Study of the biological importance of Toxoplasma gondii H2B.Z histone and function of its acetylated lysines.

AGUSTINA GANUZA; KAMI KIM; DANIELA MUÑOZ; FANNY GUZMÁN; SERGIO O. ANGEL; LAURA VANAGAS; SHEILA NARDELLI; WILLIAM J. SULLIVAN JR.

Quinidio

Congreso; Toxo XV International Congress; 2019

T.gondii and other apicomplexanparasites possess an unusual H2B variant called H2B.Z which is found innucleosomes containing H2A.Z, another histone variant.  The existence of a unique H2B variant in T.gondii offers an interesting matter of study to find new biologicalpathways and putative drug targets. H2A.Z and H2B.Z are positioned attranscription start sites (TSS) together with H3K4Me3 and both histone variantsare highly acetylated at their N-terminal tails. Mutagenesis studies wereperformed to determine the biological importance of H2B.Z, particularly therole of the N-terminal lysines. We over-expressed myc-tagged H2B.Z in Prustrain in which the acetylatable lysine residues were maintained (c-Myc-WT) or mutatedto either the neutral amino acid alanine (c-Myc-A) or the positively chargedarginine (c-Myc-R).  The resultingtransgenic parasites did not show significant differences in replication rate,but we observe changes in the rate of bradyzoite differentiation. Parasitesectopically expressing c-Myc-R/H2B.Z differentiated more compared withparental, while c-Myc-A/H2B.Z had a lower differentiation rate (1.88 and 0.74, respectively, while c-Myc-wt/H2B.Z ratewas 1.23 relative to parental taken as 1). Sincethe H2B.Z gene is essential, we generated new mutants by disruption of the endogenousH2B.Z gene using CRISPR/Cas9 in the lines over-expressing the mutant histones (c-Myc-A/KOand c-Myc-R/KO). Phenotypic analysis showed similar results to those obtainedwith the over-expressing lines: there is a higher differentiation rate in c-Myc-R/KOparasites, and lower in c-Myc-A/KO.These findings suggest that a correct acetylation of the N-terminal region ofH2B.Z is relevant for the control of parasite development.