Final Report Summary - PLASMOESCAPE (Monoallelic Gene Expression in Malaria Parasites: A Key Mechanisms in the Pathogen's Survival Strategy)
Summary
The PlasmoEscape project was designed to get a detailed molecular insight into the mechanisms that orchestrate the process of immune evasion and virulence in the human malaria parasite Plasmodium falciparum. We aimed to investigate the cellular and molecular events that control the expression of a single member of the major virulence gene family (called var gene family) that encodes 60 antigenically distinct surface adhesion molecules. Activation of a single member is associated with the relocation to a perinuclear site that remains uncharacterized. The following topics have been addressed to study the underlying mechanisms of monoallelic expression in malaria parasites: i) Develop new technologies to isolate chromatin from var expression sites; ii) Identify chromatin factors that are linked to var expression sites; iii) Validate candidate chromatin factors.
A team composed of 3-4 postdocs, one PhD student and two technicians was formed to tackle the aims of this project. The project has resulted in a number of seminal discoveries that opened distinct avenues in this field. Many have been already published in high profile journals such as Nature, Nature Biotechnology, Nature Medicine, Nature Communications and Nature Reviews in Microbiology, others are in preparation. I will highlight here the main findings.
• We demonstrated that the var intron recruits actin and other factors (putative transcriptional regulator protein Ap2) and showed that the intron has the capacity to tether a var gene to the nuclear periphery, the default location of var gene members for silencing.
• The intron produces long non-coding RNA that has been linked to the activation of var genes. We have developed a new technology based on cas9/CRISPR that allows us to perform unprecedented genome editing in P. falciparum. We have now deleted the intron of a specific var gene (marker-free) and demonstrated that the ncRNA is not necessary for the activation process. But the intron deletion leads to the default activation of this var gene in a monoallelic manner, demonstrating the key role of this genetic element in default silencing.
• We identified a GC-rich ncRNA that associates with the var expression site associating for the first time a specific chromatin component to the expression site. Importantly, overexpression of this ncRNA lead to the derepression of var gene members, demonstrating a key role in monoallelic expression.
• We have identified several histone lysine methyltransferase (HKMT) genes that are essential during the blood stage development and var gene silencing. Using drug screens, we identified for the first time small molecule inhibitors that target histone methylation and kill efficiently this parasite. We show that certain types of histone methylation are inhibited by these drugs. In order to identity the specific target HKMT, we have developed an expression protocol for recombinant active HKMT enzymes and are now screening small libraries with our inhibitors. This is a very promising new translational aspect that has emerged from the grant. We are now working in collaboration with Glaxo Smith Kline (Tres Cantos) to identify new inhibitors of HKMT and other epigenetic factors that have been identified in this project.
• While studying chromatin factors linked to silent var genes, we discovered a novel type of exonuclease called RNase II. We demonstrated a new type of gene silencing mechanism never described before. It involves the degradation of nascent RNA from var genes. The RNase II apparently is removed from the active var gene, allowing the production of full length mRNA.
Taken together, this ERC grant has allowed my team to gain in depth insight into the epigenetic events that orchestrate antigenic variation. It has lead to important new emerging research topics such as the development and application of a new genome editing tool (cas9/CRISPR), new types of posttranscriptional gene regulation and discovery of a ncRNA gene family linked to monoallelic gene expression of var genes.
The PlasmoEscape project was designed to get a detailed molecular insight into the mechanisms that orchestrate the process of immune evasion and virulence in the human malaria parasite Plasmodium falciparum. We aimed to investigate the cellular and molecular events that control the expression of a single member of the major virulence gene family (called var gene family) that encodes 60 antigenically distinct surface adhesion molecules. Activation of a single member is associated with the relocation to a perinuclear site that remains uncharacterized. The following topics have been addressed to study the underlying mechanisms of monoallelic expression in malaria parasites: i) Develop new technologies to isolate chromatin from var expression sites; ii) Identify chromatin factors that are linked to var expression sites; iii) Validate candidate chromatin factors.
A team composed of 3-4 postdocs, one PhD student and two technicians was formed to tackle the aims of this project. The project has resulted in a number of seminal discoveries that opened distinct avenues in this field. Many have been already published in high profile journals such as Nature, Nature Biotechnology, Nature Medicine, Nature Communications and Nature Reviews in Microbiology, others are in preparation. I will highlight here the main findings.
• We demonstrated that the var intron recruits actin and other factors (putative transcriptional regulator protein Ap2) and showed that the intron has the capacity to tether a var gene to the nuclear periphery, the default location of var gene members for silencing.
• The intron produces long non-coding RNA that has been linked to the activation of var genes. We have developed a new technology based on cas9/CRISPR that allows us to perform unprecedented genome editing in P. falciparum. We have now deleted the intron of a specific var gene (marker-free) and demonstrated that the ncRNA is not necessary for the activation process. But the intron deletion leads to the default activation of this var gene in a monoallelic manner, demonstrating the key role of this genetic element in default silencing.
• We identified a GC-rich ncRNA that associates with the var expression site associating for the first time a specific chromatin component to the expression site. Importantly, overexpression of this ncRNA lead to the derepression of var gene members, demonstrating a key role in monoallelic expression.
• We have identified several histone lysine methyltransferase (HKMT) genes that are essential during the blood stage development and var gene silencing. Using drug screens, we identified for the first time small molecule inhibitors that target histone methylation and kill efficiently this parasite. We show that certain types of histone methylation are inhibited by these drugs. In order to identity the specific target HKMT, we have developed an expression protocol for recombinant active HKMT enzymes and are now screening small libraries with our inhibitors. This is a very promising new translational aspect that has emerged from the grant. We are now working in collaboration with Glaxo Smith Kline (Tres Cantos) to identify new inhibitors of HKMT and other epigenetic factors that have been identified in this project.
• While studying chromatin factors linked to silent var genes, we discovered a novel type of exonuclease called RNase II. We demonstrated a new type of gene silencing mechanism never described before. It involves the degradation of nascent RNA from var genes. The RNase II apparently is removed from the active var gene, allowing the production of full length mRNA.
Taken together, this ERC grant has allowed my team to gain in depth insight into the epigenetic events that orchestrate antigenic variation. It has lead to important new emerging research topics such as the development and application of a new genome editing tool (cas9/CRISPR), new types of posttranscriptional gene regulation and discovery of a ncRNA gene family linked to monoallelic gene expression of var genes.