Transcript Nuclear compartmentalization and chromatin regulation An example of a functional cell biology analysis Data from: Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and.

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
Cavalli
Lab
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
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
PRE Heterozygous
PRE Homozygous
Weak PcG mediated
repression
Strong PcG mediated
repression
An example of Pairing Sensitive silencing: the Fab-X transgenic line
P
Fab-7
mini-white
P
Transgenic Fab-7
heterozygous
w
scalloped (sd)
Chromosome X
Transgenic Fab-7
homozygous
w
w
---> weak silencing of the
mini-white reporter gene
---> strong mini-white silencing
In the line Fab-X, the Fab-7 transgenic PRE at the homozygous state
represses an endogenous gene located close to the insertion site
P
Fab-7
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
The sd repression mediated by the Fab-7 transgene requires the presence of
the endogenous copy of Fab-7
Fab-X; Fab71
Fab-X
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
Do PRE lead to long-distance pairing?
Three dimensional 2-color FISH in whole mount embryos
Fixation
Permeabilisation
3D-Acquisition
Interphase nuclei
Dapi
Denaturation
Hybridization
Detection
DAPI counterstating
5 mm
BX-C
locus
Sd
gene
PRE associate in the nucleus through DNA sequence homology
Dapi
Sd
BX-C
Merge
WT
Percentage
of pairing
sd
BX-C
Fab-7
Fab-X
sd
BX-C
25
-
20
-
15
-
10
-
Fab-7
Fab-7
Fab-X; Fab-71
sd
5-
Fab-7
0-
BX-C
WT
3mm
Transgenic Fab-7 at sd (Chr.X)
-
Endogenous Fab-7 at the BX-C (Chr.III)
+
Fab-X Fab-X;
Fab-71
+
+
+
-
Chromatin states can be inherited through
meiosis by the following generations
Chromosome pairing and meiotic inheritance
Genetic approach: 1. Erase trans homology
by genetic crosses
2. Restore trans homology
Pairing
Strong PcG mediated
repression
No pairing
Weak PcG mediated
repression
When pairing is disrupted, silencing is weakened.
If pairing is re-established, can silencing be immediately re-acquired, or
is the derepressed state inherited?
Meiotic inheritance of sd derepression
Fab-X
Fab-X
Back cross with
Cross with
Fab-X; Fab71/+
Fab-X
Fab-X; Fab71
F0 repressed
(85-90% sd)
F1 partially derepressed
(40-45% sd)
F2 and F3
derepressed
(20-25% 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?
YES! Meiotic inheritance of sd derepression
correlates with loss of Fab-7 pairing
Dapi
Sd
BX-C
Merge
25
-
20
-
15
-
10
-
Fab-X
after
meiosis
% of pairing
Fab-X
50-
Fab-X
Fab-X
after
Meiosis
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
Polycomb, nuclear organization
and RNAi
Charlotte Grimaud
Frédéric Bantignies
Collaborators:
Utpal Bhadra - CCMB
Manika Pal-Bhadra - IICT
Hyderabad, India
RNAi and regulation of gene expression
PTGS
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
TGS
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
Fab-7 dependent silencing depends on RNAi components
Fab-X
Fab-X;piwi 1
Fab-X;AGO1 45/72
Fab-X;piwi 2
Fab-X;dcr-2 L811fsX
Fab-X;hls E1
PcG proteins form nuclear compartments called PcG bodies
Z axis image projections
PC / Heterochromatin
Do RNAi component colocalize with PcG bodies?
Specific RNAi components colocalize with PcG bodies
DAPI
PH
RNAi
proteins
merge
Colocalization
AGO1
42%
Dcr-2
62%
PIWI
52%
Hls
19%
Dcr-1
Very low
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?
PcG proteins can be recruited to Fab-7 in most RNAi mutants
DAPI
FISH
PC
merge
Fab-X
Fab-X;AGO1 KO8121/45
Fab-X;piwi 1
Fab-X ; dcr-2 L811fsX
The piwi gene
is an
exception !
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
Fab-X;piwi 2
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?
Defective maintenance of Fab-7 long-distance
interactions in RNAi mutant larvae
DAPI
w1118
+
-
Fab-X
+
+
Fab-X;dcr-2 L811fsX
+
+
Fab-X;piwi 2
+
+
sd
BX-C
merge
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
Cavalli
Lab