CavalliCavalli LabLab

Nuclear compartmentalization

and chromatin regulation

An example of a functional cell biology analysis

Data from: Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G. (2003). Inheritance of Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev 17, 2406-2420.Grimaud, C., Bantignies, F., Pal-Bhadra, M., Ghana, P., Bhadra, U., and Cavalli, G. (2006). RNAi Components Are Required for Nuclear Clustering of Polycomb Group Response Elements. Cell 124, 957-971

PgG proteins form nuclear compartments

Z axis image projectionsPC / Heterochromatin

PC protein has a speckled distribution in the cell nucleus

Different target genes may cluster at PcG foci. These associations may play a role in regulation of PcG targets

During embryonic development, chromosome homologs pair in diptera. Pairing brings homolog sequences in close physical proximity. This

pairing correlates with the strength of PcG and trxG mediated regulation

Weak PcG mediated repression

Strong PcG mediated repression

PRE Heterozygous PRE Homozygous

Chromosome pairing and PRE function

w w

w

Chromosome XPP mini-whiteFab-7 scalloped (sd)

An example of Pairing Sensitive silencing: the Fab-X transgenic line

---> strong mini-white silencing---> weak silencing of the mini-white reporter gene

Transgenic Fab-7heterozygous

Transgenic Fab-7homozygous

Chr. XPP

In the line Fab-X, the Fab-7 transgenic PRE at the homozygous state represses an endogenous gene located close to the insertion site

Fab-7

18 kb

scalloped (sd)mini-white

Heterozygous transgenic Fab-7

sd sd

sdsd

No silencing of the endogenous sd gene

Homozygous transgenic Fab-7

Strong silencing of the endogenous sd gene

Fab-X; Fab71

The sd repression mediated by the Fab-7 transgene requires the presence of the endogenous copy of Fab-7

XX

III III

Fab-X

Strong sd silencingdependent both on transgenic

and endogenous Fab-7

sd derepression when the endogenous Fab-7 element is deleted

sd sd

BX-C BX-C

Fab-7 transgene

Endogenous Fab-7

Endogenous Fab-7 deletion, named Fab-71

3D-AcquisitionFixation

Permeabilisation

DenaturationHybridization

DetectionDAPI counterstating

5 m

Interphase nuclei

Sdgene

BX-Clocus

Dapi

Do PRE lead to long-distance pairing?

Three dimensional 2-color FISH in whole mount embryos

0

5

10

15

20

25

+ + -Endogenous Fab-7 at the BX-C (Chr.III)

3m

-

-

-

-

-

-Fab-X Fab-X;

Fab-71WT

WT

Fab-X

Fab-X; Fab-71

+ +-Transgenic Fab-7 at sd (Chr.X)

Fab-7

Fab-7

Fab-7

Fab-7

sd

BX-C

sd

BX-C

sd

BX-C

Dapi Sd BX-C Merge

Percentageof pairing

PRE associate in the nucleus through DNA sequence homology

Chromatin states can be inherited through meiosis by the following

generations

When pairing is disrupted, silencing is weakened.If pairing is re-established, can silencing be immediately re-acquired,

or is the derepressed state inherited?

Chromosome pairing and meiotic inheritance

PairingStrong PcG mediated

repressionWeak PcG mediated

repressionNo pairing

1. Erase trans homology by genetic

crosses

2. Restore trans homology

Genetic approach:

Meiotic inheritance of sd derepression

F0 repressed (85-90% sd)

F1 partially derepressed(40-45% sd)

F2 and F3

derepressed(20-25% sd)

Fab-X

Fab-X; Fab71/+

Fab-X

Back cross withCross with

Fab-X; Fab71 Fab-X

Once established, the derepressed state can be inherited into a fraction of the progeny

Is it because pairing can not be immediately re-established?

YES! Meiotic inheritance of sd derepression correlates with loss of Fab-7 pairing

Dapi Sd BX-C Merge

Fab-X

Fab-X after

meiosis

Fab-X Fab-Xafter

Meiosis

0

5

10

15

20

25

% o

f pa

irin

g

-

-

-

-

-

-

Multiple copies of 3.6 Kb of homologous DNA can find each other among 180Mb of genomic chromatin

Chromatin pairing induces silencing and it is heritable

Regulation of endogenous target genes of the Pc-G and of the trx-G may involve a precise compartmentalization in the cell nucleus.

Nuclear compartmentalization is a heritable feature!

Nuclear architecture is heritable

PcG bodies

Targetgenes

Heterochromatin

Polycomb, nuclear organization and RNAi

Frédéric Bantignies

Charlotte Grimaud

Collaborators:Utpal Bhadra - CCMB

Manika Pal-Bhadra - IICTHyderabad, India

The RNAi machinery can induce post-transcriptional gene silencing by processing dsRNAs into short interfering RNAs of 21-23 nt that pair with the target mRNA and induce its degradation. The RNAi machinery is also responsible for the metabolism of endogenous noncoding transcripts, leading to the production of miRNAs. miRNAs can induce processing of mRNAs or block their transcription

PTGS

RNAi is also involved in transcriptional silencing in several species, like plants, Drosophila and S.pombe. In Drosophila, PcG components and RNAi members are required for a gene silencing process called cosuppression. We thus tested whether RNAi mutations affect the function of PREs

TGS

RNAi and regulation of gene expression

Fab-7 dependent silencing depends on RNAi components

Fab-X Fab-X;dcr-2 L811fsX Fab-X;AGO1 45/72

Fab-X;piwi 1 Fab-X;piwi 2 Fab-X;hls E1

Z axis image projectionsPC / Heterochromatin

PcG proteins form nuclear compartments called PcG bodies

Do RNAi component colocalize with PcG bodies?

Specific RNAi components colocalize with PcG bodies

DAPIRNAi

proteinsPH merge

AGO1

PIWI

Dcr-2

Hls

Dcr-1

Colocalization

42%

62%

52%

19%

Very low

Is PcG protein recruitment dependent on RNAi components in Drosophila?

RNAi components are required for the recruitment of heterochromatin components to centromeres in S. pombe

The piwi gene is an exception !

PcG proteins can be recruited to Fab-7 in most RNAi mutants

Fab-X

Fab-X;AGO1 KO8121/45

Fab-X;piwi 1

Fab-X ; dcr-2 L811fsX

DAPI FISH PC merge

w1118

Fab-X;piwi 2

Fab-X

Fab-X

DAPI sd BX-C mergePC

Fab-X;dcr-2 L811fsX

PcG protein targeting to Fab-7 in RNAi mutants

Loss of targeting

only in piwi mutants

Nuclear RNAi components are required for PcG mediated silencing of transgenes

…but not for targeting of PcG proteins at PREs

Are long-distance interactions of PREs affected in RNAi mutants?

DAPI sd BX-C merge

w1118

Fab-X

Fab-X;piwi 2

Endogenous

Fab-7Tra

nsgenic

Fab-7

+

+ +

-

+ +

+ +Fab-X;dcr-2 L811fsX

Defective maintenance of Fab-7 long-distance interactions in RNAi mutant larvae

CavalliCavalli LabLab

Summary

The Fab-7 element can establish long-distance interactions that require a novel nuclear function of RNAi components

RNAi components affect PcG-mediated gene silencing at the Fab-7 element

They are not absolutely required for PcG recruitment at this PRE or at endogenous genes

Most RNAi components are partly located in the nucleus, where they partially colocalize with PcG proteins

We predict a general role of long-range associations and of the RNAi machinery in cosuppression/paramution phenomena