UC Riverside

The human malaria parasite, one of the deadliest infectious agents in the world, still contributes significantly to the global burden of disease. In 2017, an estimated 214 million cases of infection and over 400,000 malaria-related deaths were reported, a majority of which are caused by the most lethal human malaria parasite, Plasmodium falciparum. Given the absence of an FDA-approved vaccine and parasite resistance to all current antimalarial drugs there is a desperate need for new therapeutic approaches.

Plasmodium falciparum has a complex life cycle that requires coordinated gene expression regulation to allow host cell invasion, transmission and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. Therefore, in this dissertation work, we further explore genome architecture, epigenome, proteome and transcriptome including long-non-coding RNAs (lncRNAs) to better understand the relationship between chromatin structure, genome organization and transcriptional regulation in malaria parasites.

In the first chapter, we explore genome organization in human Plasmodium parasite stages including the transmission stages from human to mosquito (gametocytes) and from mosquito to human (sporozoites). Our work demonstrates that genome organization is an important regulator for several parasite-specific gene families involved in pathogenesis and immune evasion, erythrocyte and liver cell invasion, sexual differentiation, and master regulators of gene expression. In the second chapter, we investigated genome organization in five malaria parasites and two related apicomplexan parasites with the goal to identify common features of genome organization and possible connections between genome architecture and pathogenicity. We show that in all malaria parasites, genome organization is dominated by the clustering of Plasmodium-specific gene families in 3D space. Our data highlight the importance of spatial genome organization in gene regulation and control of virulence in malaria parasites.

In the subsequent chapters, we aim to identify molecular components, specifically proteins and lncRNAs, that maintain and regulate chromatin structure in the malaria parasite. To investigate parasite proteins and protein complexes maintaining and regulating nuclear architecture, we undertook comparative genomics analysis using twelve distinct eukaryotic genomes. We identified conserved and apicomplexan parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We validated two of our candidate proteins including a novel plant-related protein that is functionally analogous to animal nuclear lamina proteins and might have a role in heterochromatin organization. Finally, we also explore the role of lncRNAs in P. falciparum. In eukaryotes, lncRNAs have been shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum, we first explored the intergenic distribution of lncRNA using deep sequencing in nuclear and cytoplasmic subcellular locations. We then validate the subcellular localization and stage-specific expression of several putative lncRNAs at single cell resolution using fluorescence in situ hybridization (FISH) technology. Additionally, we explore the genome-wide occupancy of several candidate nuclear lncRNAs using Chromatin Isolation by RNA Purification followed by deep sequencing (ChIRP-seq) technology. Data analysis revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. Discovery of these proteins and lncRNAs are the starting point for further exploration of mechanisms regulating chromatin structure and genome architecture in these deadly parasites.

Collectively, our data highlight the importance of spatial genome organization as a mechanism of transcriptional regulation in malaria parasites, and our work directly addresses one of the central outstanding questions in Plasmodium biology, namely, how a parasite with approximately 6,000 genes manages to control gene expression in a coordinated fashion using a limited number of transcription factors.