A Rapid and Efficient Method for the Extraction of Histone Proteins

Abstract

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.

Keywords: chromatin; fungal pathogens; histone-modifying enzymes; histones; post-translational modification; protein extraction; yeast.

Figure 1

Outline of the histone extraction and isolation methods. (A) The standard yeast and mammalian cell methods. (B) Single step FASP extraction method for both yeast and mammalian cells.

Figure 2

FASP method enriches for histones and other small hydrophilic proteins. (A) Protein profile of the yeast and mammalian cell extracts. (B) Physicochemical properties of proteins extracted with the standard and rapid methods (n = 3 experimental and technical replicates). (C) Proteins observed in the mass spectrometry categorized by their number and relative abundances. (D) Enrichment fold change of the proportion of core histones over the total proteins observed by mass spectrometry for each method. Histone enrichment is represented as relative to the standard method.

Figure 3

FASP method preserves histone H3 integrity. (A) Presence of the truncated N-terminal tail of H3 derived from the standard method (Std: H3 Δ1–20). Histone extraction by the FASP method illustrates the absence of N-terminal truncation. Two controls were performed to validate the disappearance of the clipping: (1) Solubility test in acidified ethanol (AE); (2) Control with a specific antibody solely recognizing the N-terminal tail of H3. A model of the targeted epitopes of both antibodies is presented to facilitate the immunoblot readout. (B) Comparison of H3-phosphoS10 levels of the standard and FASP methods by immunoblotting (left). Cells were synchronized in G2/M with nocodazole to enrich the proportion of histones containing the H3–S10 phosphorylation before blotting. To facilitate the synchronization of cells, α-factor was initially added to synchronize cells in G1. H3–S10A mutant strain was used as control for antibody specificity. FACS profiles (right) were generated to validate cell synchronization. (C) Overview of the relative occupancy of acetylation levels at different lysine sites on H3. PRM was performed on the following peptides with all the combinatorial possibilities of acetylation (Kac) versus propionylation (Kpr): K₉STGGK₁₄APR, K₁₈QLASK₂₃AAR, and FQK₅₆STELLIR/STELLIR (n = 3 experimental and technical replicates). (D) Proportion of intact histone under different conditions.

Figure 4

Profiling of H3 K56ac in different fungal pathogens. (A) Predicted Rtt109 in opportunistic pathogens. The residues highlighted in yellow are important for the catalytic activity of Rtt109. (B) Proportion of H3–K56ac in different fungal pathogens (n = 2 technical replicates, 10⁷ cells).