Toxoplasma H2A Variants Reveal Novel Insights into Nucleosome Composition and Functions for this Histone Family

Abstract

Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here we report the characterization of three H2A histones, a canonical H2A1 and variants H2AX and H2AZ. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)φ (φ denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We also show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We also found that h2ax expression, but not h2a1 and h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are actively expressed and h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this life cycle stage. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.

MYST Family Lysine Acetyltransferase Facilitates Ataxia Telangiectasia Mutated (ATM) Kinase-mediated DNA Damage Response in Toxoplasma gondii

“…H2AX is rapidly phosphorylated in response to double-stranded breaks (termed ␥H2AX), which are induced by MMS. We have established that Toxoplasma contains H2AX that becomes phosphorylated in response to double- stranded DNA breaks (12). In TgMYST-B-overexpressing parasites, however, we found high levels of basal ␥H2AX and found that the increase in ␥H2AX is dependent on the KAT activity of TgMYST-B (Fig.…”

“…Antibody to Toxoplasma tubulin (used at 1:1,000) was provided by David Sibley (Washington University, St. Louis, MO) to serve as a protein loading control. Mouse monoclonal antibody to phosphorylated H2AX was from Millipore (05-636) (12). Mouse monoclonal antibody to ATM kinase (2C1 (1A1); used at 1:2,000) was purchased from Abcam (ab78).…”

Chromatin-Mediated Epigenetic Regulation in the Malaria Parasite Plasmodium falciparum

“…The replacement of canonical histones with histone variants can influence the nucleosome stability and chromatin patterns (95,123). Interestingly, in both Trypanosoma brucei and Toxoplasma gondii, H2A.Z interacts with H2Bv and is associated with active genes (31,99). In T. brucei, variant histones are used to mark boundaries of polycistronic transcription units, and nucleosomes containing two variant histones (H2A.Z and H2Bv) are highly enriched at the putative polymerase II transcription start sites (TSS) (137).…”

The Antibiotic Monensin Causes Cell Cycle Disruption ofToxoplasma gondiiMediated through the DNA Repair Enzyme TgMSH-1

“…H2AX has been shown to mediate DNA damage repair (12). In addition, it has been shown that H2AX transcription is upregulated in T. gondii in response to oxidative DNA damage (10). Previously, Singh et al (32) demonstrated that monensin caused significant oxidative stress and DNA fragmentation in rat testicular cells.…”