Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules

Abstract

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

Fig. 1.

Antibody evaluation experimental paradigm and method of quantification. A, histones were acid-extracted from HeLa S3 cells labeled in light or heavy SILAC growth medium, and H3 was further purified by reversed phase chromatography. Light H3 was incubated with a pan-specific H3 antibody, whereas heavy H3 was incubated with a mark-specific antibody under ChIP buffer conditions. The eluates were mixed and subjected to SDS-PAGE. The H3 band was derivatized with propionic anhydride, digested with trypsin, and then rederivatized (see “Experimental Procedures” for details). The peptides were analyzed by LC-MS on an Orbitrap MS instrument. B, this example demonstrates how H3K4 mark-targeting antibodies were evaluated. XICs corresponding to all possible modification states of the peptide bearing H3K4 were made for heavy (red traces) and light (blue traces) variants of the peptide. Additionally, we made XICs for an unmodified peptide spanning residues 41–49 of H3. The area under the curve was calculated for each XIC. The heavy/light ratio of the H3(41–49) peptide was used to normalize all other heavy/light ratios, thus ensuring that the same amount of H3 was being analyzed in each experiment. In the lower right panel, we derive two types of information. Each sub-box in the tree map (upper portion) is proportional to the amount of ion current from any given modification state over the total of all ion current from all modification states at H3K4, analogous to a pie chart. The heat map (lower portion) shows the log₂ of the enrichment ratio (heavy/light) of the antibody being tested for each possible modification state of H3K4. un, unmodified; me1, monomethyl; me2, dimethyl; me3, trimethyl; ac, acetyl.

Fig. 2.

Evaluation of antibodies directed against H3K4, H3K27, and H3K9 modifications. Tree maps and heat maps for each antibody evaluated are annotated as in the lower right panel of Fig. 1B. Note that the intended target of enrichment is bounded by a yellow box in each heat map. Where necessary, we collapsed several modification combinations into a single value to represent enrichment of the desired mark (i.e., H3K9me3K14un + H3K9me3K14ac). This was done by computing an intensity-weighted average of all modification combinations that bear the mark stated in the column. The intensity of the pan-specific background was used to weight each combination. A, evaluation of H3K4 targeting antibodies. B, evaluation of H3K27me3 targeting antibodies on their ability to enrich marks on the H3.1 and H3.2 variants of H3 (Ala-31 variant). C, evaluation of H3K27me3 targeting antibodies on their ability to enrich marks on the H3.3 variant of H3 (Ser-31 variant). D, evaluation of an H3K9me3 targeting antibody.

Fig. 3.

Estimation of background frequencies of histone modifications at other sites. Background frequencies were estimated using the techniques detailed under “Experimental Procedures” and are depicted in the tree map format explained in the legend for Fig. 1B.

Fig. 4.

Hierarchical clustering of antibody enrichment histone modification data. A, enrichment ratios for all of the sites measured on H3 were clustered (Pearson correlation, complete linkage) by modification and antibody enrichment. Each row corresponds to a particular antibody enrichment strategy and is labeled with the intended target of the antibody and its catalogue number. Each column corresponds to a different histone modification, where un means unmodified. Each square is color-coded by an intensity-weighted average of all modification combinations that bear the mark stated in the column. Intensity of the pan-specific background was used to weight each combination. The yellow box depicts a core set of modification enrichments that drive clustering of antibodies together that target canonical activating marks. The blue box depicts a core set of modification enrichments that drive clustering of antibodies together that target canonical repressive marks. B, antibody enrichment data were clustered together with canonical chromatin states as determined computationally by Ernst and Kellis (13). Only rows were clustered in this case, and the data set was limited to histone modifications that were common to the two studies. Some of Ernst's states are omitted for clarity. Because the data sets are on two different scales, the color scale is arbitrary. See text for details.