterative study of histone modifications and other abundant DNA binding proteins. This kit has been optimized for cell lysis, immunoprecipitation, cross linking and DNA purification for convenient use by our customers.
Appendix:
Primer sequences mentioned in the manuscript:
References
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