Histone purification kit

Histones were extracted using the Histone Purification Kit (Active Motif). Purified histones were measured using spectrophotometer (Nanodrop).

Purified histone extracts were isolated from nuclear fractions using the Histone Purification Mini Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol. Active Motif’s Histone Purification Kit preserves phosphoryl, acetyl, and methyl post-translational modifications on histones. Briefly, an equal volume of ice-cold extraction buffer was added to the nuclear suspension. After homogenization, samples were left overnight in the extraction buffer on a rotating platform at 4°C. Next day, tubes were centrifuged at maximum speed for 5 min in a microfuge at 4°C and the supernatants, which contained the crude histone extracts, were neutralized with one-fourth volume of 5x neutralization buffer (pH 8.0). Neutralized extracts were loaded to previously equilibrated histone isolation spin columns. After three washes with histone wash buffer, we eluted histones in 100 μl of histone elution buffer and precipitated overnight by adding 4% perchloric acid. On the following day, samples were centrifuged at maximum speed for 1 hour; histone pellets were washed first with 4% perchloric acid, later with acetone containing 0.2% HCl, and finally with pure acetone, after which they were air dried. Histones were suspended in sterile distilled water and the yield of total core histone proteins was quantified by measuring the absorbance at 230 nm.

For histone purification, HFF cells were grown to confluence and infected with Pru∆ku80 parasites. Intracellular tachyzoites were treated with histone deacetylase HDAC3 inhibitor, 90 nM FR235222 for 18 hr. As an appropriate control, we treated tachyzoites with 0.1% DMSO. Histones were extracted and purified using histone purification kit (Active motif) according to manufacturer’s protocol. For western blotting, histone proteins were run on a NuPAGE 4–12% Bis-Tris polyacrylamide gels in MES-SDS running buffer (Invitrogen) and transferred to a polyvinylidene fluoride PVDF membrane (Immobilon-P; Millipore) using NuPAGE transfer buffer (Invitrogen). The blots were probed using primary antibodies: pan acetyl H4, H4K31ac and H4K31me1, followed by phosphatase-conjugated goat secondary antibodies (Promega). The expected band of histones were detected using NBT-BCIP (Amresco). Nucleosomes from T. gondii-infected cells were purified and proteins separated by SDS-PAGE. The band corresponding to H4 was excised and its protein content digested using trypsin. The resulting peptides were submitted to mass spectrometry-based proteomic analysis (U3000 RSLCnano coupled to Q-Exactive HF, Thermo Scientific). Peptides and proteins were identified using Mascot software (Matrix Science).