5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites

Abstract

In the human malaria parasite Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5' flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5' upstream region of transcribed var and during the poised state (non-transcribed phase of var genes during the 48 h asexual life cycle), 'bookmarking' this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5' flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5' flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.

Fig. 1

A. P. falciparum transcriptional var gene states during the 48 h blood stage cycle. Transcription of a single var gene starts about 4 h after the erythrocyte invasion by free parasite forms called merozoites and lasts for approximately 12–14 h (ring stage) (Kyes et al., 2000). For the remaining time of the 48 h cycle (trophozoite and schizont stage), which is mainly devoted to multiple rounds of parasite DNA replication and differentiation to merozoites, var gene transcription ends until free merozoites establish a new round of asexual blood stage cycle. Activation of a particular var member was achieved by selecting infected erythrocytes expressing a var gene able to bind to CSA, called var2csa. The two different states of transcription of an active var loci during the blood stage cycle is called here ‘ON’ for being transcribed and ‘POISED’ for being transiently silent but ready to get reactivated in the next cycle. Stable silencing (‘OFF’ state) of the var2csa gene is achieved by selecting parasites that express another var gene able to bind to CD36. B. Identification of histone H3 methyl marks in chromatin of asexual blood stage parasites. Equal amounts (∼8 μg per lane) of partially purified P. falciparum histones from mid to late trophozoites (30–34 h post invasion) or total HeLa cell extracts were separated by 15% SDS-PAGE. Various methyl modifications present on histone H3 were detected by immunoblotting with respective site-specific antibodies in equal dilutions.

Fig. 3

Trimethylated H3K9 levels at var2csa. A. Distribution of H3K9 trimethylation along the var2csa gene in CD36 (black bars) and CSA ring parasites (white bars). B. Distribution of H3K9 trimethylation along the var2csa gene in CD36 (black bars) and CSA mature parasites (white bars). C. Levels of trimethylated H3K9 (fold enrichment over the same positions in chromatin from CSA parasites) along the var2csa gene in CD36 parasites on ring stages (white bars) and mature stages (black bars). D. Levels of trimethylated H3K9 (fold enrichment over the chromatin from CSA parasites) at GBP130 and PfGam genes in CD36 parasites on ring stages (white bars) and mature stages (black bars). E. Levels of H3K9 trimethylation at GBP130 and PfGam genes in CD36 (black bars) and CSA ring parasites (white bars). qChIP values are given as DNA recovery (%input) normalized for the %input of total H3 for (A), (B) and (E). CD36/CSA ratio of the previous qChIP values is shown in (C) and (D). Regions are named as shown in Figure S1 and a simplified scheme is shown for reference. Results are the average of two or three independent experiments. Error bars denote standard deviation.

Fig. 2

Determination of the transcription initiation site of the FCR3 var2csa gene. A. Schematic representation of the 5′var2csa region. The transcription initiation site of the var2csa gene is shown by a flag at position about −1475. The primers used for the RACE assays are indicated. B. The RACE products were analysed by gel electrophoresis on a 1.5% agarose gel. C. The sequence of the 200 bp DNA fragment obtained with the RACE 5′-assays with AUAP primer (Gibco) and nested var2.3as primer is shown.

Fig. 5

Dimethylated H3K4 levels at var2csa. A. Distribution of H3K4 dimethylation along the var2csa gene in CSA (white bars) and CD36 ring parasites (black bars). B. Distribution of H3K4 dimethylation along the var2csa gene in CSA (white bars) and CD36 mature parasites (black bars). C. Levels of dimethylated H3K4 (fold enrichment over the same positions in chromatin from CD36 parasites) along the var2csa gene in CSA parasites on ring stages (white bars) and mature stages (black bars). D. Levels of dimethylated H3K4 (fold enrichment over the chromatin from CD36 parasites) at GBP130 and PfGam genes in CSA parasites on ring stages (white bars) and mature stages (black bars). E. Levels of H3K4 dimethylation at GBP130 and PfGam genes in CSA (white bars) and in CD36 ring parasites (black bars). The data are plotted as for Fig. 3.

Fig. 4

Trimethylated H3K4 levels at var2csa. A. Distribution of H3K4 trimethylation along the var2csa gene in CSA (white bars) and CD36 ring parasites (black bars). B. Distribution of H3K4 trimethylation along the var2csa gene in CSA (white bars) and CD36 mature parasites (black bars). C. Levels of trimethylated H3K4 (fold enrichment over the same positions in chromatin from CD36 parasites) along the var2csa gene in CSA parasites on ring stages (white bars) and mature stages (black bars). D. Levels of trimethylated H3K4 (fold enrichment over the chromatin from CD36 parasites) at GBP130 and PfGam genes in CSA parasites on ring stages (white bars) and mature stages (black bars). E. Levels of H3K4 trimethylation at GBP130 and PfGam genes in CSA (white bars) and in CD36 ring parasites (black bars). The data are plotted as for Fig. 3.

Fig. 6

Acetylated and methylated H3K9 levels at var2csa. A. Distribution of H3K9ac (white bars) and H3K9me3 (black bars) along the active var2csa gene in FCR3 CSA ring stage parasites. B. Distribution of H3K9ac (white bars) and H3K9me3 (black bars) along the silent var2csa gene in FCR3 CD36 ring stage parasites. The data are plotted as for Fig. 3. Note that the H3K9ac antibody gives stronger signals and that the y-axes are different for the two antibodies.

Fig. 7

Schematic presentation of histone H3 marks linked to silent or active var genes. Histone modifications of the 5′ flanking region and exon 1 of an active (var2csa^ON or var2csa^POISED) and silent var gene loci (var2csa^OFF) are shown. H3K9me3 is dynamically removed in the surrounding area of the transcription start site upon gene activation and is restored upon repression. Histone H3 marks at lysine 4 and 9 linked to active var genes peak in chromatin associated with 5′ flanking var region, supporting further the concept for a key role of this DNA element in the control of mono-allelic expression of var genes (Dzikowski et al., 2006; Voss et al., 2006). Exon 2 is transcribed via a promoter-like sequence within the var intron sequence element in silent var genes and has been linked to var gene silencing (Dzikowski et al., 2006; 2007). Chromatin associated with var intron and exon 2 has not been analysed due to the sequence homology of these regions with most members of the var gene family.