Our data suggest that both loci PFI0025c and PFD0070c may also be regulated epigenetically at the studied time points in sporozoites and gametocytes. Our experiments did not show any connection of rif transcription and cytoadherence patterns, since the transcripts found in the re-selected parasite line showed transcripts differing from those identified in our previous study. This view is also supported by a TH-302 recently published microarray-based analysis. We could also not identify any connection between the genomic context, transcribed var genes and rif transcription, at least in the samples from the experiment shown in Figure 2. The dominantly transcribed var gene is distant from transcribed rif genes and no specific pattern of transcript quantity and genomic context was identified. Lopez-Rubio and colleagues showed that var genes that are transcribed after the following reinvasion are marked with H3K4me2 in schizonts when var transcription has ceased.
In our experiments, loci with low or undetectable transcription showed few modification of this type, however, little modification seemed not predictive for absence of transcriptional activity in the same cycle. Even with a very low level of the “poised mark” modification, transcription may occur. We propose that for stronger “poised” modifications in trophozoites, higher transcript quantities are to be expected in schizonts in the same cycle, also coinciding with a higher H3K9 acetylation or low H3K9 trimethylation modification in schizonts. A significant argument against a general rule for the link of active transcription/acetylation at H3K9 and dimethylation at H3K4 is seen at the locus PFD0070c, indicating that other factors or histone modifications may have decisive influence on the transcriptional status of a locus, including perhaps the recently discovered modifications at H4 and H4K8. In any case, more biological replicates would be necessary to clarify the role of the H3K4me2 and possibly the modifications at H4 in the “poised” status of rif genes. Upon its first publication in 2007, the poised mark was only tested for the var2csa locus. During the blood stage replication of P. falciparum, its immune evasion through antigenic variation. Although a number of transcriptional regulators were annotated in the P. falciparum and some of them were already localized to the nucleus or even shown to associate to silenced chromatin such as HP1, PfKMT1, or to var promoter elements, the exact events that orchestrate the dynamics of antigenic variation and allelic exclusion are not yet resolved. Our data demonstrate for the first time that active rif loci are marked not only with the H3K9 acetylation mark, but also the poised mark H3K4me2.
Also, we showed that silenced loci may either associate to H3K9me3 modified loci or unmodified loci, at least in the stages that were looked at. It is possible that unmodified H3K9 residues are associated to an almost permanently silent locus while H3K9me3 loci were recently switched off or may become switched on. Further, the turnover dynamics are probably faster than for var loci. Additional studies targeting the exact chromatin modifications at other histone residues or at H4 comparing active or silent variant gene loci are clearly necessary in order to elucidate the mechanics of recruiting transcription factors to the respective sites.