Analysis of Histone Exchange during Chromatin Purification

Abstract

Central to the study of chromosome biology are techniques that permit the purification of small chromatin sections for analysis of associated DNA and proteins, including histones. Chromatin purification protocols vary greatly in the extent of chemical cross-linking used to prevent protein dissociation/re-association during isolation. Particularly for genome-wide analyses, chromatin purification requires a balanced level of fixation that captures native protein-protein and protein/DNA interactions, yet leaving chromatin sections soluble and accessible to affinity reagents. We have applied a relative quantification methodology called I-DIRT (isotopic differentiation of interactions as random or targeted) for optimizing levels of chemical cross-linking for affinity purification of cognate chromatin sections. We show that fine-tuning of chemical cross-linking is necessary for isolation of chromatin sections when minimal histone/protein exchange is required.

Figure 1. I-DIRT analysis of histone exchange during chromatin purification

H2B-TAP cells were grown isotopically light (¹²C₆-Arg), while non-tagged cells were grown isotopically heavy (¹³C₆-Arg). Cultures were treated with various levels of formaldehyde. Cells were harvested independently and mixed 1:1 for co-cryogenic lysis. Chromatin was sheared and then affinity purified on IgG coated Dynabeads. Co-purifying histones were resolved by SDS-PAGE and the ratios (isotopically light to heavy arginine containing histone peptides) were measured with high resolution mass spectrometry.

Figure 2. Purification of formaldehyde cross-linked chromatin

(A) DNA shearing as a function of formaldehyde (FA) cross-linking. DNA was isolated from formaldehyde treated H2B-TAP cells, resolved by electrophoresis and visualized by ethidium bromide staining. (B) Affinity purification of chromatin was monitored by western blotting for H2B-TAP. P, lysate pellet; S₁, pre-purification supernatant; S₂, post-purification supernatant; AP, affinity purified. (C) Sheared chromatin was affinity purified then histones were resolved by SDS-PAGE, visualized by Coomassie staining and excised for mass spectrometric analysis/identification.

Figure 3. In vivo chemical cross-linking prevents histone exchange of cross-linked chromatin

Shown are representative mass spectra collected with an Orbitrap mass analyzer for doubly charged peptides from histone H2B-TAP (A) and histone H4 (B). The percent isotopically light peptide (%L) is indicated. (C) Percentage isotopically light arginine containing histone peptides are reported as a function of formaldehyde (FA) cross-linking. The standard error from triplicate experiments is shown. Levels approaching ~100% indicate minimal exchange, while those at ~50% reflect rapid exchange. The average of ten rapidly exchanging/non-specific proteins is shown (non-specific).