1 EMBL, Gene Expression Programme, Meyerhofstrasse 1, 69117, Heidelberg,
Germany
2 Max-Planck-Institute für Entwicklungsbiologie, Spemannstrasse 35/III,
72076 Tübingen, Germany
Author for correspondence (e-mail:
juerg.mueller{at}embl.de)
Accepted 21 January 2004
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SUMMARY |
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Key words: Silencing, Polycomb, Polycomb response element
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Introduction |
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Recent progress towards understanding the PcG/trxG system has come from the
biochemical characterization of PcG and trxG protein complexes. Two distinct
PcG protein complexes have been characterized to date; PRC1 functions by
inhibiting chromatin remodeling by SWI/SNF complexes in in vitro assays
(Shao et al., 1999;
Francis et al., 2001
), whereas
the Esc-E(z) complex functions as a histone methyltransferase
(Cao et al., 2002
;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Müller et al., 2002
).
Similarly, the trxG proteins Trithorax and Ash1 exist in two distinct
multiprotein complexes (Papoulas et al.,
1998
; Petruk,
2001
) and both function as histone methyltransferases
(Milne et al., 2002
;
Nakamura et al., 2002
;
Beisel et al., 2002
;
Byrd and Shearn, 2003
). Thus,
it appears that both PcG and trxG proteins regulate gene expression by
modifying the structure of chromatin.
Nevertheless, silencing by Polycomb group proteins requires specific
cis-acting sequences, called Polycomb response elements (PREs). PREs were
initially identified as regulatory sequences that prevent inappropriate
activation of Hox reporter genes in a PcG protein-dependent fashion in
transgenic Drosophila embryos and larvae
(Müller and Bienz, 1991;
Simon et al., 1993
;
Chan et al., 1994
;
Christen and Bienz, 1994
).
PREs contain binding sites for Pleiohomeotic (Pho) and Pho-like (Phol), the
only known DNA-binding PcG proteins, and binding of these proteins to PREs is
crucially required for silencing in Drosophila
(Brown et al., 1998
;
Brown et al., 2003
;
Fritsch et al., 1999
;
Shimell et al., 2000
;
Busturia et al., 2001
;
Mishra et al., 2001
). Pho and
Phol do not co-purify with PRC1 or the Esc-E(z) complex, and neither PRC1 nor
the Esc-E(z) complex bind to DNA in a sequence-specific fashion. However,
formaldehyde cross-linking studies showed that components of both PRC1 and the
Esc-E(z) complex specifically associate with the chromatin of PREs in tissue
culture cells and in developing embryos and larvae
(Strutt and Paro, 1997
;
Orlando et al., 1998
;
Cao et al., 2002
). This
association is crucial for the long-term repression of Hox genes as most PcG
proteins are needed throughout development to keep Hox genes silenced
(Beuchle et al., 2001
).
Moreover, excision of a PRE from a silenced Hox reporter gene results in loss
of repression, even if the PRE is removed late in development
(Busturia et al., 1997
). Taken
together, these findings support the idea that PREs are silencer elements in
Hox genes through which PcG proteins mediate long-term repression by modifying
chromatin structure.
Although PREs function as very potent silencers within Hox reporter genes,
their ability to silence transcription in the context of other enhancers and
promoters has not been systematically tested. Several PREs have been reported
to partially repress transcription of a linked miniwhite reporter
gene (Chan et al., 1994;
Zink and Paro, 1995
;
Hagstrom et al., 1997
)
(reviewed by Kassis, 2002
). In
those studies, the effect of a PRE on miniwhite expression was
analyzed by monitoring eye pigmentation in adult flies, and repression of
miniwhite by the linked PRE was revealed by an increase in eye
pigmentation in animals that are heterozygous for PcG mutations. It is
important to note that the miniwhite reporter gene was never
completely repressed in those studies, even though this process is often
referred to as `miniwhite silencing'. A major limitation in the
interpretation of this incomplete silencing of miniwhite is the fact
that the miniwhite gene in the reporter construct also served as
transformation marker to isolate transgenic lines harboring the reporter gene
and, hence, only lines showing incomplete silencing of miniwhite were
isolated and analyzed. Thus, it has remained unclear whether PREs function as
general transcriptional silencers, or whether they only function effectively
in the context of Hox genes and require specific target sequences in enhancers
and/or promoters.
Here, using a reporter gene assay in imaginal discs, we test a PRE from the Hox gene Ultrabithorax (Ubx) for its capacity to silence reporter genes that contain enhancer and promoter sequences from genes that are normally not under PcG control. We find that the Ubx PRE very potently prevents transcription of each of the tested reporter genes, and we show that this silencing depends on PcG gene function. Excision of the PRE from the reporter gene by flp-mediated recombination results in the complete loss of repression within 12 hours of flp induction. These results imply that, after removal of the PRE, changes in the chromatin state generated by the action of PcG proteins cannot be propagated by the flanking chromatin.
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Materials and methods |
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X-gal stainings were performed as described
(Christen and Bienz,
1994).
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Results and Discussion |
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Long-term silencing requires the continuous presence of the PRE
To test the long-term requirement for the PRE for silencing of these
reporter genes, we excised the PRE during larval development and we then
monitored ß-gal expression at different time points after excision.
Forty-eight hours after induction of flp expression, all six reporter genes
show robust derepression of ß-gal, suggesting that, in each
case, removal of the PRE resulted in the loss of PcG silencing
(Fig. 4 and data not shown).
Among the different enhancer-promoter combinations used in this study, the
dppW enhancer fused to the TATA box minimal promoter
appears to direct the highest levels of lacZ expression;
>PRE>dppWTZ transformant lines consistently
show the strongest ß-gal staining after excision of the PRE (see Figs
1,
2). We therefore analyzed
>PRE>dppW-TZ transformants at 4, 8, 12 and 24 hours
after induction of flp expression to study the kinetics of this derepression.
We did not detect ß-gal signal at 4 hours or even at 8 hours after flp
induction, but 12 hours after flp induction, all discs show robust ß-gal
expression (Fig. 4). Thus, even
in the case of the most potent enhancer-promoter combination used here (i.e.
dppW enhancer and TATA box minimal promoter), we observe a
delay of 12 hours between flp induction and ß-gal expression. As the
average cell cycle length of imaginal disc cells in third instar larvae is 12
hours (Neufeld et al., 1998), this implies that most disc cells have undergone
a full division cycle within this period. Derepression of the reporter gene in
this experiment requires several steps: (1) excision of the PRE by the flp
recombinase; (2) dissociation of the PRE and PcG proteins attached to it
possibly by disrupting PcG protein complexes formed between the PRE
and factors bound at the promoter (Breiling
et al., 2001; Saurin et al.,
2001
); and (3) transcriptional activation by factors binding to
the enhancer in the construct. It is possible that one or several steps in
this process require a specific process during the cell cycle (e.g. passage
through S phase).
|
Concluding remarks
Our experiments here show that three reporter genes, each containing a
different enhancer linked to a canonical TATA box promoter, are completely
silenced by a PRE placed upstream of the enhancer. Our data suggest that PcG
proteins that act through this PRE prevent indiscriminately activation by a
variety of different transcription factors. The PcG machinery thus does not
seem to require any specific enhancer and/or promoter sequences for
repression.
Two points deserve to be discussed in more detail. The first concerns the
stability of silencing imposed by a PRE. Previous studies suggested that
transcriptional activation in the early embryo could prevent the establishment
of PcG silencing by PREs (Müller and
Bienz, 1991; Poux et al.,
1996
). More specifically, early transcriptional activation of Hox
genes by blastoderm enhancers may play an important role in preventing the
establishment of permanent PcG silencing in segment primordia in which Hox
genes need to be expressed at later developmental stages
(Poux et al., 1996
).
Importantly, none of the three enhancers used in this study is active in the
early embryo. Moreover, these enhancers probably do not contain binding sites
for specific transcriptional repressors, such as the gap repressors, which are
required for establishment of PcG silencing at some PREs in the early embryo
(Zhang and Bienz, 1992
). We
therefore imagine that, in our constructs, PcG silencing complexes assemble by
default on the 1.6 kb Ubx PRE in the early embryo and that PcG
silencing is thus firmly established by the stage when the imaginal discs
enhancers would become active. Silencing by the PRE during larval stages
therefore appears to be dominant overactivation and cannot be overcome by any
of the enhancers used here. There is other evidence in support of the idea
that PcG silencing during larval development is more stable than in embryos.
In particular, a PRE reporter gene that contains a Gal4-inducible promoter is
only transiently activated if a pulse of the transcriptional activator Gal4 is
supplied during larval development; by contrast, a pulse of Gal4 during
embryogenesis switches the PRE into an `active mode' that supports
transcriptional activation throughout development
(Cavalli and Paro, 1998
;
Cavalli and Paro, 1999
).
Furthermore, recent studies in imaginal discs suggest that there is a
distinction between transcriptional repression and the inheritance of the
silenced state; the silenced state can be propagated for some period even if
repression is lost (Beuchle et al.,
2001
). Specifically, loss of Hox gene silencing after removal of
PcG proteins in proliferating cells can be reversed if the depleted PcG
protein is resupplied within a few cell generations
(Beuchle et al., 2001
). Taken
together, it thus appears that PcG silencing during postembryonic development
is a remarkably stable process. Finally, the results reported in this study
also imply that, once PcG silencing is established, Hox genes can `make use'
of virtually any type of transcriptional activator to maintain their
expression; PcG silencing will ensure that activation by these factors only
occurs in cells in which the Hox gene should be active. The analysis of
Ubx control sequences supports this view; if individually linked to a
reporter gene, most late-acting enhancers direct expression both within as
well as outside of the normal Ubx expression domain
(Müller and Bienz, 1991
;
Castelli-Gair et al.,
1992
).
The second point to discuss here concerns the repression mechanism used by
PcG proteins. Biochemical purification of PRC1 revealed that several TFIID
components co-purify with the PcG proteins that constitute the core of PRC1
(Saurin et al., 2001;
Francis et al., 2001
).
Moreover, formaldehyde crosslinking experiments in tissue culture cells showed
that TFIID components are associated with promoters, even if these are
repressed by PcG proteins (Breiling et al.,
2001
). This suggests that PcG protein complexes anchored at the
PRE interact with general transcription factors bound at the promoter. One
possibility would be that PcG repressors directly target components of the
general transcription machinery to prevent transcriptional activation by
enhancer-binding factors. As mentioned above, three distinct activators act
through the three enhancers used here (Kim
et al., 1996
; Kim et al.,
1997
; Müller and Basler,
2000
) and, according to our results, none of them is able to
overcome the block imposed by the PcG machinery. But how do the known
activities of PcG protein complexes [i.e. histone methylation by the Esc-E(z)
complex and inhibition of chromatin remodeling by PRC1] fit into this
scenario? Both these activities may be required for the repression process by
altering the structure of chromatin around the transcription start site and
thus preventing the formation of productive RNA Pol II complexes. Other
scenarios are possible. For example, histone methylation may primarily serve
to mark the chromatin for binding of PRC1 through Pc
(Fischle et al., 2003
;
Min et al., 2003
), and PRC1
components such as Psc then perform the actual repression process
(Beuchle et al., 2001
;
Francis et al., 2001
).
Whatever the exact repression mechanism may be, our PRE-excision experiment
shows that this repression is lost within one cell generation after removal of
the PRE. This implies that changes in the chromatin generated by the action of
PcG proteins cannot be propagated by the flanking chromatin.
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ACKNOWLEDGMENTS |
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Footnotes |
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