Department of Zoology and Animal Biology, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland
Author for correspondence: (e-mail: francois.karch{at}zoo.unige.ch)
Accepted 16 July 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Boundary, Insulator, Scs, Fab-7, Bithorax complex, Polycomb, Transcription, Silencing, Drosophila
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The regulation of the BX-C homeotic genes during embryogenesis is
subdivided into two phases: initiation and maintenance. In the initiation
phase, the products of the gap and pair-rule segmentation genes are
responsible for initiating the parasegment specific expression of the BX-C
homeotic genes. These proteins interact with target sequences (called
initiation elements) in the nine cis-regulatory domains
(Simon et al., 1990;
Qian et al., 1991
;
Muller and Bienz, 1992
;
Shimell et al., 1994
).
However, the products of the segmentation genes are present only transiently
in the early embryo. Maintenance of the initial pattern requires the
trithorax-Group (trx-G) and Polycomb-Group
(Pc-G) genes. The trx-G genes function to keep the homeotic
genes on, while the Pc-G genes function to maintain the inactive
state of the homeotic genes (reviewed by
Paro, 1990
;
Simon and Tamkun, 2002
).
Experiments with reporter constructs have identified elements, called Polycomb
Response elements or PREs, in several of the BX-C cis-regulatory
domains, that appear to be targets for Pc-G action. When these PREs
are combined with a parasegment-specific initiation element, they maintain the
parasegmentally restricted expression pattern conferred on the reporter by the
initiation element (Muller and Bienz,
1991
; Busturia and Bienz,
1993
; Simon et al.,
1993
; Chan et al.,
1994
; Chiang et al.,
1995
; Poux et al.,
1996
). In addition to this maintenance activity, PREs are also
able to repress the activity of the mini-white reporter gene used to establish
transgenic lines. Usually, transgenic lines carrying the mini-white gene
harbor darker eye color when the inserts are homozygous. When they are
included in a mini-white transgene, the PREs repress or even
eliminate mini-white expression when the animals are homozygous (a
phenomenon referred to as the pairing-sensitive repression assay)
(Kassis et al., 1991
;
Chan et al., 1994
;
Gindhart and Kaufman, 1995
;
Hagstrom et al., 1997
;
Muller et al., 1999
) (for a
review, see Pirrotta and Rastelli,
1994
).
Genetic and molecular analysis has identified chromatin domain boundaries
that demarcate the cis-regulatory domains, insuring the functional
autonomy of each regulatory domain
(Gyurkovics et al., 1990;
Galloni et al., 1993
;
Mihaly et al., 1997
;
Zhou et al., 1999
;
Barges et al., 2000
) (for a
review, see Mihaly et al.,
1998
). For example, in PS11, the Fab-7 boundary protects
the active iab-6 cis-regulatory domain from the inactive
iab-7 domain, preventing inappropriate regulatory interactions
between the two domains. Immediately adjacent to the Fab-7 boundary,
lies the iab-7PRE, which is involved in maintaining inactivity of
iab-7 in parasegments anterior to PS12
(Hagstrom et al., 1997
;
Mihaly et al., 1997
).
Two classes of mutations affect the Fab-7 region. Class II
mutations, such as Fab-72, delete the boundary alone and
leave the nearby iab-7 PRE intact. They lead to a mixed gain- and
loss-of-function phenotype in PS11/A6; there are groups of cells acquiring
PS10/A5 identity, because in these cells both iab-6 and
iab-7 are inactive. The remaining cells of PS11 adopt a PS12/A7 fate,
because both iab-6 and iab-7 are active in these cells. This
mixed gain- and loss-of-function phenotype arises because there is a
competition in the fused cis-regulatory domain between positive elements in
iab-6 that ectopically activate iab-7 and negative elements
in iab-7 that ectopically silence iab-6. Class I mutations,
such as the original Fab-71 allele, are larger deletions
that remove not only the boundary but also the nearby iab-7 PRE. In
this class of mutation, the balance between gain- and loss-of-function
phenotype is shifted towards gain-of-function, and A6 is completely
transformed into A7 (see Fig.
2B) (Mihaly et al.,
1997).
|
Most chromatin domain boundaries in higher eukaryotes have been identified
by their ability to block enhancer-promoter interactions when intercalated
between them (enhancer-blocking assay) (for reviews, see
Gerasimova and Corces, 1996;
Geyer, 1997
;
Sun and Elgin, 1999
;
Bell et al., 2001
). In our
terminology, we call elements defined in the enhancer-blocking assay chromatin
insulator. In Drosophila two insulators, scs/scs'
(Kellum and Schedl, 1991
;
Kellum and Schedl, 1992
) and
gypsy (Geyer and Corces,
1992
; Roseman et al.,
1993
) have been extensively studied in the enhancer-blocking
assay. We have previously described that gypsy or a minimal scs
fragment (scsmin) cannot substitute for Fab-7; their
enhancer-blocking activity prevents the iab-5 and iab-6
cis-regulatory domains from interacting with the Abd-B target
promoter (Hogga et al., 2001
).
We describe the results of experiments in which we replace Fab-7 by a
slightly larger scs fragment that was used in enhancer-blocker experiments by
different laboratories (Kellum and Schedl,
1991
; Kellum and Schedl,
1992
; Vazquez and Schedl,
1994
; Dunaway et al.,
1997
; Krebs and Dunaway,
1998
; Parnell and Geyer,
2000
). Surprisingly, this scs fragment behaves differently than
scsmin and leads to opposite gain- and loss-of-function phenotype
depending on its orientation within the context of the Fab-7 region.
The orientation-dependent effect is due to the presence of a promoter
immediately adjacent to the scs insulator. Our results suggest that
transcription through the iab-7 PRE interferes with the maintenance
of a Polycomb repression complex on the iab-7 domain.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA techniques, fly work, antibody staining and in situ
hybridization
DNA techniques, fly work, antibody staining and in situ hybridization have
been described previously (Mihaly et al.,
1997; Hogga et al.,
2001
; Zhou et al.,
1999
). The antibody against ABD-B was kindly provided by Sue
Celniker (Celniker, 1990).
Abdominal cuticles
Adult abdominal cuticles were mounted as described elsewhere
(Mihaly et al., 1997),
examined and photographed on an Axioplan microscope with a 5x lens. Only
half cuticles are shown in Fig.
2. The dorsal surface of each abdominal segment has a rectangular
plate of hard cuticle called the tergite (only half of the tergites are
visible on the left of each panel, as well as the genitalia at the bottom).
The ventral surface of abdominal segments is composed of soft cuticle called
the pleura. On the ventral midline of the second (A2) and more posterior
segments, there are small plates of harder cuticle called sternites. In males
only six abdominal segments are visible. The tergites on A5 and A6 are
pigmented and can be therefore distinguished from more anterior tergites. On
the ventral side, the sixth sternites can be distinguished from the more
anterior sternites by its different shape and by the absence of bristles.
Homeotic transformations associated with Fab-7 are best visible in
males where most (Fab-72) or all of A6
(Fab-71) is missing (see
Fig. 2B). As A7 and A8 do not
contribute to any visible cuticle after metamorphosis in males, homeotic
transformations associated with Fab-8 are detected in females where
A7 develops as a smaller segments than the anterior segments (see
Fig. 2C).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Fig. 2A shows the phenotype
observed in homozygous males in which the promscs construct
replaces Fab-7. In these flies, A3 to A6 were transformed into a
mixture of A2-A3 identity, indicating that iab-3 through
iab-6 are affected by the promscs element. In prior
experiments, we have shown that replacement of Fab-7 by the minimal
scs insulator (scsmin) in both orientations results in a consistent
phenotype in which iab-5 and iab-6 are prevented from
interacting with Abd-B by the intervening insulator
(Hogga et al., 2001). Thus,
the extra 282 bp DNA element appeared to interfere at a distance with
iab-3 and iab-4. Interference with iab-3 and
iab-4 functions in promscs is surprising, because these
cis-regulatory domains regulate abd-A (see
Fig. 5A) and are distant from
promscs. This result implies that, promscs exerts a
negative polar effect that can spread 40 kb away into iab-3. In
addition, we have previously provided evidence that, upon insulation from
Abd-B by the intervening scsmin insulator, iab-5
is targeted instead to the abd-A gene, which it activates in a
pattern appropriate for specifying a A5-like identity (see
Hogga et al., 2001
). In
promscs, A5 is transformed into A2-A3, indicating that the negative
polar effect exerted by promscs also affects iab-5/abd-A
interaction. This phenotype was seen in four independent conversion lines and
a whole genome Southern analysis has verified that there are no large
rearrangements affecting the iab-3 through iab-5 regions of
the promscs chromosome. Finally, as heterozygous flies are wild
type, the negative polar effect of promscs on iab-3
through iab-5 is only acting in cis.
|
Fig. 2B shows the phenotype observed in homozygous males in which the same scs fragment replaces Fab-7 in the opposite orientation (scsprom). Instead of observing the loss-of-function phenotype described above, where A3, A4, A5 and A6 are transformed to a more anterior segment, we found a gain-of-function phenotype in which A6 took the identity of a more posterior segment, A7. This is similar to removal of Fab-7 entirely. Because in these conversions we removed Fab-7 by introducing the Fab-72 deletion, we confirmed by sequencing that scsprom is intact. Moreover, the same phenotype is observed in the five other independent conversion events that we recovered. The dominant gain-of-function phenotype associated with scsprom was confirmed by the observation that heterozygotes (scsprom/+) displayed the same phenotype, although not as severe. Because the dominant gain of function associated with scsprom was absent from the flies in which Fab-7 was replaced by scsmin, we conclude that the same extra 282 bp fragment (MluI-PstI) is responsible for the loss-of-function phenotype in promscs and the dominant gain-of-function phenotype in scsprom.
An anti-PRE at the edge of scsprom
Closer examination of the males shown in
Fig. 2B revealed that the
phenotype of scsprom flies was slightly different from the
phenotype generated by the Fab-72 deletion alone. As
mentioned in the Introduction, class 2 mutations such as
Fab-72, which remove only the boundary and leave an intact
iab-7PRE, caused a mixed gain- and loss-of-function phenotype: most
cells of A6 adopted A7 identity while the remaining adopted A5 identity (see
Fig. 2B). If the
scsprom construct had no effect on the region, we should have
observed a Fab-72 phenotype simply because of the removal
of the Fab-7 element. Fig.
2B shows that this was not the case: A6 was completely transformed
into A7 in homozygous scsprom flies. This phenotype is identical to
the phenotype of the class I Fab-712 allele where the
boundary and nearby iab-7PRE are deleted
(Fig. 2B). Thus, introduction
of the scsprom element converts a class II allele into a class I
allele, as if the extra 282 bp fragment (MluI-PstI)
interfered with the activity of the nearby iab-7 PRE. To test this
hypothesis, we decided to verify how scsprom affected PRE-mediated
pairing-sensitive repression of a miniwhite reporter construct (see
Introduction). Although transformants with the scs element in the
promscs orientation are pairing sensitive in 63% of the lines, when
the scs element is in the opposite orientation (scsprom) the
pairing-sensitive frequency decreases to 11%
(Fig. 1). Thus, our hypothesis
that scsprom within the BX-C contains an anti-PRE activity is
supported by these ectopic constructs. To localize this anti-PRE activity, we
analyzed the pairing-sensitivity of scsprom derivatives in which
the scsprom fragment was progressively shaved from one end (see
Fig. 1). A deletion removing
282 bp from the end (MluI-PstI deletion; scsmin)
restored pairing-sensitivity to a frequency of 47%. Because a deletion that
extended further towards the HpaI site did not significantly increase
the proportion of pairing-sensitive lines (50%), we conclude that most of the
anti-PRE element of scs is located within the MluI-PstI
fragment.
The anti-PRE associated with scsprom interferes with
iab-8 silencing
Examination of homozygous scsprom females indicated that
iab-8 was also partially activated in the sixth and seventh abdominal
segments, both of which show an identity intermediate between A7 and A8, a
phenotype that is reminiscent of Fab-8 boundary deletions
(Fig. 2C) (Barges et al.,
2001). These observations suggest that the anti-PRE activity contained in
scsprom is not only interfering with the functioning of the nearby
iab-7 PRE, but spreads across the whole iab-7 domain and
reaches iab-8.
The gain-of-function effect associated with scsprom is
post-embryonic
We also examined the Abd-B expression pattern in embryos
(Fig. 3). In scsmin,
the partial block between iab-5/iab-6 and the Abd-B promoter
was visualized by the great reduction of Abd-B expression in PS10 and
PS11 (Hogga et al., 2001). In
scsprom, because of the posterior transformation of A6 into A7, we
were expecting to monitor a reiteration of the PS12/A7 Abd-B
expression pattern in PS11/A6 (Galloni et
al., 1993
). This is not what we found. In PS10 and PS11, the
expression patterns were similar to the patterns detected in
scsmin. In PS12, the expression pattern was much lower than in wild
type and was similar to the pattern detected in PS11. In fact, this expression
pattern resembled that of an iab-7 deletion mutant
(Galloni et al., 1993
). This
should lead to a transformation of PS12 into PS11, exactly the opposite of the
phenotype that we observe in adults. Unfortunately, there are no
distinguishing morphological landmarks in embryos and larvae that definitively
identify these two parasegments earlier in development. It is not clear why,
in the adult, the readout of scsprom results in a gain-of-function
phenotype, whereas in the embryo, Abd-B expression resembles loss of
function. We believe these findings indicate that iab-7 misexpression
is more pronounced in the pupa, when the adult structures are forming,
strongly supporting the idea that scsprom affects primarily the
maintenance phase. Bender and Fitzgerald have described similar mutations that
affect the maintenance phase of iab-2 silencing in PS6/A1
(Bender and Fitzgerald, 2002
).
In their case, the identity of the affected abdominal segment PS6/A1 could be
easily recognized in embryos and larvae from PS7/A2. Intriguingly, the
dominant gain-of-function phenotype associated with their mutations was also
only detectable in adult.
|
The region responsible for the orientation-dependent effect contains
a promoter
We have previously shown that scs contains a chromatin insulator element
(scsmin) that is able to interfere with long-range
enhancer-promoter interactions (Hogga et
al., 2001). In addition to the insulator, the 1.2 kb scs fragment
contains at one extremity an element that, in conjunction with
scsmin, is able to induce a gain-of or loss-of-function phenotype
when replacing Fab-7. When facing iab-7, this element
destabilizes iab-7 and iab-8 silencing in A6. However, when
facing iab-6, this element exerts a negative polar effect on
iab-5, iab-4 and iab-3. These two gain- and loss-of-function
effects are difficult to reconcile. Previously, Avramova and Tikhonov
(Avramova and Tikhonov, 1999
)
discovered that scs contained a promoter and challenged the idea that scs was
a neutral chromatin domain boundary. Their finding supported the promoter
decoy hypothesis of Geyer (Geyer,
1997
), in which insulation is achieved by a promoter-trapping
mechanism. Although the promoter described by Avramova and Thikonov (Avramova
and Thikonov, 1999) maps to the other side of scs (see
Fig. 1), promoter trapping
could account for the polar effect on iab-5, iab-4 and
iab-3. In this scheme, insertion of a promoter at the iab-6
edge of Fab-7 would attract the nearby cis-regulatory
elements and divert them from their normal abd-A and/or
Abd-B promoter (see Fig.
5A). In this case, transcription of sequences near the promoter
should be detected in parasegments affected by promscs (and
scsprom). In order to test this hypothesis, we performed
whole-mount in situ hybridization on embryos of the promscs and
scsprom lines. We synthesized strand-specific probes from the
iab-6 (probe A; Fig.
4) or iab-7 DNA (probe B;
Fig. 4) flanking the scs
element. Using both probes on promscs, scsprom and
scsmin lines, we found that the promoter described by Avramova and
Thikonov (Avramova and Thikonov, 1999) remained silent in the context of the
BX-C. There was, however, a promoter that became active in the context of the
BX-C at the other side of the scs fragment.
Fig. 4 shows the results
obtained with a probe from the iab-6 side of promscs
(probe A). In wild type (or Fab-72), transcription was
detected from a very early stage throughout embryogenesis in PS13 and PS14.
The expression profile of this transcript was reminiscent of a transcript that
originates from a promoter localized just downstream from the Abd-B
transcription unit (Zhou et al.,
1999
). In promscs embryos, however, we detected
additional transcripts in PS10/A5, PS11/A6 and PS12/A7. Interestingly, we
failed to detect these transcripts when the truncated version of
promscs (scsmin) replaced Fab-7, indicating
that the MluI-PstI fragment contains the promoter (or
sequences necessary for its activity). Appearance of PS10-specific transcripts
may explain the polar effect of promscs on iab-5 if we
assume that iab-5 activity is trapped by the scs promoter and thus
diverted from the abd-A promoter [in the promscs context,
iab-5 is insulated from Abd-B by the intervening
scsmin insulator, see Hogga et al.
(Hogga et al., 2001
)]. A
similar mechanism would explain the polar effect on iab-4 and
iab-3 (Fig. 5A).
However, we failed to detect transcription in PS9 and PS8, where both
regulatory domains are active (Fig.
4). The discrepancy between the observed pattern of embryonic
transcription and the adult phenotype may reflect a higher affinity of the
promoter to iab-3,4 in adults.
|
It is not entirely clear which regulatory domain activates transcription in
PS12. In the promscs context, the scs insulator is located between
the promoter and the iab-7 cis-regulatory domain. As reported by
Hogga et al. (Hogga et al.,
2001), the insulator alone does not completely impair interactions
between the distal iab-5/iab-6 cis-regulatory domains and their
Abd-B target promoter (see also
Fig. 3). Thus, in
promSCS, iab-7 may not be completely insulated from the
promoter and activates transcription in PS12. As an alternative explanation we
believe that transcription in PS12 in promscs embryos results from
activation by the more anterior cis-regulatory domains
(iab-3, iab-4, iab-5 or iab-6), which once
activated in a given parasegment, remain active in the more posterior
parasegments, as first proposed in Ed Lewis' model
(Lewis, 1978
). It should be
noted that although promoter-trapping is an attractive explanation accounting
for the polar effect of promscs on the iab-3, iab-4,
iab-5 and iab-6, we cannot exclude the possibility that
transcription through these cis-regulatory domains is the cause of their
inactivation.
Transcription through the iab-7PRE may interfere with
iab-7 silencing
In scsprom, the edge of scs containing the promoter is facing
the iab-7 cis-regulatory domain. Not surprisingly, we detected
intense transcription in PS12 with a probe from the iab-7 edge (probe
B, Fig. 4). The same probe
failed to detect any transcript in wild type or in embryos in which
Fab-7 is replaced by scsmin. These results indicate that
the PS12-specific transcript very likely originates from the same promoter
that is firing in PS10-12 in promscs. We also detected equally
intense transcription in PS11. Thus, the fragment harboring anti-PRE activity
when associated with scsprom, contains a promoter that fires
transcription across the iab-7PRE with which it interferes. As
mentioned above, the anti-PRE activity contained in scsprom not
only interferes with the functioning of the nearby iab-7PRE, but
spreads across the whole iab-7 domain and reaches iab-8.
Notably, the same transcription pattern in PS11 and PS12 is observed with a
strand-specific probe originating from iab-8 (data not shown, see
Fig. 4).
Fig. 5B suggests how the
transcription from scsprom might cause the posterior transformation
in the sixth and seventh abdominal segments (PS11 and PS12). The promoter in
scsprom is activated in PS11 and PS12
(Fig. 4), sending transcripts
across the iab-7 and iab-8 regulatory regions. In PS11, both
of these regulatory regions are normally silent, but the act of transcription
apparently reverses the silencing, causing the cells in PS11 to differentiate
in the same way as those of PS12 or PS13. Likewise, in PS12, transcription
across the iab-8 region activates it, transforming PS12 cells towards
PS13 character. We do not observe transcription from scsprom in
PS13. In this parasegement, however, the iab-8 promoter is activated,
giving rise to leftwards transcription
(Zhou et al., 1999). It is
possible that this leftwards transcription interferes with rightwards
transcription from scsprom. It is perhaps surprising that the
transcription from scsprom begins in PS11, as the PS12-specific
regulatory region (iab-6) is separated from the promoter of
scsprom by the scs insulator. However, the insulator in an
scsmin conversion at the same site does not completely insulate
iab-6 from the Abd-B target promoter
(Hogga et al., 2001
) (see also
Fig. 3), making it likely that
iab-6 can activate the scsprom across the insulator. It is
also possible that the function of an insulator depends on neighboring
sequences. If, for example, insulator function is enhanced by a nearby PRE,
then partial loss of the iab-7PRE function might weaken the
scsprom insulator.
There are precedents where transcription has been suggested to play a role
in chromatin remodeling. For example, the human ß-globin locus is
subdivided into three chromatin domains, each of which become more accessible
to nuclease digestions upon gene activation
(Ashe et al., 1997;
Gribnau et al., 2000
;
Plant et al., 2001
).
Interestingly, large intergenic transcripts delineate each of these domains
and chromatin remodeling of each domain is preceded by its transcription.
Another example has been reported by Rank et al.
(Rank et al., 2002
). Using a
transgenic context, they found that transcription across a PRE could interfere
with silencing. Finally, in the accompanying paper, Fitzgerald and Bender
provide evidence that transcription across the iab-2 cis-regulatory
domains in PS6/A1 interferes with iab-2 silencing, resulting in the
posterior transformation of PS6/A1 into PS7/A2
(Bender and Fitzgerald, 2002
).
In this case, the identity of the affected abdominal segment can easily be
recognized in embryos and larvae. Despite the existence of intense
transcription of iab-2 in embryos, the dominant gain-of-function
phenotype associated with iab-2 misexpression is only detectable in
the adult. Thus, as in our case, transcription across the iab
regulatory regions appear to interfere with silencing during the late
maintenance phase, when the adult structures are forming.
If transcription can interfere with Pc-G silencing, what are the mechanisms
responsible for this activity? Factors that affect RNA polymerase II (RNAPII)
transcript elongation have been shown to have an effect on chromatin. For
example, it has been suggested that histone acetyl transferases (HAT) such as
PCAF (Cho, 1998) or ELP3 (Wittschieben, 1999) assist RNAPII in
relieving inhibition caused by nucleosome arrays. Although active chromatin
requires acetylation of specific lysine residues in the H3 and/or H4 histone
tails (Moazed, 2001), the
recent purification of Pc complexes suggests that histone deacetylation is
required for establishing a stable long-term Pc-G silencing complex
(Saurin et al., 2001
;
Tie et al., 2001
). In the case
of scsprom, perhaps the frequent passage of RNAPII and its
associated histone acetylation activities though the PREs interferes with the
assembly of the Pc-G silencing. Involvement of acetylated histones in
antagonizing PcG-dependent silencing is supported by the findings of Cavalli
and Paro (Cavalli and Paro,
1999
) showing that high levels of acetylated histone H4 are
associated with non-repressive PRE sequences. Alternatively, it has been
recently found that variant histone H3.3 was deposited on active chromatin
during transcription, providing a mechanism for the immediate activation of
genes that are silenced by histone modification
(Ahmad and Henikoff, 2002
). It
may be possible that transcription across iab-7 (and also
iab-8) results in deposition of new nucleosome marked by H3.3,
interfering thereby with the maintenance of silencing by the Pc-G complex.
Concluding remarks
It has been known for a long time that the large cis-regulatory
regions of the bithorax complex are transcribed
(Lipshitz et al., 1987;
Sanchez-Herrero and Akam,
1989
; Cumberledge et al.,
1990
). In blastoderm stage embryos, the iab-2 though
iab-8 regions can be divided into three domains, each transcribed in
a region that extends from a specific anterior limit to the posterior limit of
the segmented part of the embryo. These domains are only broadly defined but
their order on the chromosome reflects the anterior limit of expression for
each of them. In the light of our data, it is tempting to speculate that
transcription of the iab domains convey a regulatory signal,
preventing assembly of the Polycomb-repressing complex on the
iab domains that need to remain active. If this is true, transcripts
should appear in the anteriormost parasegments/segments where each
cis-regulatory domain in activated. However, so far, we have not seen
transcripts in every regulatory region, which would account for the sequential
activation of each regulatory domain. Moreover, this model predicts that the
iab-7 PRE and iab-7 domains should be transcribed from PS12,
where iab-7 is first active. So far transcripts across the
iab-7 domain have only been detected in PS13 and 14 (this study)
(Zhou et al., 1999
). Thus, it
remains unclear whether intergenic transcription plays a role in wild-type
animals to create and/or maintain open chromatin, or whether the existence of
intergenic transcripts is the consequence of an open structure. However, our
experiments strongly suggest that forced transcription through an inactive
cis-regulatory domain interferes with the maintenance of silencing,
highlighting an incompatibility between transcription and Pc-G mediated
silencing. This activity probably reflects a fundamental mechanism to protect
an actively transcribed gene from being inactivated by the Pc-G proteins that
are present in all cells.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmad, K. and Henikoff, S. (2002). The histone variant H3.3 marks chromatin by replication-independent nucleosome assembly. Mol. Cell 9,1191 -1200.[Medline]
Ashe, H. L., Monks, J., Wijgerde, M., Fraser, P. and Proudfoot,
N. J. (1997). Intergenic transcription and transinduction of
the human beta-globin locus. Genes Dev.
11,2494
-2509.
Avramova, Z. and Tikhonov, A. (1999). Are scs and scs' `neutral' chromatin domain boundaries of the locus? Trends Genet. 15,138 -139.[CrossRef][Medline]
Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Müller,
M., Shanower, G., Schedl, P., Gyurkovics, H. and Karch, F.
(2000). The Fab-8 boundary defines the distal limit of
the bithorax complex iab-7 domain and insulates iab-7 from
initiation elements and a PRE in the adjacent iab-8 domain.
Development 127,779
-790.
Bell, A. C., West, A. G. and Felsenfeld, G.
(2001). Insulators and boundaries: versatile regulatory elements
in the eukaryotic genome. Science
291,447
-450.
Bender, W. and Fitzgerald, D. P. (2002).
Transcription activates repressed domains of the Drosophila bithorax
complex. Development
129,4923
-4930.
Bender, W. and Hudson, A. (2000). P element
homing to the Drosophila bithorax complex. Development
127,3981
-3992.
Busturia, A. and Bienz, M. (1993). Silencers in abdominal-B, a homeotic Drosophila gene. EMBO J. 12,1415 -1425.[Abstract]
Cavalli, G. and Paro, R. (1999). Epigenetic
inheritance of active chromatin after removal of the main transactivator.
Science 286,955
-958.
Celniker, S. E., Sharma, S., Keelan, D. J. and Lewis, E. B. (1990). The molecular genetics of the bithorax complex of Drosophila: cis-regulation in the Abdominal-B domain. EMBO J. 9,4277 -4286.[Abstract]
Chan, C. S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J. 13,2553 -2564.[Abstract]
Chiang, A., O'Connor, M. B., Paro, R., Simon, J. and Bender,
W. (1995). Discrete Polycomb-binding sites in each
parasegmental domain of the bithorax complex.
Development 121,1681
-1689.
Cho, H., Orphanides, G., Sun, X., Yang, X. J., Ogryzko, V.,
Lees, E., Nakatani, Y. and Reinberg, D. (1998). A human RNA
polymerase II complex containing factors that modify chromatin structure.
Mol. Cell. Biol. 18,5355
-5363.
Cumberledge, S., Zaratzian, A. and Sakonju, S. (1990). Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex. Proc. Natl. Acad. Sci. USA 87,3259 -3263.[Abstract]
Dunaway, M., Hwang, J. Y., Xiong, M. and Yuen, H. L. (1997). The activity of the scs and scs' insulator elements is not dependent on chromosomal context. Mol. Cell. Biol. 17,182 -189.[Abstract]
Duncan, I. (1987). The bithorax complex. Annu. Rev. Genet. 21,285 -319.[CrossRef][Medline]
Galloni, M., Gyurkovics, H., Schedl, P. and Karch, F. (1993). The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 12,1087 -1097.[Abstract]
Gerasimova, T. I. and Corces, V. G. (1996). Boundary and insulator elements in chromosomes. Curr. Opin. Genet. Dev. 6,185 -192.[CrossRef][Medline]
Geyer, P. K. (1997). The role of insulator elements in defining domains of gene expression. Curr. Opin. Genet. Dev. 7,242 -248.[CrossRef][Medline]
Geyer, P. K. and Corces, V. G. (1992). DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6,1865 -1873.[Abstract]
Gindhart, J. G., Jr and Kaufman, T. C. (1995).
Identification of Polycomb and trithorax group responsive elements in the
regulatory region of the Drosophila homeotic gene Sex combs reduced.
Genetics 139,797
-814.
Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. and Fraser, P. (2000). Intergenic transcription and developmental remodeling of chromatin subdomains in the human beta-globin locus. Mol Cell 5,377 -386.[Medline]
Gyurkovics, H., Gausz, J., Kummer, J. and Karch, F. (1990). A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation. EMBO J. 9,2579 -2585.[Abstract]
Hagstrom, K., Muller, M. and Schedl, P. (1997).
A Polycomb and GAGA dependent silencer adjoins the Fab-7
boundary in the Drosophila bithorax complex.
Genetics 146,1365
-1380.
Hogga, I., Mihaly, J., Barges, S. and Karch, F. (2001). Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex. Mol. Cell 8,1145 -1151.[Medline]
Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I. Celniker, S., Crosby, M. and Lewis, E. B. (1985). The abdominal region of the bithorax complex. Cell 43, 81-96.[Medline]
Kassis, J. A., VanSickle, E. P. and Sensabaugh, S. M.
(1991). A fragment of engrailed regulatory DNA can mediate
transvection of the white gene in Drosophila. Genetics
128,751
-761.
Kellum, R. and Schedl, P. (1991). A position-effect assay for boundaries of higher order chromosomal domains. Cell 64,941 -950.[Medline]
Kellum, R. and Schedl, P. (1992). A group of scs elements function as domain boundaries in an enhancer- blocking assay. Mol. Cell. Biol. 12,2424 -2431.[Abstract]
Krebs, J. E. and Dunaway, M. (1998). The scs and scs' insulator elements impart a cis requirement on enhancer-promoter interactions. Mol Cell 1, 301-308.[Medline]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[Medline]
Lipshitz, H. D., Peattie, D. A. and Hogness, D. S. (1987). Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev. 1, 307-322.[Abstract]
Martin, C. H., Mayeda, C. A., Davis, C. A., Ericsson, C. L., Knafels, J. D., Mathog, D. R., Celniker, S. E., Lewis, E. B. and Palazzolo, M. J. (1995). Complete sequence of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92,8398 -8402.[Abstract]
Mihaly, J., Hogga, I., Gausz, J., Gyurkovics, H. and Karch,
F. (1997). In situ dissection of the Fab-7 region of the
bithorax complex into a chromatin domain boundary and a Polycomb-response
element. Development
124,1809
-1820.
Mihaly, J., Hogga, I., Barges, S., Galloni, M., Mishra, R. K., Hagstrom, K., Muller, M., Schedl, P., Sipos, L., Gausz, J., Gyurkovics, H. and Karch, F. (1998). Chromatin domain boundaries in the Bithorax complex. Cell. Mol. Life Sci. 54, 60-70.[CrossRef][Medline]
Moazed, D. (2001). Common themes in mechanisms of gene silencing. Mol. Cell 8, 489-498.[Medline]
Muller, J. and Bienz, M. (1991). Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo. EMBO J. 10,3147 -3155.[Abstract]
Muller, J. and Bienz, M. (1992). Sharp anterior boundary of homeotic gene expression conferred by the fushi tarazu protein. EMBO J. 11,3653 -3661.[Abstract]
Muller, M., Hagstrom, K., Gyurkovics, H., Pirrotta, V. and
Schedl, P. (1999). The mcp element from the Drosophila
melanogaster bithorax complex mediate long-distance regulatory interactions.
Genetics 153,1333
-1356.
Parnell, T. J. and Geyer, P. K. (2000).
Differences in insulator properties revealed by enhancer blocking assays on
episomes. EMBO J. 19,5864
-5874.
Paro, R. (1990). Imprinting a determined state into the chromatin of Drosophila. Trends Genet. 6, 416-421.[CrossRef][Medline]
Peifer, M., Karch, F. and Bender, W. (1987). The bithorax complex: control of segmental identity. Genes Dev. 1,891 -898.
Pirrotta, V. and Rastelli, L. (1994). White gene expression, repressive chromatin domains and homeotic gene regulation in Drosophila. BioEssays 16,549 -556.[Medline]
Plant, K. E., Routledge, S. J. and Proudfoot, N. J.
(2001). Intergenic transcription in the human beta-globin gene
cluster. Mol. Cell. Biol.
21,6507
-6514.
Poux, S., Kostic, C. and Pirrotta, V. (1996). Hunchback-independent silencing of late Ubx enhancers by a Polycomb Group Response Element. EMBO J. 15,4713 -4722.[Abstract]
Qian, S., Capovilla, M. and Pirrotta, V. (1991). The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes. EMBO J. 10,1415 -1425.[Abstract]
Rank, G., Prestel, M. and Paro, R. (2002). Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigematic switch. Mol. Cell. Biol. (in press).
Roseman, R. R., Pirrotta, V. and Geyer, P. K. (1993). The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J. 12,435 -442.[Abstract]
Sanchez-Herrero, E. (1991). Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos. Development 111,437 -449.[Abstract]
Sanchez-Herrero, E. and Akam, M. (1989). Spatially ordered transcription of regulatory DNA in the bithorax complex of Drosophila. Development 107,321 -329.[Abstract]
Sanchez-Herrero, E., Vernos, I., Marco, R. and Morata, G. (1985). Genetic organization of Drosophila bithorax complex. Nature 313,108 -113.[Medline]
Saurin, A. J., Shao, Z., Erdjument-Bromage, H., Tempst, P. and Kingston, R. E. (2001). A Drosophila Polycomb group complex includes Zeste and dTAFII proteins. Nature 412,655 -660.[CrossRef][Medline]
Shimell, M. J., Simon, J., Bender, W. and O'Connor, M. B. (1994). Enhancer point mutation results in a homeotic transformation in Drosophila. Science 264,968 -971.[Medline]
Simon, J. A. and Tamkun, J. W. (2002). Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev. 12,210 -218.[CrossRef][Medline]
Simon, J., Chiang, A., Bender, W., Shimell, M. J. and O'Connor, M. (1993). Elements of the Drosophila bithorax complex that mediate repression by Polycomb group products. Dev. Biol. 158,131 -144.[CrossRef][Medline]
Simon, J., Peifer, M., Bender, W. and O'Connor, M. (1990). Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis. EMBO J. 9,3945 -3956.[Abstract]
Sun, F. L. and Elgin, S. C. (1999). Putting boundaries on silence. Cell 99,459 -462.[Medline]
Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E. and Harte, P.
J. (2001). The Drosophila Polycomb Group proteins ESC and
E(Z) are present in a complex containing the histone-binding protein p55 and
the histone deacetylase RPD3. Development
128,275
-286.
Vazquez, J. and Schedl, P. (1994). Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions. EMBO J. 13,5984 -5993.[Abstract]
Wittschieben, B. O., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., Li, Y., Allis, C. D., Tempst, P. and Svejstrup, J. Q. (1999). A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell 4,123 -128.[Medline]
Zhou, J., Ashe, H., Burks, C. and Levine, M.
(1999). Characterization of the transvection mediating region of
the abdominal- B locus in Drosophila. Development
126,3057
-3065.