A Rapid and Efficient Method for the Extraction of Histone Proteins.

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.

Histone H4K20 monomethylation enables recombinant nucleosome methylation by PRMT1 in vitro.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to specific arginine residues of histones and nonhistone proteins. There are nine members in the PRMT family (PRMT1 to PRMT9), and PRMT1 is a dominant member catalyzing majority of arginine methylation in the cell. However, none of the PRMTs is active with recombinant nucleosome as substrate in vitro. Here, we report the discovery of the first in class novel crosstalk between histone H4 lysine 20 (H4K20) monomethylation on nucleosome by SETD8 and histone H4 arginine 3 (H4R3) methylation by PRMT1 in vitro. Full kinetic characterization and mass spectrometry analysis indicated that PRMT1 is only active with recombinant nucleosomes monomethylated at H4K20 by SETD8. These data suggests that the level of activity of PRMT1 could potentially be regulated selectively by SETD8 in various pathways, providing a new approach for discovery of selective regulators of PRMT1 activity.