School of BioSciences - Theses

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    Exploring the roles of bromodomain protein 2 (PfBDP2) and acetylated histone variant PfH2A.Z and PfH2B.Z in Plasmodium falciparum chromatin biology
    Azizan, Mohd Suffian ( 2022)
    The eukaryotic Plasmodium falciparum parasite employs multiple levels of gene regulation to alter its morphology throughout its complex life cycle to survive different environments it encounters in mosquitoes and humans. Thus, epigenetic mechanisms that control gene regulation are integral to the parasite’s survival. Histone lysine acetylation is the post-translational modification of histones, generally associated with eukaryotic transcriptional activation. This epigenetic modification is recognised by bromodomain, and bromodomain-containing proteins (BDPs) can recruit transcription factors to promote gene expression. In the P. falciparum genome, eight bromodomain-containing proteins were identified but only two have been functionally characterised. This thesis detailed the works done on the uncharacterised P. falciparum bromodomain protein 2 (PfBDP2). I explored cellular and epigenomic localisations of PfBDP2 in the asexual schizont-stage P. falciparum parasites via biochemical assays and native chromatin immunoprecipitation, which revealed that PfBDP2 is a nuclear protein expressed throughout the asexual cycle and is enriched within the 5' intergenic region of invasion genes in schizonts. PfBDP2 is also enriched within heterochromatin, particularly across the promoters of silent multigene families and across var gene introns. This points to PfBDP2 being a chromatin protein with dual epigenetic roles, associated with both gene expression and silencing. This thesis also reports a growth delay upon the conditional disruption of PfBDP2 expression in vitro, indicating its importance for normal growth during the asexual intraerythrocytic cycle. The inability to recover stable PfBDP2 knockouts following knockout induction suggests that PfBDP2 may be essential to blood-stage growth and could be druggable by novel antimalarials. Finally, this thesis also detailed the epigenomic localisation of two unique Apicomplexan histone variants, PfH2A.Z and PfH2B.Z, and their acetylated cognates, PfH2A.Zac and PfH2B.Zac, previously shown to be the binding targets of PfBDP2. PfSir2A is a histone deacetylase shown to anti-correlate with PfH2A.Z deposition at heterochromatin in the P. falciparum genome, fulfilling a critical role in the maintenance of chromatin structure. Using crosslinked chromatin immunoprecipitation, the genome-wide localisation of the histone variants was mapped in both wild-type and PfSir2A-KO parasites. There was an enrichment of both PfH2A.Zac and PfH2B.Zac across all euchromatic intergenic regions of P. falciparum genome in the wild-type parasites. This pattern was similar in PfSir2AKO parasites but differed in the aberrant, increased enrichment of the acetylated histone variants within heterochromatin, signalling a breakdown of heterochromatin-euchromatin boundaries. This indicates an important structural function of the two histone variants in maintaining chromatin boundaries in P. falciparum. Additionally, PfH2A.Zac and PfH2B.Zac appeared to be enriched at specific loci around var genes, most importantly var introns, suggesting a role in var gene regulation and lending further evidence to the acetylated histone variants being in vivo binding targets of PfBDP2.