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Plasmodium telomeres: a pathogens’ perspective

Plasmodium telomeres: a pathogens’ perspective. Plasmodium falciparum life cycle. Knob structure. Cooke et al., 2OOO. B. TELOMERE ASSOCIATED REGION. var. rep 20. Exon I. Exon II. C. ATS. DBL- a. DBL- e. DBL- b. DBL- g. CIDR. cytoplasmic region. 1kb.

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Plasmodium telomeres: a pathogens’ perspective

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  1. Plasmodium telomeres: a pathogens’ perspective

  2. Plasmodium falciparum life cycle

  3. Knob structure Cooke et al., 2OOO

  4. B TELOMERE ASSOCIATED REGION var rep 20 Exon I Exon II C ATS DBL-a DBL-e DBL-b DBL-g CIDR cytoplasmic region 1kb sub-telomeric organization A Antigen genes Antigen genes Telomere

  5. HB3 3D7 Dd2 powerful mechanism for generating diversity • Approx. 50 var genes per haploid genome • Tremendous diversity in var gene repertoires • in field isolates

  6. Does the nuclear architecture of P. falciparum have a role in var gene recombination?

  7. Fluorescent labled DNA probes (>1-2 kb) Nucleus (DAPI) Fluorescent In Situ Hybridisation (FISH) Slide

  8. virulence factor genes telomere TAS ectopic recombination SEXUAL STAGES Telomere Clustering in P. falciparum : ASEXUAL STAGES

  9. Epigenetic var gene regulation

  10. Internal region 14 linear chromosomes Molecular mechanism of var gene expression and switching

  11. Trophozoites Schizonts Does the sub-nuclear architecture have a role in var gene regulation ? var a var b var c var gene expression var d var e Rings

  12. Chromatin architecture in P. falciparum

  13. Chromosome 2 painting 130 Kb 170 Kb Nuclear periphery 10 Kb 150 Kb The chromosome ends are compact and concentrated at the nuclear perifery forming clusters.

  14. A B Nuclear compartmentalization of chromosome 2

  15. Sir 2-4 Mlp complex Model of a chromosome end Rap 1 nuclear pore Taz 1 Rif 1 nuclear membrane Ku 70 telomere

  16. TELOMERE FISH Immunolocalisation Sir 2-13 Sir 2-14 Sir 4 Sir 3 Ku 70 Nop I

  17. heterochromatin active inactive -ac -ac Histone hyperacetylation alters chromatin -ac Typical chromosome end of Plasmodium falciparum Sir complex in Saccharomyces cerevisiae

  18. B A Localization of Sir2 by Immuno-Electron Microscopy The Sir2 protein localizes to the electron-dense heterochromatic region at the nuclear periphery of P. falciparum parasites. Panels (A) and (B) show developing merozoites in a late-schizont-stage parasite. Scale bars in (A) and (B) are 250µm.

  19. Sir 2 in P. falciparum

  20. Confirm presence of Sir 2 in P. falciparum FISH +IF IF Pf-Sir2-13 FISH Telomere Merged +DAPI Merged

  21. HRP1 GBP130 Rep 20 Telomere Input Anti-acetyl histone H4 Anti-Sir2 Anti-rabbit Confirm presence of Sir 2 in P. falciparum CHIP anti-PfSir2 Y Y Y Y Sonication Fixation Telomere A chromosomal gradient of PfSir2 and acetylated histones

  22. A B C Heterochromatin model in P. falciparum antigen genes TAREs var gene Telomere 1 3 6 (rep20) 2 4 5 10 kb

  23. CHIP using Anti-acetyl histone H4 Expressed blood stage gene ON Single copy at telomeric location OFF ON 22 copies at telomeric location OFF OFF Non expressed blood stage gene OFF Non expressed blood stage gene OFF

  24. Conclusions: We have identified the first telomere binding proteins in P. falciparum, PfSir2 PfSir 2 binds to the telomere, spreads as far as the rep20 Element and is therefore a neighbour of the var gene promoter. • Specific histone modifications can be targeted to specific gene • promotor regions (activated chromatin domains)

  25. Nuclear compartimentalization DAPI IF Merged anti-Pfsir2-13 anti-acetyl histone H3 anti-acetyl histone H4

  26. 3D model of P. falciparum nuclei P. falciparum nucleus is sub compartmentalised.

  27. Collaborators Institut Pasteur France Artur Scherf Stuart Ralph Spencer Shorte Centro de Investigación y de Estudios Avanzados del IPN México Rosaura Hernandes

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