PcG, trxG and the maintenance of gene expression

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,
2406-2420.
2
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
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
4
An example of Pairing Sensitive silencing the
Fab-X transgenic line
Chromosome X
Fab-7
mini-white
P
scalloped (sd)
P
Transgenic Fab-7 heterozygous
Transgenic Fab-7 homozygous
w
w
w
---gt strong mini-white silencing
---gt weak silencing of the mini-white reporter
gene
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
Chr. X
Fab-7
mini-white
P
scalloped (sd)
P
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 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
7
Do PRE lead to long-distance pairing?
Three dimensional 2-color FISH in whole mount
embryos
3D-Acquisition
Fixation Permeabilisation
Interphase nuclei
Dapi
Denaturation Hybridization Detection DAPI
counterstating
5 mm
Sd gene
BX-C locus
8
PRE associate in the nucleus through DNA sequence
homology
Dapi
Sd
BX-C
Merge
WT
Percentage of pairing
sd
-
25
Fab-7
BX-C
-
20
Fab-X
Fab-7
sd
-
15
Fab-7
BX-C
-
10
Fab-X Fab-71
-
5
Fab-7
sd
-
BX-C
0
Fab-X
Fab-X Fab-71
WT
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
1. Erase trans homology by genetic crosses
Genetic approach
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
Back cross with
Cross with
Fab-X Fab71/
Fab-X
Fab-X Fab71
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
-
0
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