Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA
* Author for correspondence (e-mail: pschedl{at}molbio.princeton.edu)
Accepted 8 July 2004
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SUMMARY |
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Key words: Fab-7, Drosophila, Bithorax, Boundary, Insulator, Abd-B
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Introduction |
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Several constitutively active boundary elements have been found in the
Drosophila Antennapedia (ANT-C) and bithorax (BX-C) complexes, and
these elements are crucial for the developmental functions of the homeotic
genes in each complex (Gyurkovics et al.,
1990; Mihaly et al.,
1998
; Zhou and Levine,
1999
; Barges et al.,
2000
; Belozerov et al.,
2003
). BX-C contains three homeotic genes, Ultrabithorax
(Ubx), abdominal-A (abd-A) and Abdominal-B
(Abd-B), which are responsible for specifying segment identity in the
posterior parasegments (PS) 5-14 of the fly. Parasegment identity depends upon
which of these homeotic genes is activated and upon its precise pattern of
expression. Transcriptional activity is controlled by a large
300 kb
cis-regulatory region that is subdivided into nine parasegment
specific cis-regulatory domains: abx/bx, bxd/pbx and
iab2-iab8 (Duncan,
1996
; Mihaly et al.,
1998
). Each cis-regulatory domain directs the expression of one of
the BX-C homeotic genes in a pattern appropriate for specifying a particular
parasegment. The domains are sequentially activated going from anterior to
posterior parasegments. For example, in PS11, Abd-B expression is
controlled by the iab-6 domain. Although iab-6 is active in
this parasegment, the adjacent domain, iab-7 and its neighbor
iab-8, are silenced. In PS12, iab-7 is activated and it
directs Abd-B expression, while iab-8 remains silent. The
activity state of the BX-C cis-regulatory domains is set early in
embryogenesis by the products of the gap and pair-rule genes. The gap and
pair-rule gene products are only transiently expressed and by stage 11 of
embryogenesis, BX-C regulation switches from the initiation to the maintenance
phase. Maintenance depends upon trithorax group (trxG) and
Polycomb group (PcG) genes
(Simon, 1995
;
Simon and Tamkun, 2002
). Genes
in the trx group are required to maintain the homeotic genes in their
active state, whereas genes in the Pc group function to silence
homeotic gene expression.
The most thoroughly characterized of the BX-C boundary elements is
Fab-7. It is located in between the iab-6 and iab-7
cis-regulatory domains (Fig.
1A) and, like the other boundaries in BX-C, it functions to ensure
the genetic autonomy of the two flanking cis-regulatory domains.
Mutations that inactivate Fab-7 lead to the fusion of the
iab-6 and iab-7 domains, and this disrupts the specification
of PS11 (Gyrukovics et al., 1990; Galloni
et al., 1993; Mihaly et al.,
1997
). In most Fab-7 mutant PS11 cells, positive
regulatory elements in iab-6 inappropriately activate the iab-7
cis-regulatory domain. As a consequence, Abd-B expression in
these cells is driven by iab-7 not iab-6, and they assume a
PS12 identity. In the remaining mutant PS11 cells negative elements in
iab-7 inappropriately silence iab-6 (and iab-7).
When iab-6 is silenced Abd-B expression is driven by
iab-5 and the cells assume a PS10 identity.
|
The minimal Fab-7 boundary defined in the ftz:hsp70-lacZ
and wEN:mini-white enhancer blocking assays is 1.2 kb in
length. As shown in Fig. 1A, it
extends from the minor nuclease hypersensitive site (*) on the
proximal side to the iab-7 PRE (which corresponds HS3)
(Hagstrom et al., 1997;
Mishra et al., 2001
) on the
distal side and includes two major chromatin-specific nuclease hypersensitive
regions, HS1 and HS2 (Karch et al.,
1994
). The largest hypersensitive region, HS1, contains six
consensus GAGA factor binding sites arranged in three pairs, 1-2, 3-4 and 5-6.
The ubiquitously expressed GAGA factor is encoded by the
Trithorax-like (Trl) gene
(Farkas et al., 1994
), and it
is thought to function in the formation and/or maintenance of the nucleosome
free regions of chromatin associated with a variety of cis-acting
elements in flies, including enhancers, promoters, Polycomb Response Elements
(PRES) and boundaries (Lehmann,
2004
). Chromatin immunoprecipitation experiments demonstrate that
GAGA is associated with the Fab-7 boundary in vivo
(Strutt et al., 1997
).
Moreover, the GAGA-binding sites in HS1 are important for boundary function.
In previous studies, we found that the enhancer blocking activity of the
minimal 1.2 kb boundary is compromised in both the embryo and adult when GAGA
sites 1-5 are mutated (Schweinsberg et
al., 2004
). Although this finding indicates that GAGA (or another
protein that recognizes the GAGA consensus) is required for Fab-7
boundary activity throughout development, the GAGA sites are not functionally
equivalent. We found that when only the centromere proximal pair, 1-2, are
mutated, blocking of the ftz UPS stripe enhancer in the ectoderm of
early embryos by the minimal Fab-7 boundary is weakened, but there is
no apparent effect on the blocking of either the ftz NE enhancer in
the CNS of older embryos or the w enhancer in adults. By contrast,
mutation of the central pair, 3-4, weakens blocking of the w enhancer
in the eye, but has little effect on the blocking of the ftz
enhancers in embryos.
One interpretation of these results is that the constitutive boundary activity of the Fab-7 element is generated by sub-elements whose activities are developmentally restricted. In the studies reported here, we have tested this hypothesis. We show that, unlike other well characterized boundaries, the constitutive activity of Fab-7 is generated by combining a series of subelements that function at different stages of development. This unexpected finding indicates that chromatin domains are not always static units, but instead may be redefined by inactivating or activating a boundary element such that the chromatin domain can expand to include new genes or regulatory sequences, or alternatively contract eliminating genes or regulatory sequences.
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Materials and methods |
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Results |
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Although all three deletions share the same distal breakpoint, they differ
in their proximal breakpoints. The DNA segment removed in the largest
deletion, P14.1, is more than 1 kb in length
(Fig. 1B) and it corresponds
closely to the minimal Fab-7 boundary defined in transgene assays. By
contrast, the P6.1 and P18.1 deletions are only 510 bp and
594 bp, respectively. As both of the smaller deletions still have HS1 GAGA
sites 1-2, as well as more proximal Fab-7 sequences, we wondered
whether they retained an ability to block the iab-6 cis-regulatory
domain from activating the Ubx-lacZ reporter in early embryos. As
this earlier time point had not been examined in the studies of Mihaly et al.
(Mihaly et al., 1997), we
decided to compare the pattern of ß-galactosidase expression in germband
extended blt embryos and in the various deletion mutant embryos. As
illustrated by representative embryos in
Fig. 1B, the anterior limit of
ß-galactosidase expression in P6.1 and P18.1 embryos is
PS12, just like the parental blt control that has an intact
Fab-7 boundary. By contrast, the anterior limit of
ß-galactosidase expression in the larger P14.1 deletion is PS11.
As reported by Mihaly et al. (Mihaly et
al., 1997
) for the activity of the Ubx-lacZ reporter in
the CNS, when we examined ß-galactosidase expression in the ectoderm of
older germband retracted embryos, we found that the anterior limit for
P6.1 and P18.1 embryos was PS11, just like the larger
deletion P14.1 (not shown). These findings indicate that the
sequences retained in P6.1 and P18.1, which include the GAGA
site pair 1-2, are sufficient to confer boundary activity during the
initiation phase of BX-C regulation, but not later in development when
regulation has switched to the maintenance mode. It should be pointed out that
in our experiments and those of Mihaly et al., ß-galactosidase expression
in PS11 of germ band retracted P6.1 and P18.1 embryos is not
as robust as it is in the PS14.1 deletion. As all three deletions
have indistinguishable Fab-7 mutant phenotypes in the adult, we
presume that the smaller deletions have lower levels of ß-galactosidase
expression in PS11 in germband retracted embryos because the early boundary
activity of P6.1 and P18.1 is lost gradually rather than
abruptly, perhaps reflecting a depletion of some maternal product.
Proximal HS1 has boundary function in the early embryo but not later in development
The results described above suggest that sequences in the proximal part of
Fab-7 have boundary function during the early stages of
embryogenesis, but not later in embryogenesis or in subsequent stages of
development. To investigate this possibility further, we tested whether
sequences from the large HS1 nuclease hypersensitive region that are retained
in both the P6.1 and P18.1 deletions have boundary activity
in transgene assays. For this purpose, we tetramerized a 235 bp HS1
sub-fragment called pHS1. As illustrated in
Fig. 2, the proximal end of
pHS1 corresponds to the proximal edge of the HS1 nuclease hypersensitive
region while the distal end corresponds to the proximal breakpoint of the 594
bp deletion P18.1. This fragment includes GAGA sites 1-2 plus a 100
bp sequence that is 95% identical between D. melanogaster and D.
virilis (V in Fig.
2). We placed the pHS1x4 tetramer either in the blocking position
in between the ftz enhancers and the hsp70 promoter or, as a
control, in the non-blocking position upstream of the enhancers. Transgenic
ftz:hsp70-lacZ embryos were stained with X-gal and compared with
control transgenes carrying either the minimal 1.2 kb Fab-7 fragment,
five binding sites for the Su(Hw) insulator protein, or a random DNA control
fragment with no enhancer blocking activity
(Fig. 2).
In the ftz:hsp70-lacZ assay (Fig. 2B), the ability of the minimal 1.2 kb Fab-7 boundary to block the stripe (UPS) and the neurogenic (NE) enhancers is intermediate between that of an element containing 5 (Fig. 2C) and 12 (not shown) (see Hagstom et al., 1996) binding sites for the ubiquitously expressed insulator protein, Suppressor of Hairywing, Su(Hw). As illustrated in Fig. 2D, the pHS1x4 tetramer has no effect on ß-galactosidase expression when placed in the non-blocking position (NB pHS1x4) upstream of the two enhancers and the level of ß-galactosidase stripe and CNS expression resembles that observed in the random DNA control. By contrast, in the blocking position, pHS1x4 insulates the hsp70 promoter from the ftz UPS enhancer about as well as the 1.2 kb Fab-7 boundary, and little if any stripe ß-galactosidase expression is observed (Fig. 2D; Table 1). This result indicates that sequences derived from the proximal side of HS1 can confer boundary function during the early stages of embryogenesis. As boundary function is lost in the Fab-7 P6.1 and P18.1 deletions at later stages in embryogenesis, we anticipated that pHS1x4 would be less effective in blocking the ftz NE enhancer in the CNS of germband retracted embryos. This is the case. As shown in Fig. 2D and Table 1, blocking of the NE enhancer by pHS1x4 is reduced compared with the minimal 1.2 kb Fab-7 boundary (Fig. 2C,D; Table 1). In three of the pHS1X4 lines, the level of ß-galactosidase in the CNS is the same as the random DNA control, while in the three other lines it is comparable with (or higher than) the ftz transgene carrying 5 su(Hw)-binding sites in the blocking position.
We also examined the enhancer blocking activity of pHS1x4 in the
wEN:mini-white assay. The minimal 1.2 kb Fab-7
boundary blocks the w enhancer when interposed between the enhancer
and w promoter; however, unlike other fly boundaries, including the
BX-C boundary Fab-8, Fab-7 blocking activity in this assay is
sensitive to chromosomal position effects and is observed in only 50% of
the lines (Hagstrom et al.,
1996
). The reason for this position dependence is not currently
understood. Unlike the minimal boundary, pHS1x4 has no apparent boundary
function in the wEN:mini-white assay and out of
almost 40 independent transgenic lines, only two were classified as `blocking'
(Fig. 3). This result is
consistent with the Fab-7 mutant phenotypes seen in
Fab-7P6.1 and Fab-7P18.1 flies, and
suggests that as was the case in the CNS of germband retracted embryos, pHS1
has little if any boundary function in adults.
|
Distal HS1 has boundary function in the CNS and in adults
We next tested a tetramerized 291 bp fragment that corresponds roughly to
the distal half of HS1 (dHS1) (Fig.
4). Though dHS1x4 blocks the UPS enhancer, its insulating activity
is reduced compared with either pHS1x4 or the 1.2 kb Fab-7 fragment.
Most of the dHS1x4 transgenic lines (Table
1) have blocking activity approximately equivalent to that of
Su(Hw)5 (Fig. 2C,
Fig. 4). However, the boundary
activity of dHS1x4 in the embryonic CNS is close to that of the intact
Fab-7 element and most of the lines show a substantial reduction in
ß-galactosidase expression in the CNS
(Table 1). We also examined the
boundary activity of dHS1x4 in the wEN:mini-white assay.
Although the intact Fab-7 element blocks shows blocking in only about
50% of the lines, blocking is observed in over 80% of the dHS1x4 lines
(Fig. 3). These findings
demonstrate that the distal region of HS1 is able to strongly block the NE and
white enhancers but is significantly compromised in its ability to
block the UPS enhancer.
We next divided dHS1 into a 198 bp fragment, dHS1A, that contains the
central pair of GAGA sites (3-4) and a 114 bp fragment, dHS1B, that contains
the most distal GAGA sites (5-6). The boundary activity of dHS1Ax4 in the
embryo resembles but is not quite as strong as the larger dHS1x4 tetramer.
Thus, unlike dHS1x4, many of the dHS1Ax4 lines exhibit neither UPS nor NE
enhancer blocking activity (Fig.
4). However, dHS1Ax4 retains the very strong w enhancer
blocking activity (91%) observed with dHS1x4
(Fig. 3). These findings map an
element that functions to block the w enhancer to dHS1A. Moreover,
the strong w enhancer blocking activity of dHS1Ax4 would be
consistent with the deleterious effects of mutations in GAGA sites 3-4 on the
boundary activity of the 1.2 kb Fab-7 fragment in the
mini-white assay (Schweinsberg et
al., 2004).
A very different result is obtained for dHS1Bx4. Like pHS1, dHS1Bx4 has little if any enhancer blocking activity in the wEN:mini-white assay. However, in the embryo, dHS1Bx4 exhibits a weak position dependent enhancer blocking activity for both the UPS and NE enhancers (Fig. 4; Table 1). These findings indicate that sequences on the distal edge of HS1 have boundary activity during embryogenesis, but not at later stages.
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Discussion |
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The idea that the constitutive boundary function of Fab-7 depends
upon combining subelements whose activity is developmentally restricted is
supported by our analysis of three Fab-7 deletions generated by
imprecise excisions of blt that retain an intact transposon. The
largest of these, P14.1, removes a DNA segment closely corresponding
to the minimal Fab-7 element defined in enhancer blocking transgene
assays. This deletion has no discernable boundary activity at any stage of
development and the blt Ubz-lacZ reporter is active in PS11 from
early embryogenesis onwards. The two smaller deletions, P6.1 and
P18.1, retain all of the sequences in pHS1 (plus sequences proximal
to pHS1, which are important for the boundary function of the minimal 1.2 kb
Fab-7 element). Because the pHS1 sequence (when multimerized) confers
boundary activity in transgene assays during the early stages of
embryogenesis, one might expect that these two smaller deletions will retain
at least some boundary function in early embryos, and indeed they do. In both
deletions, the anterior limit of Ubx-lacZ expression is initially
PS12 just like wild-type Fab-7. However, as these two deletions lack
sequences on the distal side of HS1 that confer enhancer blocking activity in
the embryonic CNS and the adult eye in transgene assays, they might be
expected to have little boundary function at later stages of development.
Indeed, Mihaly et al. (Mihaly et al.,
1997) and we have found that lacZ expression from the
blt transposon in both deletion mutants spreads into PS11 in germband
retracted embryos. In addition, the Fab-7 mutant phenotype of the two
smaller deletions in adult flies is indistinguishable from that of the larger
deletion. These findings indicate that although functionally autonomous
iab-6 and iab-7 cis-regulatory domains can be
established by the P6.1 and P18.1 mutants, the
Fab-7 boundary sequences remaining in these mutants are unable to
sustain autonomy as development proceeds. This would suggest that the process
of establishing an autonomous domain is not irreversible and that boundary
elements must remain continuously active in order to maintain independent
units of genetic activity. Conversely, the properties of dHS1 or dHS1A would
suggest that functionally independent domains can be established de novo by
activating a previously inactive boundary element.
A number of models could potentially account for the developmentally
restricted activity of the different subelements from Fab-7. One idea
is that the boundary function of each subelement is enhancer and/or promoter
specific. Although we can not exclude this possibility, we note that the
Fab-7 boundary itself shows no evidence of enhancer or promoter
specificity. In transgene assays and also in the context of BX-C itself, the
boundary is able to block a wide range of enhancer-promoter combinations in
many different tissues and cell types from early embryogenesis through to the
adult (Galloni et al., 1993;
Hagstrom et al., 1996
;
Zhou et al., 1996
). Another
idea is that the subelements have target sequences for DNA-binding proteins
and/or accessory factors whose expression or activity is developmentally
restricted. In this model, the boundary function of the pHS1 multimer, the two
deletions P6.1 and P18.1, and perhaps also dHS1B in early
embryos would depend upon factors that are either deposited in the egg during
oogenesis or expressed only in early stages of embryogenesis. In this case,
one would expect that boundary activity would be lost when the complement of
these factors is depleted as the embryo develops. Consistent with the idea
that pHS1 function depends upon maternal factors, we have found that UPS
blocking by the 4xpHS1 multimer is compromised in progeny of mothers
heterozygous for several 3rd chromosome deficiencies (A. DeBourcy, C. Summers
and S.E.S., unpublished). Conversely, because blocking by dHS1 (or dHS1A) is
weak in early embryos, but then becomes stronger, it would be reasonable to
think that its boundary activity depends more crucially upon factors that are
zygotically expressed rather than of maternal origin. In this context, it is
interesting to note that the interval in which the pHS1 subelement is active
as a boundary corresponds roughly to the initiation phase of BX-C regulation,
while it is not active once regulation switches to the maintenance mode. The
converse seems to be true for the dHS1 subelement, which appears to become
activated as BX-C regulation switches from initiation to maintenance.
These overlapping patterns of activity suggest that one reason why Fab-7 might be composed of different subelements is that this would permit the use of boundary factors that are specialized with respect to their interactions with, in one case, initiation phase gap and pair-rule transcription factors, and, in the other case, with the maintenance phase trithorax and Polycomb group proteins. More generally, the fact that the boundary activity of the Fab-7 subelements is developmentally restricted suggests a hitherto unexpected plasticity in boundary function. This plasticity indicates that the activity of some boundary elements is likely to be subject to tissue or stage-specific regulation. If this is the case, the genes and regulatory elements included within a chromosomal domain, which is the unit of autonomous genetic activity, could change from one tissue or stage to the next by turning boundary function on or off. This would afford a novel mechanism of high order genetic regulation.
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ACKNOWLEDGMENTS |
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