Fluctuating Heterochromatin Disguises Malaria from its Host

Malaria cells are often symbolized as criminals or thieves. You see criminals disguising themselves and using different methods to avoid getting detected, similarly, the malaria parasites in the red blood cells show particular proteins on the surface of the blood cell to hide from the immune system, and this is why the human body isn’t able to fight off the malaria parasites so easily. The malaria parasites, also known as the Plasmodium falciparum, if detected, start to switch the identity of the protein that is on the surface, and this process is called the antigenic variation. (Li, et al., 2019) moreover, in order to assist the parasite that hides from the immune system, this protein on the surface also goes on to improve the binding process of this infected blood cell with the cardiovascular system. This binding leads to serious malaria symptoms and causes the infected red blood cell to not get sent to the spleen where it can be eradicated. 

The surface proteins are encoded by almost 60 unique genes that are known as var genes, and these genes are grouped into many different sorts and are denoted by upsA-E. This is based on the structure and shape of the sequence that promotes it. The Malaria parasites only show one var gene at once. Several types of research have shown that epigenetic regulation, which encapsulates variation in the chromatin assembly and histone changes comes with var gene expression control. However, the whole mechanism is not very well learned as of yet. (Jiang, et al., 2013) It is interesting to know that two DNA helicases seen in the Plasmodium Falciparum called PfWRN and PfRecQ1 are proven to be involved in maintaining the stability of the genome and thereby regulates gene expression. A question is raised that are these DNA helicases involved in regulating var gene expression? A lot of scientists have checked how the loss of PfWRN and PfRecQ1 impacts the var gene expression in Plasmodium Falciparum. 

Scientists first examined the PfWRN and PfRecQ1 in the Plasmodium Falciparum using the CRISPR/Cas9 method. Once the global gene expression was evaluated, the changes in the knockout cell lines were compared to the wild type parasites, and it was seen that in PfRECQ1 knockout cells, the whole family of var genes did not show any movement, and it was silenced. In order to further check and validate this finding, the scientists checked the expression of all the var genes separately by RT-qPCR. It was found that the loss of PfRecQ1 finished the expression of all the var genes, whereas the loss of PfWRN did not have any impact on the var gene silencing. Interestingly, when the expression of PfRECq1 was added back in the knockout cell line, it was observed that in the two independent experiments, the parasites showed different var genes as compared to the var gene that was seen in the parent cell line. Moreover, the newly found var gene came from a different type. These were the upsB and upsE as compared to the initially active var gene which was upsC1. The results from the study carried out established that PfRecQ1 expression is mandatory to show and maintain the expression of the active var gene. Also, its regulation cannot be stopped to a specific type. 

After this, the scientists tried to see the mechanism through which the PfRcQ1 impacts the var gene expression. This is because the var gene expression is related to the spatial agreement in the perinuclear space. This space is between the inner and outer parts of the nucleus membrane. The scientists also checked the spatial distribution of var genes alterations after the loss of PfRecQ1. The DNA-Fluorescence was used and In-situ Hybridization (DNA-FISH) was also used. The scientists examined the localization of the saved repetitive sequence Rec20 since it flanks the var DNA sequences. In the wild-type cells, the scientists proved that the Rec20 was seen in around 4-7 clusters in the perinuclear area. However, in the PfRecQ1 and PfWRN knockout cells, there was a notable clumping of Rec20 regions in lesser independent rec20 clusters. The increased clumping tells us that both the helicases are significant in the spatial distribution of the var genes in the perinuclear space. 

Furthermore, the chromatin mark that is repressive is famous for repressive var gene expression. Therefore, the scientists tried finding out if PfRecQ1 or PfWRN are involved in maintaining chromatin structure. Scientists discovered that H3K9me3 is full of telomeric particles and var genes in wild-type cells. Scientists saw no difference in the global distribution of H3K9me3 in both the helicase knockout cell line. (Lopez, et al., 2007). That being said,  the alterations in H3K9me3 in the transcription start sites (TSS) are necessary for regulating gene expression, therefore, scientists used ChIp-qPCR in four unique var genes. It was found that the var gene was specially silenced in the PfrecQ1 knockout cells and it increased the levels of H3K9me3, whereas others did not. The results prove that the loss of PfRecQ1 resulted in silencing the var gene that was previously active by increasing the H3Kme3. This supports the importance of PfRecQ1 at the Transcription State Sites. 

Lastly, the scientists further questioned if the loss of PfRecQ1 or PfWRN has an impact on the relative nuclear localization of the various types of var genes. By the use of DNA-FISH, scientists examined the localization of upsA1, upsB1, upsC1, and upsC2. The var gene that was active in the wild-type parasites was found. Also, there was no change in the co-localization of upsC1 with upsA1 or upsB1 on the loss of PfRecQ1. However, upon the loss of PfRecQ1, upsC1 localized the upsC2, which suggests that when upsC1 is silenced, it goes in the transcriptionally silent area with the same type of var genes. 

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References

Jiang, Mu, Zhang, Ni, Srinivasan, Rayavara, Riberio. (2013). PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum. Nature, 499(7457), 223-227.

Li, Yin, Sun, Cheng, Gilbert, Miao, Jiang. (2019). DNA helicase RecQ1 regulates mutually exclusive expression of virulence genes in Plasmodium falciparum via heterochromatin alteration. Proc Natl Acad Sci U S A.

Lopez, Gontijio, Nunes, Issar, Rivas, H., & Scherf. (2007). flanking region of var genes nucleates histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites. Mol Microbiol, 66(6), 1296-306.