Characterisation of Entamoeba histolytica anti-silencing function 1 as a histone chaperone

Anti-silencing function 1 (ASF1) is a highly conserved histone chaperone essential for the dynamics of nucleosome structure, facilitating assembly and histone exchange during important cellular processes such as replication, repair, and transcription. Although ASF1 has been well characterised in model organisms, its properties in protozoan parasites remain poorly explored. This study investigates the structural and functional adaptation of ASF1 from the early-diverging eukaryotic pathogen Entamoeba histolytica that causes amoebiasis in humans. Phylogenetic and sequence analyses place EhASF1 in a distinct clade and indicate that it contains a remarkably acidic C-terminal extension, like yeast ASF1, but different in length and composition compared to metazoan homologs. Recombinant EhASF1 was expressed successfully and purified, and a predominant β-sheet secondary structural composition was confirmed by circular dichroism spectroscopy. A variety of biophysical approaches, such as size-exclusion chromatography, sedimentation velocity analytical centrifugation (SV-AUC), and small-angle X-ray scattering, revealed EhASF1 to be a monomer in solution with an elongated, flexible structure. Interaction studies indicated that EhASF1 has selective binding specificity to histone H3/H4 dimer, wherein isothermal titration calorimetry established a 1:1 stoichiometric interaction with a micromolar binding affinity. Further, an in vitro nucleosome assembly assay established that EhASF1 can promote the deposition of histones onto DNA and thereby confirming its function as a histone chaperone. This study establishes EhASF1 as the first characterised histone chaperone from E. histolytica, reporting conserved chromatin assembly mechanisms and illuminating the evolution of histone chaperones from ancient eukaryotic pathogens.