Quantitative analysis of histone exchange for transcriptionally active chromatin
© Byrum et al; licensee BioMed Central Ltd. 2011
Received: 20 May 2011
Accepted: 7 July 2011
Published: 7 July 2011
Genome-wide studies use techniques, like chromatin immunoprecipitation, to purify small chromatin sections so that protein-protein and protein-DNA interactions can be analyzed for their roles in modulating gene transcription. Histone post-translational modifications (PTMs) are key regulators of gene transcription and are therefore prime targets for these types of studies. Chromatin purification protocols vary in the amount of chemical cross-linking used to preserve in vivo interactions. A balanced level of chemical cross-linking is required to preserve the native chromatin state during purification, while still allowing for solubility and interaction with affinity reagents.
We previously used an isotopic labeling technique combining affinity purification and mass spectrometry called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) to identify the amounts of chemical cross-linking required to prevent histone exchange during chromatin purification. New bioinformatic analyses reported here reveal that histones containing transcription activating PTMs exchange more rapidly relative to bulk histones and therefore require a higher level of cross-linking to preserve the in vivo chromatin structure.
The bioinformatic approach described here is widely applicable to other studies requiring the analysis and purification of cognate histones and their modifications. Histones containing PTMs correlated to active gene transcription exchange more readily than bulk histones; therefore, it is necessary to use more rigorous in vivo chemical cross-linking to stabilize these marks during chromatin purification.
Keywordscross-linking histone post-translational modification chromatin affinity purification
Eukaryotic genomes are highly organized into transcriptionally active (euchromatic) and silent (heterochromatic) chromatin regions. Conversion of chromatin between the two major forms is regulated in part through interactions between chromatin-modifying enzymes and nucleosomes. Nucleosomes are the fundamental unit of chromatin and consist of approximately 147 base pairs of DNA wrapped around an octameric core of the histones H2A, H2B, H3, and H4 . Chromatin structure plays a key role in the regulation of gene activity and its mis-regulation is a theme characteristic of many types of disease and cancer . The N-terminal tails of histones, which protrude outside of the nucleosome core , are subject to many sites and types of post-translational modifications (PTMs), which, in turn, help regulate biological processes through altering nucleosome stability or the function of chromatin-associated complexes [3, 4]. For example, acetylation of histone lysine residues on the N-terminal tail has been correlated to active gene transcription either by countering the negative charge of the DNA backbone, or through the recruitment or stabilization of bromodomain-containing proteins [3, 5, 6].
A major emphasis in the field of chromatin biology is the understanding of how histone PTMs and protein-protein interactions are associated with specific gene loci to regulate gene transcription. Current technologies like ChIP (chromatin immunoprecipitation), affinity purification of protein-histone complexes for proteomic analysis, and more recent technology that allows for the purification of chromosome sections for proteomic analysis are used to study protein interactions on chromosomes [7–10]. One pitfall of these technologies is the challenge of purifying cognate histones (i.e., preserving the in vivo associated histones during isolation of chromatin). To overcome this pitfall, we have previously reported how to monitor and prevent dynamic exchange of histones during chromatin purification . In vivo chemical cross-linking reagents, such as formaldehyde, can be used to prevent histone exchange during the purification of chromatin sections . However, there is a balanced level of chemical cross-linking needed to trap protein-protein and protein-DNA interactions, while still allowing for the solubility of chromatin for purification and access of affinity reagents .
We have recently published a quantitative approach using I-DIRT, an isotopic labeling technique utilizing affinity purification and mass spectrometry, to measure levels of histone exchange in purified chromatin sections . Here we describe a bioinformatic analysis, which expands on this published work, reporting the significance of proper cross-linking to capture histones with transcription activating PTMs during chromatin purification. In this work, we are able to gain new insights into the dynamic exchange of histones and post-translationally modified histones.
Detailed methods are described in Byrum et al. 2011. Briefly, Saccharomyces cerevisiae HTB1::TAP-HIS3 BY4741 (Open Biosystems) cells grown in isotopically light media and cells from an arginine auxotrophic strain (arg4::KAN BY4741, Open Biosystems) cultured in isotopically heavy media (13C6 arginine) were grown to midlog phase (3.0 × 107 cells/mL) and cross-linked using either 0%, 0.05%, 0.25%, or 1.25% formaldehyde (FA). The cells were harvested, mixed 1:1 by cell weight (isotopically light cells: heavy cells), and lysed under cryogenic conditions. The cell powder was resuspended in affinity purification buffer (20 mM HEPES pH 7.4, 300 mM NaCl, 0.1% tween-20, 2 mM MgCl2, and 1% Sigma fungal protease inhibitors) and the DNA sheared to ~1 kb sections. Small chromatin sections containing TAP tagged H2B histones were affinity purified on IgG-coated Dynabeads and the eluted proteins were resolved with a 4-20% Tris-Glycine gel. Following colloidal Coomassie-staining, histone gel bands were excised, trypsin digested, and tryptic peptides were subjected to tandem mass spectrometric analysis with a coupled Eksigent NanoLC-2D and Thermo LTQ-Orbitrap mass spectrometer . The histone purification experiments were performed in triplicate.
The isotopically light and heavy arginine containing histone peptides were identified using a Mascot (version 2.2.03) database search. Peptide identification can be made with mass spectrometric database searching software other than Mascot with equivalent results. The search parameters included: precursor ion tolerance 10 ppm, fragment ion tolerance 0.6 Da, fixed modification of carbamidomethyl on cysteine, variable modification of oxidation on methionine and acetyl on lysine, and 2 missed cleavages possible with trypsin. The Mascot results were uploaded into Scaffold 3 (version 3.00.01) for viewing the proteins and peptide information. A false discovery rate of 1% was used as the cut off value for arginine containing histone peptides. The monoisotopic peak intensity (I) values for each arginine containing peptide were extracted using Qual Browser (version 2.0, Thermo). The percent light for each peptide was calculated as IL/(IL + IH). The average of all peptides identified for each percentage of cross-linking was calculated along with the standard error. The number of unique identified peptides was: bulk H3 (26, 14, 9, and 8), H3K9acK14ac (7, 4, 8, and 8), bulk H4 (25, 8, 8, and 13), and H4K12acK16ac (7, 4, 5, and 3) for 0%, 0.05%, 0.25% and 1.25% FA, respectively. Percent light peptide reported here differs from the Byrum et al report  as we have separated PTM containing and unmodified peptides in the current report.
Results and Discussion
We have previously published the application of I-DIRT technology to determine the level of histone dissociation/re-association during chromatin purification . In this report, we have applied additional bioinformatic analyses to study the dynamics of exchange for histones containing transcription activating PTMs. As demonstrated in the histone exchange analysis shown in Figure 3, we show that chromatin marked for gene transcription is susceptible to the loss of histones during purification and therefore requires sufficient levels of in vivo chemical cross-linking to preserve the native chromatin composition. The technique reported in Byrum et al. 2011 and further analyzed here is relevant for a variety of genome-wide studies, and should be considered when preservation of in vivo chromatin content is essential for functional analyses, especially when examining transcriptional processes.
(isotopic differentiation of interactions as random or targeted)
This work was funded by NIH R01DA025755, P20RR015569, P20RR016460 and F32GM093614.
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