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First published online November 21, 2008
doi: 10.1242/10.1242/dev.024554
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
* Author for correspondence (e-mail: jaynes{at}jci.tju.edu)
Accepted 8 October 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Polycomb group, Trithorax group, Epigenetics, Pleiohomeotic, Even-skipped, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Muller and Kassis, 2006;
Ringrose, 2007;
Schuettengruber et al., 2007;
Schwartz and Pirrotta, 2007).
Among the genetically identified Drosophila PcG proteins, only
Pleiohomeotic (Pho) and its homolog Pho-like (Phol) have sequence-specific
DNA-binding activity (Brown et al.,
2003; Brown et al.,
1998; Fritsch et al.,
1999). They, together with other DNA-binding proteins
(Americo et al., 2002;
Brown et al., 2005;
Muller and Kassis, 2006;
Ringrose and Paro, 2007;
Schuettengruber et al., 2007),
have been implicated in targeting each of the known PcG complexes
(Cao et al., 2002;
Czermin et al., 2002;
Kuzmichev et al., 2002;
Shao et al., 1999;
Wang et al., 2004a) to their
target genes. In mammals, PcG proteins are involved in maintenance of the stem
cell niche (Buszczak and Spradling,
2006; Sparmann and van
Lohuizen, 2006) and in oncogenesis
(Sparmann and van Lohuizen,
2006).
Trithorax group (trxG) proteins oppose the action of the PcG
(Kennison, 2004), form
complexes on target DNA (Badenhorst et al.,
2002; Bantignies et al.,
2000; Fyodorov et al.,
2004; Papoulas et al.,
1998; Petruk et al.,
2001) and maintain an active state, by modifying histones and
remodeling chromatin. PcG and trxG proteins are considered to be distinct from
other regulators in that they can mediate heritable maintenance of the
transcriptional state from one cellular generation to another
(Grimaud et al., 2006;
Muller and Kassis, 2006;
Ringrose and Paro, 2007;
Schuettengruber et al., 2007;
Schwartz and Pirrotta, 2007).
Target sequences that mediate the functions of these proteins are termed PcG-
and trxG-responsive elements (PREs and TREs, respectively) or, collectively,
maintenance elements (MEs) (Brock and van
Lohuizen, 2001), because they often overlap. Dissection of these
target elements has proven to be difficult, because of their complexity, and
because the associated activities manifest themselves fully only in a
developmental context. Consequently, how PcG and trxG complexes are targeted
to DNA, and how this targeting varies with the transcriptional state, is just
beginning to be elucidated.
Transgenes carrying PREs/TREs in Drosophila have been seen to
share a property known as pairing-sensitive silencing (PSS)
(Gindhart and Kaufman, 1995;
Kassis, 2002). Transgenic
flies carrying the mini-white gene typically have eye colors ranging
from yellow to orange in a white mutant background. Normally, flies
that are homozygous for such a transgene have a darker eye color than
heterozygotes, as the genetic dose of mini-white is doubled. However,
with transgenes carrying PRE/TREs, the eye color is often lighter in
homozygotes than in heterozygotes. PSS is also often accompanied by a
reduction in mini-white activity from heterozygous transgenes,
causing a relatively faint eye color, or a variegated pattern of eye
coloration. This phenomenon was first reported for a pairing-sensitive element
(PSE) near the engrailed (en) transcription start site
(Kassis, 1994;
Kassis et al., 1991). It was
also shown that the en PSE can function as a PRE in embryos, in that
it can restrict expression of a transgenic reporter gene driven by the
Ultrabithorax (Ubx) regulatory region bithoraxoid
(bxd) to the posterior part of the embryo, where Ubx is
normally expressed, in a PcG-dependent manner
(Americo et al., 2002). More
generally, although PREs usually exhibit PSS activity, PSEs may not be
sufficient to function as a PRE (Kassis,
1994; Kassis,
2002).
Genome-wide predictions of possible Drosophila PREs using
sequences common to known PREs, including binding sites for the PcG and trxG
proteins Pho/Phol, GAGA factor (GAF) and Zeste, identified 167 candidates,
including the even skipped (eve) promoter region
(Ringrose et al., 2003). Other
studies using two different techniques to determine in vivo protein binding
(ChIP on chip and DamID) identified other candidate PREs with some, although
not extensive, overlap (Negre et al.,
2006; Schwartz et al.,
2006; Tolhuis et al.,
2006). These studies suggested that the eve locus is
bound by the Polycomb (Pc) protein
(Schwartz et al., 2006;
Tolhuis et al., 2006).
Earlier, mapping of binding sites for Pc and Polyhomeotic (Ph) on polytene
chromosomes identified the cytological location of eve
(Sinclair et al., 1998).
Consistent with this, eve is ectopically expressed in the embryonic
nervous system in mutants of the PcG gene ph
(Smouse et al., 1988).
Here, we show that the Drosophila eve gene is regulated by a
Pho-dependent maintenance element located 9 kb downstream of the promoter.
This element maintains repression in cells where eve is turned off
during early development, but maintains the active state in other cells. Both
negative and positive maintenance depend on Pho binding, and on pho
gene activity. The element shares properties with other PREs, including
binding sites for several factors. Dissection suggests that although GAF is
involved, Zeste and Dsp1 may be dispensible, and that Grainyhead/Elf1
(Bray and Kafatos, 1991), as
well as unknown factors, may also contribute. Thus a `core' DNA-binding
component, differentially modified or regulated by the existing
transcriptional state, mediates epigenetic maintenance of that state.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
construct the PSS assay transgenes of Fig.
1, eve DNA from +8.9 to +9.2 kb (PRE300) was cloned
upstream of the Glass-binding sites followed by mini-white. DNA from
-275 bp to +116 bp was similarly used to make PSEpro. The mini-white
P-element-based GFP-RR construct was made by fusing eve DNA from -275
bp to +99 bp with the EGFP-coding region, driven by the RR regulatory DNA
(+7.9 to +9.2 kb), which consists of the neuronal RP2+a/pCC enhancer and
PRE300 (Fujioka et al., 2003).
For 
C31 recombinase-mediated cassette exchange (RMCE)
(Bateman et al., 2006;
Groth et al., 2000), the
piB-GFP construct (Bateman et al.,
2006) was first modified to increase the number of unique cloning
sites (attB
2; details available on request). Then, GFP-RR described
above was transferred to this vector. For the PRE assays of
Fig. 2, a P-element based
bxd-Ubx-Z vector (with a bxd enhancer fragment inserted
upstream of the -3.1Ubx-βgal fusion in Uβghz)
(Muller and Bienz, 1991) was
modified by inserting PRE300 (with or without the Pho site mutated) into the
XbaI site, oriented such that the PRE300 3' end was closer to
the Ubx promoter. For RMCE, the PRE300 (with or without the Pho site
mutated)-bxd-Ubx-Z region was transferred to attB2. PRE300 was
also replaced by a 500 bp 
phage DNA fragment as non-PRE negative
control in this context (500-bxd-Ubx-Z). Binding site
mutations used in this study are shown in
Fig. 3.
Transgenic and genetic analysis
P-element-based transgenic lines were established as previously described
(Fujioka et al., 2000;
Rubin and Spradling, 1982).
RMCE was performed as previously described
(Bateman et al., 2006). The
attP target lines used were chosen at chromosomal locations that were not
suggested to have PREs nearby (Negre et
al., 2006; Schwartz et al.,
2006; Sinclair et al.,
1998; Tolhuis et al.,
2006). The attP target lines 25C, 52D
(Bateman et al., 2006) and 78C4
(obtained in our laboratory) were used for inserting GFP-RR. For RMCE of
bxd-Ubx-Z constructs, 25C, 52D, 78C4 and 95E5 (obtained in our
laboratory) lines were tested. 52D and 95E5 were used for further analysis.
For staining with anti-β-gal (ICN, 1:1000 dilution), embryos were
collected for 6 hours at room temperature (RT), then incubated at 17°C for
15 hours. The antibody was visualized using biotin-conjugated anti-rabbit IgG
followed by peroxidase-conjugated streptavidine, with DAB as a substrate. For
Fig. 4, each central nervous
system (CNS) was dissected out in phosphate-buffered saline, placed on a
poly-L-lysine-coated cover slip, and covered with a non-coated cover slip
using another cover slip as a spacer to prevent flattening.
For pho mutant analysis of PSS, six PRE300-carrying lines
(including two GFP-RR lines) that showed PSS were crossed to generate
PRE300;pho1/ciD. Larvae from these stocks were
grown at room temperature, and pharate adults were dissected to analyze their
eye color. For pho and phol mutant analysis of positive
maintenance in the CNS, a mini-white P-element-based GFP-RR line was
used to produce GFP-RR;phol81A/TM6B,Tb,
GFP-RR;pho1/ciD or
GFP-RR;phol81A/TM6B,Tb;pho1/+, which
were then self-crossed. Homozygous phol larvae were tentatively
identified as non-Tubby, and confirmed post hoc by PCR. In all cases,
homozygous pho and pho/+ larvae and pupae were identified
post hoc by PCR analysis of their DNA
(Brown et al., 2003). GFP-RR
at 52D was crossed into mutant backgrounds to produce
GFP-RR(52D);E(z)61/TM6B,Tb,
GFP-RR(52D);trx1/TM6B,Tb, and
GFP-RR(52D);Trl13C/TM6B,Tb. Homozygous mutant
larvae from these lines were identified by their non-Tb phenotype.
For E(z)61, eggs were collected at 18°C for 24 hours,
kept at 18°C until near hatching, then moved to 29°C until the third
larval instar. For trx1, eggs were collected similarly,
then moved to 25°C until the third larval instar. For
Trl13C, eggs were collected similarly, and kept at
18°C until the third larval instar.
To test effects of PcG mutations on eve PRE activity, lines
homozygous for bxd-Ubx-Z-derived transgenes (at target site 52D) were
each crossed with ph503/FM7,B or
Pc4/TM3,Sb. Then, ph503/+;bxd-Ubx-Z/+
(non-B) females were crossed with homozygous bxd-Ubx-Z
males, while bxd-Ubx-Z/+;Pc4/+ (non-Sb) males and
females were intercrossed. Resulting embryos were collected as described
above. Homozygous mutant embryos were identified by ectopic lacZ
expression in anterior regions of embryos, which were not present in a
wild-type background. For ph503, the ectopic lacZ
phenotype of hemizygous mutant embryos was confirmed by double-staining for
ectopic eve expression in the CNS
(Smouse et al., 1988).
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) analysis was performed as follows.
Dechorionated embryos were crosslinked with 2% formaldehyde for 15 minutes at
room temperature, washed and stored at -80°C. About 200 µl of embryos
were homogenized in 1 ml Buffer A (350 mM sucrose, 15 mM HEPES pH 7.6, 10 mM
KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA). Homogenates were
centrifuged at 600 g for 5 minutes to pellet nuclei, which
were then washed gently with 1 ml Buffer A and repelleted. Pellets were
resuspended in 1 ml Buffer A containing 0.2% Triton-X100, incubated for 5
minutes on ice, then layered on top of 3 ml 800 mM sucrose (in Buffer A) in 15
ml conical-bottom tubes, and centrifuged at 250 g for 10
minutes. The pellets were subjected to ChIP analysis essentially as described
previously (Wang et al.,
2004b), using Pho-specific antiserum
(Brown et al., 1998). PCR
primer sequences will be provided on request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This region also caused repression of mini-white
expression in heterozygotes, making it difficult to use a standard
mini-white-containing P-element vector to generate transgenic lines,
because complementation of the white- phenotype was
extremely weak at most chromosomal locations (data not shown). In such a case,
it is likely that insertions at many sites are missed, resulting in a biased
sampling of potential insertion sites. To overcome this problem, three tandem
Glass activator-binding sites, which are active specifically in the eye disc,
were introduced just upstream of the mini-white promoter to increase
expression (Fujioka et al.,
1999). This Glass site vector was also used in the analysis of the
en PRE (Americo et al.,
2002), where it was shown that the presence of Glass sites does
not affect the percentage of PRE-carrying transgenic insertions that exhibit
PSS. As expected, with the Glass-binding sites, the range of eye colors of
heterozygous transgenic flies shifted toward the dark end of the spectrum
(Fig. 1, compare `with Glass'
with `without Glass'; note that none of these lines carry a PSE). Among 53
independent lines carrying the Glass vector without the PSE, 43 were
homozygous viable and none showed PSS (Fig.
1, `with Glass').
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The eve promoter region (from about -700 to -100 bp), but not the
eve 3' region, was previously identified in a genome-wide
prediction of PRE/TREs based on a clustering of Pho-, GAF- and Zeste-binding
sites (Ringrose et al., 2003).
We discovered that the eve promoter region from -275 to +166 bp also
has PSS activity. Fifty-three percent of transgenic lines carrying this region
showed PSS (PSEpro, Fig. 1).
Although the combination of PRE300 with the eve promoter caused more
repression of heterozygous mini-white expression than did either one
alone, PSS activity itself was not increased relative to that of PRE300
(PRE300 + PSEpro, Fig. 1).
eve PSS activity depends on a Pho binding site, and requires pho
As the eve 3' PRE showed stronger activity than the
promoter, we focused on the 3' PRE in further experiments. Previous
dissections of PSS regions from the en gene
(Americo et al., 2002), the MCP
silencer (Busturia et al.,
2001) and the iab-7 PRE
(Hagstrom et al., 1997;
Mishra et al., 2001) (both
from the bithorax complex) showed that activity depended on both Pho-
and GAF-binding sites. PRE300 contains one perfect match to the Pho-binding
site consensus and three close matches to the GAF consensus. Mutating the
single Pho site (Fig. 3)
strongly reduced PSS activity from 73% to 11% (PRE300Pho,
Fig. 1). This was accompanied
by a shift in the spectrum of heterozygous eye colors back to that obtained
without the PSE, suggesting that the cis-repressive activity of PRE300 is
entirely dependent on the Pho site. Mutating two or all three of the GAF sites
(Fig. 3) caused a progressive
decrease of PSS activity, but only to 53%
(Fig. 1: PRE300gaga1,
gaga12 and gaga123). Mutating GAF sites also did not have as
much of an effect on heterozygous eye color as did mutating the Pho site.
These data suggest that the PSS activity of PRE300 depends only weakly on GAF
binding sites, whereas, like other PSEs examined in detail, it is strongly
dependent on the Pho-binding site.
In order to test whether this Pho-binding-site dependence reflects an
actual dependence on Pho binding, or whether it might be due to some
overlapping DNA-binding activity, we placed several PRE300-carrying lines in a
pho1 mutant background. As pho1
mutants die as pharate adults, it was necessary to dissect pupae in order to
examine their eye color. Compared with a wild-type background, the homozygous
eye color was darker in pho1 mutants in all six lines
examined. This effect was very strong in one line
(Fig. 2E,F), whereas the other
lines showed less complete, albeit still clear, derepression
(Fig. 2C,D; data not shown).
Incomplete derepression is expected in the pho mutants, because the
pho-like gene (phol) provides a partially redundant function
with that of pho. It is not possible to carry out this analysis in
phol81A; pho1 double mutants, because they fail
to pupariate (Brown et al.,
2003). The convergence of these genetic data with the
Pho-binding-site dependence of PSS strongly suggests that Pho is directly
involved in silencing through PRE300.
The involvement of known factors in eve PSS activity
Among known PREs/TREs, binding sites for Pho/Phol, GAF/Pipsqueak, Sp1/KLF,
Zeste and Dsp1 were found to be clustered
(Brown et al., 2005;
Muller and Kassis, 2006;
Ringrose, 2007;
Schuettengruber et al., 2007).
PRE300 contains all of these binding sites, including two Dsp1 sites, three
GAF sites, two Sp1/KLF sites, one Zeste site and one Pho site
(Fig. 3). It has also been
shown that the grainyhead (grh) gene product
(Bray and Kafatos, 1991), which
was originally identified as a transcriptional activator termed Elf-1
(Bray et al., 1989), physically
interacts with RING, a PcG gene product
(Tuckfield et al., 2002).
Importantly, Grh/Elf-1 was shown to cooperatively interact with Pho on the
iab-7 PRE in vitro, and to genetically interact with pho
(Blastyak et al., 2006). PRE300
also contains a reasonable match to the Grh consensus site
(Fig. 3). In order to test
whether these binding sites are required for its PSS activity, PRE300 was
split into two overlapping parts (PRE300-5' and PRE300-3'). As the
Pho site is necessary for the activity, this site was included in both parts
(Fig. 3). Surprisingly,
PRE300-5', which includes all of these sites except for one Sp1/KLF, one
GAF and the Grh site, showed PSS activity in only 5% of transgenic lines
(Fig. 1). By contrast,
PRE300-3', which contains only the Pho site, one Sp1/KLF site, the Grh
site and one of the GAF sites, showed almost full PSS activity (69% versus 73%
for PRE300, Fig. 1). These data
suggest that only a subset of known binding sites are required for the PSS
activity of PRE300, and further suggest that other, unidentified factors may
well be involved.
|
). We directly tested for PRE activity of eve PRE300
by combining it in a transgene with a bxd-Ubx-Z reporter gene. This
reporter is expressed throughout the ectoderm and nervous system in late-stage
embryos (Muller and Bienz,
1991). However, when a PRE is inserted into this reporter, it can
prevent activation of lacZ expression in parasegments 1-5, similar to
the endogenous Ubx PRE from the bxd region
(Americo et al., 2002;
Brown et al., 2005;
Busturia and Bienz, 1993;
Hagstrom et al., 1997;
Horard et al., 2000;
Muller and Bienz, 1991). In
two out of four P-element-based transgenic lines carrying PRE300 in this
reporter, we observed an anteriorly restricted lacZ pattern (similar
to Fig. 2H, data not shown).
Similarly, studies of other PREs in this reporter found that not all
transgenic lines showed repression (Americo
et al., 2002; Brown et al.,
2005; Busturia and Bienz,
1993; Hagstrom et al.,
1997; Horard et al.,
2000; Muller and Bienz,
1991), presumably owing to the effects of different chromosomal
contexts. In order to further analyze the PRE activity of PRE300, we
incorporated the bxd-Ubx-Z reporter into a vector for RMCE
(Bateman et al., 2006;
Groth et al., 2004), which
allows the analysis of multiple transgenic constructs at the same chromosomal
location. We initially tested PRE300-bxd-Ubx-Z inserted at four
different chromosomal target sites, two of which were used in a previous study
(Bateman et al., 2006) and two
others that we developed. All four targeted lines showed an anteriorly
restricted pattern (Fig. 2H,N,
compare with Fig. 2G,M, which
carry non-PRE DNA as a negative control, and data not shown). We tested the
effect of mutating the Pho site in PRE300 in three of these chromosomal
contexts, and all three showed significant anterior derepression
(Fig. 2I,O; data not shown). We
further tested one of these anteriorly restricted lines to see whether the
repression depends on PcG genes, using the alleles Pc4 and
ph503. Repression was lost or significantly reduced in
each of these mutants (compare Fig.
2K,L with 2J). In
addition to a strong effect in ph hemizygotes
(Fig. 2), based on the number
of embryos in the population showing anteriorly expanded expression, there
appears to be a partial loss of repression in ph503
heterozygotes (from ph503 heterozygous mothers) as well
(data not shown). So, the PRE activity of eve PRE300 depends on both
the Pho site and on PcG gene function, and thus has similar characteristics to
other known PREs.
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) was used to drive heterologous gene expression (GFP or
lacZ), expression was normal in embryos, but was not maintained to the third
larval instar (data not shown). However, when the 3' end point was
extended to +9.2 kb (to generate the `RR' construct)
(Fujioka et al., 2003),
expression was fully maintained (Fig.
4A). The region required for this maintenance contains PRE300.
Although Pho, as a PcG gene, is known only to be involved in maintaining the
repressed state, its ortholog in mammals, YYI, is known to be involved in
activation of some target genes (Gordon et
al., 2006). We tested whether Pho affects positive maintenance
activity. When the same Pho-binding site mutation that knocked down PSS
(Fig. 3) was introduced into
the RR construct, CNS maintenance activity was reduced or eliminated. Out of
the 11 independently inserted transgenes tested, eight gave normal expression
in the embryonic CNS. Of these eight, three completely failed to maintain
expression to the third larval instar (data not shown, similar to
Fig. 4F), and the other five
showed relatively weak and variable expression of GFP in third instar larvae
(Fig. 4B and data not shown).
As the Pho dependence of positive maintenance seemed to be affected somewhat
by the chromosomal context, we confirmed these results by examining expression
in a pair of lines, one with the Pho site mutated, inserted at the same
chromosomal location using RMCE. At this target site, although GFP expression
in third instar larvae was somewhat weaker than in the random insertion line
shown in Fig. 4A, expression in
third instar larvae was nonetheless abolished when the Pho site was mutated
(Fig. 4E,F). Thus, the
maintenance of CNS expression by PRE300 requires the Pho-binding site.
|
;
Hagstrom et al., 1997;
Hodgson et al., 2001;
Horard et al., 2000;
Mishra et al., 2001), and to
facilitate binding of Pho to a chromatinized PRE
(Mahmoudi et al., 2003).
Moreover, mutations in GAF sites affect the PSS activity of PRE300
(Fig. 1). Therefore, we tested
the effects of GAF site mutations using both conventional P-element
transformation and RMCE. When all three GAF sites were mutated, GFP expression
depended strongly on the insertion site. Although two out of 11 random
insertion lines showed similar levels of expression in third instar larvae as
transgenes carrying wild-type PRE300, four out of 11 lines showed weak
expression, and five out of 11 lines showed ectopic expression throughout the
CNS, such that we could not discern the intensity in RP2 and a/pCC neurons.
When RMCE was used to compare directly the activity of wild-type and triply
GAF-mutated PRE300, there was no apparent change in GFP expression (data not
shown). Thus, mutating sites for GAF can have either a positive or a negative
effect on CNS maintenance by PRE300. The ectopic expression observed with the
GAF-mutated element also suggests that wild-type PRE300 may have a negative
maintenance activity in cells of the CNS where eve is not normally
expressed, in addition to its role in positive maintenance.
As stated above in relation to PSS, it is important to determine whether
the Pho-binding site dependence reflects an actual dependence on Pho binding,
or whether it might be due to some overlapping DNA-binding activity. In order
to test this, the GFP-RR transgene was placed in a phol81A,
pho1 double mutant background. Indeed, relative to a wild-type
background, GFP expression was clearly reduced
(Fig. 4C, compare with 4A). In
pho1 single mutants, there was a reduction in most larvae
(Fig. 4D), but the difference
versus wild type was not as clear-cut as in the double mutant. In
phol81A single mutants, there was no apparent effect (not
shown). This is consistent with the fact that pho1 mutants
die as pharate adults, whereas phol81A mutants survive to
adulthood (Brown et al.,
2003). These data indicate that Pho and Phol are indeed involved
in the maintenance not only of repression, but also of activation. Consistent
with PRE300 having a negative maintenance activity in the CNS in addition to
its role in positive maintenance, as suggested above, we observed widespread
ectopic expression (seen as a higher background of GFP fluorescence) in some
PRE300-carrying lines both in pho/phol double mutants and when the
Pho site was mutated (e.g. Fig.
4C and Fig. 4F,
respectively).
As a preliminary test of the involvement of other PcG genes in positive maintenance by PRE300, we tested the allele E(z)61, as homozygous larvae survive to the third larval instar. However, we did not see any effects on positive maintenance (data not shown). We also tested some trxG alleles that survive to the third larval instar. Larvae homozygous for trx1 and Trl13C did not show any effects on CNS expression of our GFP reporter (data not shown). Larvae heterozygous for trxB11, TrlR65 and TrlR85 were also tested, and, again, no clear effects were observed (data not shown). However, because these alleles have significant maternal and zygotic contributions that allow them to survive long enough to be assayed, further analysis will be required to determine definitively whether these genes are involved along with pho and phol in positive maintenance by PRE300.
Pho binds to the eve PRE in vivo
The convergence of genetic and biochemical evidence presented above
strongly suggests that Pho acts directly through the binding site in PRE300.
To confirm that Pho interacts with this region, and with this site, in vivo,
we performed ChIP assays. First, we surveyed the eve locus for Pho
binding using a series of primer sets for detection of immunoprecipitated
fragments by PCR. As shown in Fig.
5 (B and E, primer set 13), Pho was detected specifically in the
region of PRE300, both in early and later-stage embryos. Pho was also detected
in the promoter region where binding sites were previously predicted,
primarily in early embryos (Fig.
5B, primer set 6), as well as in the region of enhancers driving
mesodermal and anal plate expression (Fig.
5B,E, primer set 8). This confirms that Pho interacts with the
endogenous eve locus. Furthermore, it does so specifically with the
region that our transgenic analysis implicated in maintenance of both the
repressed and activated state. To test the involvement of the functionally
important Pho-binding site, we tested transgenes carrying PRE300, either with
the normal sequence, or with the Pho site mutated. Again, ChIP analysis
indicated that Pho interacts with the transgenic copy of PRE300
(Fig. 6A). Furthermore, the
signal is reduced with the mutant Pho site, relative to both the endogenous
eve signal and to the background controls
(Fig. 6D, compare to
Fig. 6A). This indicates that
the site is important for Pho binding in vivo, and the loss of binding
correlates with the loss of both repression and activation that occurs when
the site is mutated.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have suggested that PRE-containing P-element-based transgenes have a tendency to insert near endogenous PREs, and that this can bias reporter gene expression. Here, we applied both P-element analysis and the RMCE system to compare the effects of mutating binding sites. Our data reveal that there is also variation in PRE effects using RMCE into different target sites. Therefore, it would seem important to test several target sites when using RMCE, to ensure that results are not specific to one chromosomal location. Furthermore, where sensitivity to position effects is high, such as with our GAF site-mutated PRE, it remains valuable to use the standard methodology to probe a variety of insertion sites.
Surprisingly, we found that the eve PRE is also required for
positive maintenance of expression in the larval CNS, and that this activity
requires both the Pho-binding site and pho gene function. Together,
these data strongly suggest that Pho is directly involved in positive
maintenance of gene activity. This is surprising because Pho has heretofore
been associated only with direct repression of target genes, by recruiting the
PRC2 complex and other PcG proteins (Wang
et al., 2004b). However, recent studies have blurred the
distinction between PcG genes and trxG genes, as some members of each class
appear to have dual functions (Grimaud et
al., 2006). Furthermore, PREs usually reside in close proximity to
TREs (Ringrose and Paro,
2007), and an element from the promoter region of
engrailed that mediates PSS and can act as a PRE was recently shown
to have an activating role in its natural context
(Devido et al., 2008). Recent
studies of the Ubx locus have indicated that PcG proteins are present
at PREs in both the off and the on state, and that binding of Ash1 prevents
silencing by the PRC complex in cells where Ubx is expressed,
suggesting that silencing is actively prevented
(Papp and Muller, 2006). A
similar situation may pertain to Pho function in the eve locus.
Because trxG proteins are known to be involved in positive regulation by
other maintenance elements, we were interested in whether they are involved in
pho-dependent positive maintenance by the eve PRE. We were
also interested in whether other PcG proteins are involved. Because our
positive maintenance assay requires survival to the third larval instar, we
have so far been able to test only weak alleles of trx, Trl and
E(z), neither of which showed discernable effects in our assays. At
this point, we can not definitively say whether other trxG or PcG proteins are
involved in the positive maintenance function of the eve PRE.
However, our observation that a consensus Grh binding site is present in the
more active half of PRE300 suggests the involvement of Grh. Indeed, Grh has
been shown to interact genetically with Pho, and to facilitate cooperative
interaction with Pho in vitro (Blastyak et
al., 2006).
Consistent with the broad overexpression of eve seen in the CNS of
ph mutants (Smouse et al.,
1988), the eve PRE may silence expression in many cells
by forming a silencing complex. In wild-type embryos, in the subset of CNS
cells where eve is expressed, the same Pho-dependent DNA binding
platform may recruit a distinct complex that maintains the active state.
Consistent with this model, we have found that expression driven by the
eve RP2+a/pCC enhancer fades prematurely in late stage embryos in
ph mutants, at the same time that endogenous eve is broadly
overexpressed (M.F. and J.B.J., unpublished). It will be interesting to
determine the composition of Pho-dependent complexes in cells where
eve is on, and in those where eve is off.
How can a region 9 kb away from the basal promoter affect the state of gene
expression? There are accumulating data suggesting that locus-wide regulation
occurs through direct interactions of the promoter with enhancers and locus
control regions. For example, a recent study showed that silencing by the
bxd PRE directly affects the activity of the transcriptional
machinery at the promoter (Dellino et al.,
2004). In the eve locus, there are PSEs both at the
3' end of the locus and at the promoter. Both contain clusters of
binding sites typical of a PRE/TRE. It has been suggested that PRE-containing
transgenes have a tendency to insert near endogenous PREs
(Chiang et al., 1995;
Fauvarque et al., 1995), which
might be expected if they mediate long-range interactions. Putting these ideas
together, the eve 3' PRE may physically interact with the
promoter region in a Pho-dependent manner. This may serve to keep eve
on in some cells and to keep it off in others, depending on whether activating
or repressive complexes mediate the association.
|
ACKNOWLEDGMENTS |
|---|
C31 system; and Hugh W. Brock, Paul Schedl, Judy A.
Kassis and the Bloomington Stock Center for mutant strains. We also thank Judy
A. Kassis, Alex Mazo and Hugh W. Brock for comments on the manuscript. This
work was supported by NSF-IOB0416760, NSF-MCB0818118 and NIH-R01GM050231
awards to J.B.J. and M.F. |
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