Plasmodium telomeres: a pathogens’ perspective

Plasmodium falciparum life cycle

Knob structure

Cooke et al., 2OOO

Telomere

Antigengenes

Antigengenes

A

TELOMERE ASSOCIATED REGION

rep 20

Exon I Exon II

varB

sub-telomeric organization

1kb

ATScytoplasmic

region

DBL- CIDR DBL- DBL- DBL-C

Approx. 50 var genes per haploid genome

Tremendous diversity in var gene repertoires in field isolates

HB3HB3

Dd2Dd23D73D7

powerful mechanism for generating diversity

Does the nuclear architecture of P. falciparum have a role in

var gene recombination?

Fluorescent In Situ Hybridisation (FISH)

Nucleus (DAPI)

Fluorescent labled DNA probes (>1-2 kb)

Slide

Telomere Clustering in P. falciparum :

ASEXUAL STAGES SEXUAL STAGES

TAS telomere

ectopic recombination

virulence factor genes

Epigenetic var gene regulation

14 linear chromosomes

Internal region

Molecular mechanism of var gene expression and switching

Does the sub-nuclear architecturehave a role in var gene regulation ?

Rings Trophozoites Schizonts

var

ge

ne e

xpre

ssio

n

var a

var e

var d

var c

var b

Chromatin architecture in P. falciparum

10 Kb

130 Kb 170 Kb

Chromosome 2 painting

150 Kb

Nu

clea

r p

erip

her

y

The chromosome ends are compact and concentrated at the nuclear perifery forming clusters.

Nuclear compartmentalization of chromosome 2

A

B

telomere

Rap 1

Taz 1

Rif 1

Sir 2-4

Ku 70

Model of a chromosome end

Mlp complex

nuclear

membrane

nuclearpore

Immunolocalisation

TELOMERE FISH

Sir 2-13 Sir 4Sir 2-14

Sir 3 Nop IKu 70

Sir complex in Saccharomyces cerevisiae

inactiveactive

-ac

-ac

-ac

heterochromatin

Histone hyperacetylation alters chromatin

Typical chromosome end of Plasmodium falciparum

C:\Documents and Settings\Utilisateur\Bureau\Chromosome_Coil.mov

A B

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.

Localization of Sir2 by Immuno-Electron Microscopy

Sir 2 in P. falciparum

FISH +IF

IF Pf-Sir2-13

FISHTelomere

Merged Merged+DAPI

Confirm presence of Sir 2 in P. falciparum

Confirm presence of Sir 2 in P. falciparum

CHIP

HRP1

GBP130

Rep 20

Telomere

Input Anti-Sir2 Anti-rabbitAnti-acetylhistone H4

TelomereY Y Y Y

anti-PfSir2

FixationSonication

A chromosomal gradient of PfSir2 and acetylated histones

Telomere var gene

TAREs

1 2 3 4 5 6 (rep20)

10 kb

A

B

C

antigen genes

Heterochromatin model in P. falciparum

Expressed blood stage gene

Non expressed blood stage gene

Non expressed blood stage gene

CHIP using Anti-acetyl histone H4

22 copies at telomeric location

Single copy at telomeric location ON

OFF OFF

OFF

OFF

OFF

ON

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)

anti-acetyl histone H3

anti-acetyl histone H4

anti-Pfsir2-13

MergedDAPI IF

Nuclear compartimentalization

3D model of P. falciparum nuclei

P. falciparum nucleus is sub compartmentalised.

Collaborators

Stuart Ralph

Institut PasteurFrance

Artur Scherf

Rosaura Hernandes Centro de Investigación y de Estudios Avanzados del IPN

México

Spencer Shorte