Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

- We developed an antibody against recombinant histone H2Ba of Toxoplasma gondii.
- Co-IP using α-rH2Ba showed that H2Ba shares nucleosome with H2A.X, not with H2A.Z.
- We obtained a stable line of tachyzoites expressing Myc-H2AX.
- Co-IP assays using α-c-Myc confirmed that H2Ba shares nucleosome with H2A.X.
- We determined the genomic location of H2Ba in promoters of silent genes and TAS.

Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12 kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers.

Nucleosomes composed of H2A.X/H2Ba histones are present in promoters of inactive genes, as well as telomeric regions; H2A.Z/H2B.Z nucleosomes are present at promoters of active genes.122