Periodic Reporting for period 2 - MALSWITCH (Uncovering the Mechanisms Behind Adaptive Gene Expression Switching in Malaria Parasites)
Reporting period: 2023-01-01 to 2024-06-30
During its complex life cycle the malaria causing parasite Plasmodium falciparum must swiftly adapt to changes in its environment, evade the immune system of its host and tightly control the switches between cell cycle stages to ensure an ongoing infection and successful transmission. To counter these challenges, the parasite has evolved a strategy of clonally variant gene expression, which controls the essential biological functions like antigenic variation, or sexual commitment, that form the basis of these adaptive regulatory mechanisms. Heritable epigenetic silencing of the underlying specialized gene families ensures the limited expression of only a subset of these genes at any time. Switching the expression of individual clonally variant genes enables the parasite to rapidly adapt to the changes in its environment, evade the immune system and switch its cell cycle to the development of mosquito transmissible gametocyte stages. Expression switching of these clonally variant genes therefore represents a key strategy for parasite survival and underlies the evolutionary success of this deadly pathogen. However, the molecular players and their interplay in organizing the changes in chromatin structure, which control these gene expression switching events have so far been largely elusive.
We could previously identify the parasite specific protein gametocyte development 1 (GDV1) as a factor that is responsible for inducing gene expression switching of the master regulator of sexual commitment, the transcription factor Ap2-G. This provides us with a unique experimental tool, which will allow us to investigate the molecular mechanism underlying this process. For this we will 1) investigate changes in local chromatin structure as well as spatial chromatin organization of the ap2-g locus during switching, 2) use CRISPR/Cas derived proximity-based labelling methodology to reveal and investigate the molecular machinery controlling the switching events and 3) elucidate the role of long non-coding RNAs in translating environmental signals into the onset of GDV1 expression and ap2-g switching. Altogether, the outlined experiments will deliver a systematic identification and characterization of the molecular mechanisms controlling epigenetic gene expression switching during sexual commitment in P. falciparum. This will contribute to a better understanding of the molecular mechanisms driving adaptation of this deadly parasite and in the long run might enable to the design of intervention strategies that P. falciparum is unable to adapt to.
We could previously identify the parasite specific protein gametocyte development 1 (GDV1) as a factor that is responsible for inducing gene expression switching of the master regulator of sexual commitment, the transcription factor Ap2-G. This provides us with a unique experimental tool, which will allow us to investigate the molecular mechanism underlying this process. For this we will 1) investigate changes in local chromatin structure as well as spatial chromatin organization of the ap2-g locus during switching, 2) use CRISPR/Cas derived proximity-based labelling methodology to reveal and investigate the molecular machinery controlling the switching events and 3) elucidate the role of long non-coding RNAs in translating environmental signals into the onset of GDV1 expression and ap2-g switching. Altogether, the outlined experiments will deliver a systematic identification and characterization of the molecular mechanisms controlling epigenetic gene expression switching during sexual commitment in P. falciparum. This will contribute to a better understanding of the molecular mechanisms driving adaptation of this deadly parasite and in the long run might enable to the design of intervention strategies that P. falciparum is unable to adapt to.
During the reporting period we were able to identify a new long non-coding RNA that is involved in regulating the environmentally induced expression of GDV1 and thus the onset of sexual commitment. We could show that disruption of this lncRNA gene leads to an insensitivity of the parasites to external commitment signals and a loss of GDV1 expression. We successfully generated various transgenic parasite lines for the adaptation of CRISPR/Cas based RNA-knockdown approaches, as well as for proximity- labelling based chromatin proteomics approaches. These parasite lines are currently used to fully implement these methodologies in P. falciparum and investigate the various aspects of gene expressing switching of the ap2-g locus.
We can identify a cascade of regulatory long non-coding RNAs as central regulators, which translate environmental signals into the onset of sexual commitment in malaria parasites. With this we can provide first mechanistic insights on a lncRNA based regulatory mechanism controlling cell cycle switching in malaria parasites. With the successful setup and adaptation of novel RNA and chromatin proteomics centered methodologies we will now be able in the next steps to identify the molecular machinery at the center of epigenetic control of gene expression switching of the ap2-g locus and the onset of sexual commitment through environmental signals.