epigenetic silencing of virulence gene families in malaria ... · epigenetic silencing of virulence...

Epigenetic silencing of virulence gene familiesin malaria parasites

José Juan López-Rubio

Institut Pasteur, ParisBiologie des Interactions

Hôte-Parasite (BIHP)

Plasmodium falciparum life cycle

Malaria claims more than a million lives each year, followed by a long list of afflictions

An important difficulty in drugs searching and vaccines development is our limited understanding of the parasite

“Back to the basics” approach asking for more laboratory research (Callaway, 2007)

Encoded by the multigene var gene familyThe best characterized family of variant antigens

PfEMP1

Plasmodium expresses only a single gene at a time

14 chromosomes of P. falciparum

var gene

Genome organization of var gene family

var gene regulation

Two genetic elements of var genes are associated with transcriptional control (‘ups’ and ‘intron’)

These genetic elements are not sufficientto control mono-allelic expression

Mono-allelic expression is independent of antigen production

Superimposed control mechanism

EPIGENETICS

var gene

Epigenetic

var gene regulation

non coding RNA

DNA methylation

nuclear spatial organization

chromatin structure (histone modifications and chromatinbinding proteins)

epigenetic topics

non coding RNA

intron structure highly conserved in all var genes

intron RNA?

promoter activity?

other non coding RNA

Calderwood MS,... ,Deitsch KW J Biol Chem 2003

Voss TS, ..., Cowman AF Nature 2006

Frank M, ...Deitsch K J Biol Chem 2006

Dzikowski R, ... Deitsch KW EMBO Rep 2007

Epigenetic

DNA methylation

non coding RNA

DNA methylation (NO in Plasmodium falciparum)



epigenetic topics

Epigenetic

Chromatin structure and nuclear spatial organization

non coding RNA

DNA methylation (NO in Plasmodium falciparum)



epigenetic topics

Different histone modifications

ChIP chromatin immunoprecipitation

Semi-quantitative PCR, a screening array or quantitative PCR

Microarrays

Histone marks regulate var gene monoallelic-expression

H3K9 acetylation and methylation:antagonisticcompetition between them influences transcription activity

H3K4me2: memory histone mark for active var gene

H3K9me3: memory histone mark for inactive var genes

Lopez-Rubio et al., Mol Micro 2007

Genome wide analysis of histone marks with ChIP on Chip

Nimblegen

Customer DNA microarrays including non-coding regions for P. falciparum(AT rich genome)

ChIP on Chip

is the H3K9me3 enrichment for silenced var loci universal for all classes of silenced var?

are these histone modifications universal marks of transcriptional activation or silencing in the P. falciparum genome?

var2csa

chr 1

chr 2

chr 3

chr 4

chr 5

chr 6

chr 7

ChIP on CHIP H3K9me3

chr 8

chr 9

chr 10

chr 11

chr 12

chr 13

chr 14

200 kb

Only subtelomeric and some central chromosome regions are enriched in H3K9me3

Lopez-Rubio et al., Cell Host & Microbe 2009

ChIP on Chip with other histone marks

The enrichment of H3K9me3 presents a particular pattern in P. falciparum


chr 1

chr 2

chr 3

chr 4

chr 5

chr 6

chr 7

chr 8

chr 9

chr 10

chr 11

chr 12

chr 13

chr 14

200 kb

H3K9me3 is restricted to var genes and genes associated with var loci


chr 1

chr 2

chr 3

chr 4

chr 5

chr 6

chr 7

chr 8

chr 9

chr 10

chr 11

chr 12

chr 13

chr 14

200 kb



chr 1

chr 2

chr 3

chr 4

chr 5

chr 6

chr 7

chr 8

chr 9

chr 10

chr 11

chr 12

chr 13

chr 14

200 kb


H3K9me3 is restricted to large variant surface antigen families:

var, rif, stevor and Pfmc-2TM multigene families


H3K9me3 is restricted to differentially transcribed virulence gene families

Silent members: H3K9me3 enrichedActive members: no H3K9me3 enrichment

H3K9me3 dataHomology analysisTranscriptional data

Identification of putative gene families differentially

transcribed that could be implicated in virulence

common silent epigenetic mechanism for virulence gene families


H3K9 methylation is performed by KMT1 (histone methyltransferase 1)

PfKMT1 Dapi

There is a unique locus for KMT1 in P. falciparum (Pf08_0012)

PfKMT1 locates largely at the periphery of P. falciparum nuclei


Nuclear localization of H3K9me3 enriched genes

Liliana Mancio-Silva

FISH probe

10 genes

var genes localize at the nuclear periphery forming foci

var ups A var ups Bvar ups C

varDapi


FISH experiments show that H3K9me3 enriched loci (var and others) localize at nuclear periphery

Nuclear localization of H3K9me3 enriched genes (Liliana)

Nuclear zones

1

3

2



Model for nuclear organization of H3K9me3 enriched domains

Loss-of-function studies are necessary

PfSir2 and H3K9 methylation

histone deacetylase

implicated in telomere position effect

its inactivation leads to upregulation of several var genes and genes associated with var loci

is present in the promoter region of silent var2csa

Sir2 histonemethylase

acts as a H3K9 deacetylase in vitro

PfSir2

Inactivation of Sir2 affects H3K9me3 enrichments

Inactivation of Sir2 affects H3K9me3 enrichments

H3K9me3 enrichment is modulated by Sir2

lack of Sir2

Sir2 histonemethylase

Mainly var type A

other histone deacetylases should be implicated

Sir2 B ?

Conclusions

H3K9me3enriched

H3K9me3 is restricted to large variant surface antigen families suggesting a common silencing mechanism

There is a link between H3K9me3 enrichmentand perinuclear localization. PfKmt1 and H3K9me3 may be determinants for the spatial organization of falciparum nuclei

Inactivation of PfSir2 affects H3K9me3 at specific regions in a discontinuous manner

Nucleus

var loci and associated genes

Perinuclear epigenetic repressive factory:PfSir2PfKmt1other proteins (histone deacetylases)

Hypothesis

switch

Enhancer-mediated allelic exclusion

Enhancer

Silent var

Active var

chromosome conformation capture

Aknowledgments

BIHP membersArtur Scherf


Loïc Riviere

Perspectives

Improved gene-knockdown systems for malaria parasites

Analysis of the role of other histone deacetylases (Sir2B, clr6, clr3)

ChIP-on-chip for other histone modifications, PfKmt1, Sir2, HP1)

identification of insulators

Clonally variant gene families are enriched in H3K9me3

Subtelomeric Gene families nonenriched in H3K9me3



P. falciparum blood stage cell-cycle: 48Hours

20

0

40

60

80

100

0 6 12 18 24 30 36 42 48

Var

gen

e tr

ansc

rip

tion

Ring Troph Schizont

Time (hr)var « poised »var « on »0 6 12 18 24 30 36 42 48

Ring Troph Schizont

var « on » var « poised »

Selection of monomorphic parasites

population expresses var2csa

ChIP chromatin immunoprecipitation

positions analyzed at var2csa gene

Nuclear localization of H3K9me3 enriched genes


H3K9me3 enriched loci localize at the nuclear periphery

chr 2

chr 9

chr 10

chr 11

chr 12

chr 13

chr 14

= H3K9me3 enriched region without any var, rif, stevor,...

locus Dapi

epigenetic silencing of virulence gene families in malaria ... · epigenetic silencing of virulence...

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