1. 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,
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,

2. PgG proteins form nuclear compartments
PC protein has a speckled distribution in the cell nucleus
Z axis image projections
PC / Heterochromatin
Different target genes may cluster at PcG foci. These associations may play a role in regulation of PcG targets

3. Chromosome pairing and PRE function
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

4. An example of Pairing Sensitive silencing: the Fab-X transgenic line
mini-white P
scalloped (sd)
Chromosome X
Transgenic Fab-7
heterozygous
Transgenic Fab-7
homozygous w w w
---> weak silencing of the mini-white reporter gene
---> strong mini-white silencing

5. 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 P
mini-white P
scalloped (sd)
Chr. X
18 kb
Heterozygous transgenic Fab-7
Homozygous transgenic Fab-7 sd sd sd sd
No silencing of the endogenous sd gene
Strong silencing of the endogenous sd gene

6. The sd repression mediated by the Fab-7 transgene requires the presence of the endogenous copy of Fab-7
Fab-X
Fab-X; Fab71 sd sd
Fab-7
transgene X X
BX-C
BX-C
Endogenous Fab-7
III
III
Endogenous Fab-7
deletion, named Fab-71
Strong sd silencing
dependent both on transgenic and endogenous Fab-7
sd derepression when the endogenous Fab-7 element is deleted

7. Do PRE lead to long-distance pairing?
Three dimensional 2-color FISH in whole mount embryos
3D-Acquisition
Fixation
Permeabilisation
Interphase nuclei
Denaturation
Hybridization
Detection
DAPI counterstating
Dapi
5 mm
BX-C
locus Sd
gene

8. PRE associate in the nucleus through DNA sequence homology
Dapi Sd
BX-C
Merge WT
Percentage
of pairing sd -
Fab-7 25
BX-C - 20
Fab-X sd
Fab-7 - 15
Fab-7
BX-C - 10
Fab-X; Fab-71 -
Fab-7 5 sd -
BX-C WT
Fab-X
Fab-X;
Fab-71
3mm
Transgenic Fab-7 at sd (Chr.X) - + +
Endogenous Fab-7 at the BX-C (Chr.III) + + -

9. Chromatin states can be inherited through meiosis by the following generations

10. Chromosome pairing and meiotic inheritance
Genetic approach:
1. Erase trans homology by genetic crosses
2. Restore trans homology
Strong PcG mediated repression
Weak PcG mediated repression
Pairing
No pairing
When pairing is disrupted, silencing is weakened.
If pairing is re-established, can silencing be immediately re-acquired, or is the derepressed state inherited?

11. Meiotic inheritance of sd derepression
Fab-X
Fab-X
Cross with
Back cross with
Fab-X; Fab71/+
Fab-X; Fab71
Fab-X
F2 and F3
derepressed
(20-25% sd)
F0 repressed
(85-90% sd)
F1 partially derepressed
(40-45% sd)
Once established, the derepressed state can be inherited into a fraction of the progeny
Is it because pairing can not be immediately re-established?

12. YES! Meiotic inheritance of sd derepression correlates with loss of Fab-7 pairing
Dapi Sd
BX-C
Merge - 25
Fab-X - 20 - 15
% of pairing
Fab-X after meiosis - 10 - 5 -
Fab-X
Fab-X
after
Meiosis

13. Nuclear architecture is heritable
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!
Heterochromatin
Target
genes
PcG bodies

14. Polycomb, nuclear organization and RNAi Manika Pal-Bhadra - IICT
Charlotte Grimaud
Frédéric Bantignies
Collaborators:
Utpal Bhadra - CCMB
Manika Pal-Bhadra - IICT
Hyderabad, India

15. RNAi and regulation of gene expression
The RNAi machinery can induce post-transcriptional gene silencing by processing dsRNAs into short interfering RNAs of 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

16. Fab-7 dependent silencing depends on RNAi components
Fab-X
Fab-X;AGO1 45/72
Fab-X;dcr-2 L811fsX
Fab-X;piwi 1
Fab-X;piwi 2
Fab-X;hls E1

17. PcG proteins form nuclear compartments called PcG bodies
Z axis image projections
PC / Heterochromatin
Do RNAi component colocalize with PcG bodies?

18. Specific RNAi components colocalize with PcG bodies
proteins
DAPI PH
merge
Colocalization
AGO1
42%
Dcr-2
62%
PIWI
52%
Hls
19%
Dcr-1
Very low

19. RNAi components are required for the recruitment of heterochromatin components to centromeres in S. pombe
Is PcG protein recruitment dependent on RNAi components in Drosophila?

20. PcG proteins can be recruited to Fab-7 in most RNAi mutants
DAPI
FISH PC
merge
Fab-X
Fab-X;AGO1 KO8121/45
The piwi gene is an exception !
Fab-X;piwi 1
Fab-X ; dcr-2 L811fsX

21. PcG protein targeting to Fab-7 in RNAi mutants
DAPI sd
BX-C PC
merge
w1118
Fab-X
Fab-X
Fab-X;dcr-2 L811fsX
Loss of targeting only in piwi mutants
Fab-X;piwi 2

22. …but not for targeting of PcG proteins at PREs
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?

23. Defective maintenance of Fab-7 long-distance interactions in RNAi mutant larvae
Endogenous
Fab-7
Transgenic
Fab-7
DAPI sd
BX-C
merge
w1118 + -
Fab-X + +
Fab-X;dcr-2 L811fsX + +
Fab-X;piwi 2 + +

24. Summary
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
The Fab-7 element can establish long-distance interactions that require a novel nuclear function of RNAi components
We predict a general role of long-range associations and of the RNAi machinery in cosuppression/paramution phenomena