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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.
PgG proteins form nuclear compartments
PC protein has a speckled distribution in the cell nucleus
early embryos
Z axis image projections
PC / Heterochromatin
Digital image reconstruction
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