plasmodium telomeres: a pathogens’ perspective. plasmodium falciparum life cycle
TRANSCRIPT
Plasmodium falciparum life cycle
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
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
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
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.
telomere
Rap 1
Taz 1
Rif 1
Sir 2-4
Ku 70
Model of a chromosome end
Mlp complex
nuclear
membrane
nuclearpore
Sir complex in Saccharomyces cerevisiae
inactiveactive
-ac
-ac
-ac
heterochromatin
Histone hyperacetylation alters chromatin
Typical chromosome end of Plasmodium falciparum
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
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.