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A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria

Abstract

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1)1,2. PfEMP1, encoded by the 60-member var gene family3,4,5,6, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning7,8. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

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Figure 1: WR sensitivity of upsC promoter transfectants.
Figure 2: Epigenetic regulation of upsC.
Figure 3: Nuclear localization of silenced and activated upsC loci.
Figure 4: Effect of upsC activation on mono-allelic var transcription.

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Acknowledgements

We are grateful to A. Gemmill and J. Baum for statistical advice. This work was supported by the National Health and Medical Research Council and the Wellcome Trust. A.F.C. and B.S.C. are International Fellows of the Howard Hughes Medical Institute. T.S.V. is supported by fellowships from the Swiss National Science Foundation and the Roche Research Foundation. A.J.M. is supported by an Australian Postgraduate Award. J.G.B. was supported by the NHMRC and the Miller Fellowship of WEHI. We also thank G. Kelly and A. Raiko for sample collection and processing in Papua New Guinea.

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Correspondence to Alan F. Cowman.

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Supplementary information

Supplementary Discussion

This details the interaction between upsC and the intron and the interference of the activated plasmid-encoded upsC promoter with mutually exclusive var gene transcription.

Supplementary Methods

Here we discuss in detail the northern experiments and densitometry presented in Figs. 2 and 4.

Supplementary Figure Legends

This file contains the legends to Supplementary Figures 1–6.

Supplementary Figure 1

This figure integrates the main findings of this work into a proposed model of var gene transcription.

Supplementary Figure 2

This figure shows plasmid maps and Southern analysis of genomic DNA isolated from transgenic parasites to determine plasmid structure and copy number in transgenic parasites before and after WR-treatment.

Supplementary Figure 3

This figure shows that hdhfr and bsd transcription levels in the control transfectants pHBcam, pHBcamR and pHBcamRI before and after treatment with WR were unchanged and unaffected by the presence of rep20 repeats and the var gene intron.

Supplementary Figure 4

This figure shows Southern blot analysis of digested genomic DNA to confirm the integrated and episomal forms of pHBupsC in 3D7/upsC parasites and to map the integration site of pHBupsC on chromosome 12.

Supplementary Figure 5

This figure shows the endogenous var knock-down effect caused by upsC activation in 3D7/upsCR and 3D7/upsCRI parasites.

Supplementary Figure 6

This figure compares the binding of IgG in each of 20 sera from malaria-exposed adults to the surface of WR-unselected and WR-selected 3D7/upsC iRBCs.

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Voss, T., Healer, J., Marty, A. et al. A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria. Nature 439, 1004–1008 (2006). https://doi.org/10.1038/nature04407

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