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In-depth functional analysis of heterochromatin protein 1 in Plasmodium falciparum

Bui, Thi Ngoc Hai. In-depth functional analysis of heterochromatin protein 1 in Plasmodium falciparum. 2019, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_13712

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Abstract

The most severe form of malaria, caused by Plasmodium falciparum, is an enormous burden for the endemic countries without any commercial vaccine available currently. P. falciparum possesses a complex life cycle, changing from different hosts and environmental conditions, and hence requires intensive and essential mechanisms for regulation of gene expression to survive and take advantage from external environments. In recent years, epigenetic control of gene expression emerged as an important mechanism to regulate expression of genes involved in virulence and sexual differentiation. Among epigenetic histone marks, histone 3 lysine 9 trimethylation (H3K9me3), which is considered the hallmark of heterochromatin formation, is recognized by an evolutionary conserved silencing factor called heterochromatin protein 1 (HP1). HP1 is a small protein consisting of three domains: two conserved domains at the N and C-terminus, namely the chromo domain (CD) and chromoshadow domain (CSD), respectively, and a variable hinge or linker domain in between them. The CD recognizes and binds to H3K9me3 to establish heterochromatin while the CSD interacts with a large number of effector proteins. HP1 possesses a number of posttranslational modifications, among which phosphorylation is highly important in regulating its functions. P. falciparum encodes only one HP1 isoform in contrast to most other eukaryotes that contain two to five HP1 paralogs. Although HP1 has been well studied in model eukaryotes, the detailed functions of each PfHP1 domain as well as PfHP1 phosphorylation in P. falciparum are poorly understood. My PhD project aimed to investigate PfHP1 function in detail using CRISPR/Cas9-based gene editing combined with the DiCre/loxP system for conditional mutagenesis. The two specific objectives were (1) to uncover the roles of the individual PfHP1 domains in PfHP1 function and (2) to understand if and how phosphorylation of PfHP1 regulates its function. Both objectives focused on the three known PfHP1-dependent cellular processes: (1) the maintenance of heritable silencing and mutually exclusive expression of var genes, (2) mitotic progression of asexual blood stage parasites, and (3) sexual commitment.
In the first project, I aimed to analyse the functional contribution and conservation of the CD, Hinge and CSD domains of PfHP1 using the approach mentioned above to generate PfHP1 truncation and hybrid mutant lines. I found that nuclear localization ability of PfHP1 is underlined in the N-terminal 29 amino acids (1-29) and the C-terminal 76 amino acids (191-266). The former polypeptide is supposed to have weak ability of nuclear delivery while the later one is necessary for fully targeting PfHP1 to nucleus. I also showed that an intact PfHP1 containing all three structural domains is required for heterochromatin localization and the normal asexual growth pathway of blood stage malaria parasites. Third, I successfully generated a PfHP1 knock out cell line with more pronounced phenotype that allowed massive production of more than 82% of sexual commitment rate once induced and suggested a PfHP1-independent regulatory pathway of var gene silencing during gametocytogenesis. Finally, I showed that the HP1 Hinge and CSD domains are functionally conserved between human – and rodent malaria parasites.
In the second project, I aimed to address (1) the kinases phosphorylating PfHP1 and their target residues and (2) the role of selected phosphorylated residues in regulating PfHP1 function using similar approach mentioned above to generate conditional PfHP1 phosphomutant cell lines. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated native PfHP1 showed that PfHP1 is phosphorylated mostly in the CD and hinge domains. In vitro kinase assays revealed that PfHP1 is a substrate of P. falciparum CK2 (PfCK2). LC-MS/MS analysis showed that PfCK2 targets three clustered serine residues within the PfHP1 hinge region in vitro. By generating conditional PfHP1 phosphomutant cell lines using CRISPR/Cas9-mediated genome editing and the DiCre/LoxP system, I found that PfCK2-dependent phosphorylation of PfHP1, and phosphorylation of the PfHP1 hinge domain in general, is dispensable for proper PfHP1 localization, gene silencing, parasite growth and sexual conversion.
In summary, for the first time, in-depth function and functional conservation of PfHP1domains as well as phosphorylation of PfHP1 have been studied in detail in the three main PfHP1-dependent processes (var gene silencing, mitotic proliferation, sexual commitment). The experimental pipeline established in this study provides an elegant approach to interrogate function of other essential proteins. In addition, the study also provides a tool for robust production of gametocytes used in further studies on gametocyte biology or high-throughput anti-malaria gametocyte drug screen. This study is highly relevant to study pathogenesis and transmission as well as underlying epigenetic mechanisms in some other unicellular pathogenic eukaryotes since they also share common features such as changing living environments and mutually exclusive expression of surface antigens.
Advisors:Voss, Till and Pradel, Gabriele
Faculties and Departments:09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology (MPI) > Gene Regulation (Voss)
UniBasel Contributors:Voss, Till
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13712
Thesis status:Complete
Number of Pages:1 Online-Ressource (x, 157 Seiten)
Language:English
Identification Number:
Last Modified:20 Oct 2020 04:30
Deposited On:12 Oct 2020 14:38

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