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.
Highlights
Frequencies of Other Histone Modifications on H3—We estimated the total amounts of other well known histone modifications on H3 in HeLa using the same techniques as employed above (Fig. 3)
We have devised a system that measures the performance of an antibody directed toward a specific histone post-translational modification relative to the performance of a nonspecific antibody directed against the histone in general. In this manner we can: 1) measure the fold enrichment over background for the intended target of the mark-specific antibody, 2) measure the bias of the mark-specific antibody for or against any other potential post-translational modifications at the same site on the histone protein, and 3) estimate the background levels of discrete post-translational modifications at a site by using the observed abundance in the mass spectrometer. This is accomplished by the use of SILAC technology to isotopically label histone proteins in cell culture [14]
The enrichment ratios that we report are computed by weighting each state (i.e., H3K27me3/H3K36un or H3K27me3/H3K36me2) by its ion current relative to the total of all states bearing the same H3K27 modification
Summary
ChIP, chromatin immunoprecipitation; SILAC, stable isotope labeling of amino acids in cell culture; XIC, extracted ion chromatogram; IP, immunoprecipitation; AIMS, accurate inclusion mass screening. If an antibody were to purify the population of all H3K4me3-bearing histones to homogeneity, we could definitively answer the question as to whether the bivalent state—H3K4me3/H3K27me3—is ever represented on the same histone protein molecule. This is only true if the antibody reagent can perform this purifying role, and assessment of an antibody’s performance in this regard has been difficult. We use stable isotope labeling of amino acids in cell culture (SILAC) to quantitatively assess the specificity of various ChIP grade antibodies and extend their use to identify co-occurring marks on histone protein molecules
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