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

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.     

Analysis of epigenetic modifications of chromatin at specific gene loci by native chromatin immunoprecipitation of nucleosomes isolated using hydroxyapatite chromatography

Chromatin immunoprecipitation (ChIP) is routinely used to examine epigenetic modification of histones at specific genomic locations. However, covalent modifications of histone tails can serve as docking sites for chromatin regulatory factors. As such, association of these regulatory factors with chromatin could cause steric hindrance for antibody recognition, resulting in an underestimation of the relative enrichment of a given histone modification at specific loci. To overcome this problem, we have developed a native ChIP protocol to study covalent modification of histones that takes advantage of hydroxyapatite (HAP) chromatography to wash away chromatin-associated proteins before the immunoprecipitation of nucleosomes. This fast and simple procedure consists of five steps: nuclei isolation from cultured cells; fragmentation of chromatin using MNase; purification of nucleosomes using HAP; immunoprecipitation of modified nucleosomes; and qPCR analysis of DNA associated with modified histones. Nucleosomes prepared in this manner are free of contaminating proteins and permit an accurate evaluation of relative abundance of different covalent histone modifications at specific genomic loci. Completion of this protocol requires approximately 1.5 d.     

Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.     

Histone modification and chromatin remodeling during NER

Here we review our developments of and results with high resolution studies on global genome nucleotide excision repair (GG-NER) in Saccharomyces cerevisiae. Technologies were developed to examine NER at nucleotide resolution in yeast sequences of choice and to determine how these related to local changes in chromatin. We focused on how GG-NER relates to histone acetylation for its functioning and we identified the histone acetyltransferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. Factors influencing this Gcn5-mediated event are considered which include Rad16, a GG-NER specific SWI/SNF factor and the yeast histone variant of H2AZ (Htz1). We describe results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then consider the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the NER of UV-induced cyclobutane pyrimidine dimers throughout entire yeast genome. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage before it is returned to its pre-damaged status to maintain epigenetic codes.     

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.     

Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter

We have analyzed the histone modification status of the PHO5 promoter from yeast by the ChIP technology and have focused on changes occurring upon activation. Using various acetylation-specific antibodies, we found a dramatic loss of the acetylation signal upon induction of the promoter. This turned out to be due, however, to the progressive loss of histones altogether. The fully remodeled promoter appears to be devoid of histones as judged by ChIP analyses. Local histone hyperacetylation does indeed occur, however, prior to remodeling. This can explain the delay in chromatin remodeling in the absence of histone acetyltransferase activity of the SAGA complex that was previously documented for the PHO5 promoter. Our findings shed new light on the nucleosomal structure of fully remodeled chromatin. At the same time, they point out the need for novel controls when the ChIP technique is used to study histone modifications in the context of chromatin remodeling in vivo.     

Hoxc Gene Collinear Expression and Epigenetic Modifications Established during Embryogenesis Are Maintained until after Birth

The Hox genes, which are organized into clusters on different chromosomes, are key regulators of embryonic anterior-posterior (A-P) body pattern formation and are expressed at specific times and in specific positions in developing vertebrate embryos. Previously, we have shown that histone methylation patterns are closely correlated with collinear Hox gene expression patterns along the A-P axis of E14.5 mouse embryos. Since histone modification is thought to play a crucial mechanistic role in the highly coordinated pattern of collinear Hox gene expression, we examined the maintenance of the spatial collinear expression pattern of Hoxc genes and the corresponding histone modifications during embryogenesis and in early postnatal mice. Hox expression patterns and histone modifications were analyzed by semi-quantitative RT-PCR and chromatin immunoprecipitation (ChIP)-PCR analyses, respectively. The spatiotemporal expression patterns of Hoxc genes in a cluster were maintained until the early postnatal stage (from E8.5 through P5). Examination of histone modifications in E14.5 and P5 tissues revealed that level of H3K27me3 is only a weak correlation with collinear Hoxc gene expression in the trunk regions although diminished in general, however the enrichment of H3K4me3 is strongly correlated with the gene expression in both stages. In summary, the initial spatiotemporal collinear expression pattern of Hoxc genes and epigenetic modifications are maintained after birth, likely contributing to the establishment of the gene expression code for position in the anatomic body axis throughout the entire life of the organism.