Epigenetic memory at malaria virulence genes

Abstract

During its red blood cell stage, the malaria parasite Plasmodium falciparum can switch its variant surface proteins (P. falciparum erythrocyte membrane protein 1) to evade the host immune response. The var gene family encodes P. falciparum erythrocyte membrane protein 1, different versions of which have unique binding specificities to various human endothelial surface molecules. Individual parasites each contain approximately 60 var genes at various locations within their chromosomes; however, parasite isolates contain different complements of var genes, thus, the gene family is enormous with a virtually unlimited number of members. A single var gene is expressed by each parasite in a mutually exclusive manner. We report that control of var gene transcription and antigenic variation is associated with a chromatin memory that includes methylation of histone H3 at lysine K9 as an epigenetic mark. We also discuss how gene transcription memory may affect the mechanism of pathogenesis and immune evasion.

Fig. 1.

Gene transcription memory at var loci. The pie charts present relative levels of transcription of each var gene from the transgenic P. falciparum clone C7G12 as discussed in ref. . Under blasticidin pressure, only varBSD is highly expressed. After 10 weeks without the drug, subpopulations of parasites expressing other var genes have expanded, but varBSD remains the dominant gene detected. The analyzed data for var genes and expression values from ref. are presented in SI Table 1.

Fig. 2.

Histone H3K9 trimethylation at the var loci. (A) Enrichment of H3K9me3 at var genes on chromosome IV. The amount of H3K9me3 was measured by ChIP and presented as the fold difference from the value at the active varBSD. The OFF var genes shown here are located both within subtelomeric regions and in an internal cluster; these particular genes were chosen for study because their amplification by real-time PCR gave specific amplicons. (B) Location of the H3K9me3 enrichment at the OFF varBSD gene. The data are presented as percent input, which is the percent ratio of precipitated DNA compared with the total amount of DNA added into the reaction. The coding region of varBSD is shown as a rectangle. The location of each region is depicted in the diagram. The BSD/Plasmodium berghei hsp86 (Pb) 3′UTR cassette was introduced between the endogenous var promoter and the var intron, replacing exon 1 (13). The ability to study sequences further upstream of the gene was compromised because of sequence homology with numerous other var genes across the upstream region. (C) Nature of H3K9 methylation at the OFF PFD0625w var gene. The data are presented as the fold difference from the signal obtained from nonhistone antibodies (PFI1780w). Both H3K9me1 and H3K9me2 antibodies have low enrichment even when compared with nonhistone antibodies.

Fig. 3.

Temporal pattern of the transcription memory mark H3K9me3 at a var gene. (A) Diagram presenting the transcription kinetics for var RNA (dotted line) and PfEMP1 (dashed line) over the course of the P. falciparum cell cycle (modified from ref. 25). The transcription peak for var genes is ≈10–17 h, after which the signal drops significantly. (B) H3K9me3 signal at different states of transcriptional activity. The H3K9me3 signal is shown as the percent input from P. falciparum lines with ON and OFF varBSD transcription. Synchronized parasites were analyzed at 10–17 h (expressing a single var gene) and at 40–47 h (no var expression).