Hidden in Plain Sight: How Altering Heterochromatin Disguises Malaria from its Host
Like a criminal adopting a disguise to evade detection, malaria parasites inside red blood cells express specific proteins on the blood cell surface to hide from the immune system. When the malaria parasites, Plasmodium falciparum, are eventually detected, they simply switch the identity of the surface protein in a process called antigenic variation . In addition to helping the parasite hide from the immune system, this surface protein also leads to increased binding of the infected red blood cell to the wall of the cardiovascular system. This binding both causes more severe malaria symptoms and helps the infected red blood cell avoid being sent to the spleen where it would be destroyed .
These surface proteins are encoded by about 60 different genes called var genes, which are grouped into different types, denoted upsA-E, based on the structure of their promoter sequences . Parasites only express one var gene at a time. Research has demonstrated that epigenetic regulation, including changes in chromatin assembly and histone modifications, is involved in var gene expression control, but the complete mechanism is not well understood . Interestingly, two DNA helicases found in P. falciparum called PfRecQ1 and PfWRN are known to be involved in maintaining genome stability and in regulating gene expression . To ask if these DNA helicases might be involved in regulating var gene expression, Li and colleagues assessed how the loss of PfRecQ1 and PfWRN effect var gene expression in P. falciparum parasites.
The authors first knocked out PfRecQ1 and PfWRN in P. falciparum using CRISPR/Cas9. After assessing the global gene expression changes in the knockout cell lines compared to wild type parasites, they saw that in PfRecQ1 knockout cells, the entire family of var genes was silenced. To confirm this result, the authors examined the expression of each var gene individually by RT-qPCR. They found that loss of PfRecQ1 abolished expression of all var genes, while loss of PfWRN had no effect on var gene silencing. Intriguingly, when the authors added back expression of PfRecQ1 into the knockout cell line, they saw that in two independent experiments, the parasites now expressed a different var gene than the var gene that was expressed in the parent cell line. Furthermore, the newly expressed var gene was from a different type (upsB and upsE) than the initially active var gene (upsC1). These results indicate that PfRecQ1 expression is necessary to maintain expression of the active var gene and that its regulation is not constrained to a specific type.
Next, the authors aimed to determine the mechanism by which PfRecQ1 affects var gene expression. Because var gene expression is related to its spatial arrangement in the perinuclear region (the space between the inner and outer membrane of the nucleus) , the authors asked if the spatial distribution of var genes changes upon loss of PfRecQ1. Using DNA-Fluorescence In-situ Hybridization (DNA-FISH), the authors assessed the localization of the conserved repetitive sequence Rec20 because it flanks var DNA sequences. In wild type cells, the authors found that Rec20 was found in about 4-7 clusters in the perinuclear region. In both PfRecQ1 and PfWRN knockout cells, however, there was significantly more clumping of Rec20 regions resulting in fewer independent Rec20 clusters. This increased clumping suggests that both helicases are important for the spatial distribution of the var genes in the perinuclear region.
Additionally, the repressive chromatin mark, H3K9me3 is known to repress var gene expression , so the authors asked if PfRecQ1 or PfWRN are involved in maintaining chromatin structure. Through ChIP-seq of H3K9me3, the authors found that H3K9me3 is enriched at telomeric regions and var genes in wild type cells. They saw no difference in global H3K9me3 distribution in either helicase knockout cell line. However, because changes in H3K9me3 specifically at transcription state sites (TSS) are more important in regulating gene expression, the authors used ChIP-qPCR at four different var genes. They found that the var gene that was specifically silenced in PfRecQ1 knockout cells had increased levels of H3K9me3, while the others did not. This result indicates that loss of PfRecQ1 results in silencing of the previously active var gene through increased H3K9me3. Supporting the importance of PfRecQ1 at TSS, the authors found that PfRecQ1 localizes to the TSS of all genes, including var genes.
Finally, the authors asked if loss of PfRecQ1 or PfWRN affects the relative nuclear localization of the different types of var genes. Using DNA-FISH, the authors assessed the localization of upsA1, upsB1, upsC2, and finally, upsC1, the var gene that is active in the wild type parasites and loses expression in PfRecQ1 knockout parasites. Upon loss of PfRecQ1, there was no change in the co-localization of upsC1 with upsA1 or upsB1. However, when PfRecQ1 was lost, upsC1 newly co-localized with upsC2, suggesting that when upsC1 is silenced due to loss of PfRecQ1, it moves to a transcriptionally silent region with var genes of its same type.
This work demonstrates that the DNA helicase PfRecQ1 is an important regulator of var gene expression. The authors speculate that as a helicase, PfRecQ1 may be important for keeping the active var gene in an open state and thus accessible to transcription machinery. Because loss of PfRecQ1 abolishes expression of all var genes, PfRecQ1 may be a good target for the development of new therapeutics against malaria parasites, unmasking them to the watchful eye of the immune system.
Original article: Li Z, Yin S, Sun M, Cheng X, Wei J, Gilbert N, Miao J, Cui L, Huang Z, Dai X, Jiang L (2019). DNA helicase RecQ1 regulates mutually exclusive expression of virulence genes in Plasmodium falciparum via heterochromatin alteration. Proc Natl Acad Sci U S A, pii: 201811766. doi: 10.1073/pnas.1811766116.
 Scherf A, Lopez-Rubio JJ, Riviere L (2008). Antigenic variation in Plasmodium falciparum. Annu Rev Microbiol, 62:445-70. doi: 10.1146/annurev.micro.61.080706.093134.
 Jiang L, Mu J, Zhang Q, Ni T, Srinivasan P, Rayavara K, Yang W, Turner L, Lavstsen T, Theander TG, Peng W, Wei G, Jing Q, Wakabayashi Y, Bansal A, Luo Y, Ribeiro JM, Scherf A, Aravind L, Zhu J, Zhao K, Miller LH (2013). PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum. Nature, 499(7457):223-7. doi: 10.1038/nature12361.
 Claessens A, Harris LM, Stanojcic S, Chappell L, Stanton A, Kuk N, Veneziano-Broccia P, Sterkers Y, Rayner JC, Merrick CJ (2018). RecQ helicases in the malaria parasite Plasmodium falciparum affect genome stability, gene expression patterns and DNA replication dynamics. PLoS Genet, 14(7):e1007490. doi: 10.1371/journal.pgen.1007490.
 Lopez-Rubio JJ, Gontijo AM, Nunes MC, Issar N, Hernandez Rivas R, Scherf A (2007). 5′ flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites. Mol Microbiol, 66(6):1296-305.