Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

Abstract

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.

A View on the Role of Epigenetics in the Biology of Malaria Parasites

“…Studies on specific gene families, including var and others, also support this view [32]–[37]. P. falciparum clonally variant genes are located in bistable chromatin domains [36], which can be found in two alternative conformations, permissive (euchromatin) or repressive (heterochromatin).…”

Sexual development in Plasmodium parasites: knowing when it's time to commit

“…These include the repetitive interspersed family (rif), subtelomeric variable open reading frame (stevor) and P. falciparum Maurer's cleft 2 transmembrane (pfmc-2tm) multigene families, which also encode surface antigens. Mutually exclusive expression has not been demonstrated for any of these families, but they do show some similarities with var gene regulation 148,149 . Epigenetic regulation of genes involved in red blood cell invasion and in solute transport has also been demonstrated 150 , showing that epigenetic mechanisms are probably important in regulating a wide range of processes in the parasite.…”

Chromatin modifications associated with activation and repression of stevor multigene family of plasmodium falciparum during the trophozoite stage

“…This work was further continued using ChIP experiments coupled with quantitative (q) PCR. This work showed that active rifin had H3K9ac and silent rifin genes were enriched with H3K9me3 with some silent rifin having no modifications (Cabral et al 2012). However, in this study, due to technical difficulties with designing specific primers in the 5' UTR regions of the 6 rifins studied (out of 160), they only focused their mapping to the coding regions.…”

“…All stevor oligos in each immunoprecipitation were then calibrated to the input DNA signal using the formula 2 ΔCt = (Ct oligo Ab−Ct oligo input) , where Ab is the respective antibody used for ChIP (Cabral et al 2012;Petter et al 2013). The plotted enrichment was calculated as the ratio of immunoprecipitated Ab oligo signal/immuno-precipitated H3 oligo signal (Lopez-Rubio et al 2007;Cabral et al 2012;Petter et al 2013). H3 was used as to normalize between the biological replicates and also as an internal control for the experiments (Srivastava et al 2014).…”