|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 28 November 2007
doi: 10.1242/dev.010744
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Biology Department M/S 314, University of Nevada, Reno, 1664 N. Virginia
Street, Reno, NV 89557, USA.
2 College of Osteopathic Medicine of the Pacific, Western University of Health
Sciences, 309 E 2nd Street, Pomona, CA 91766, USA.
3 Biology Department, Harvey Mudd College, 301 Platt Boulevard, Claremont, CA
91711, USA.
* Author for correspondence (e-mail: drewell{at}hmc.edu)
Accepted 10 October 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: cis-regulation, Enhancer, Drosophila, Bithorax, Abdominal-B, Promoter
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; West et al.,
2002). Tissue-specific silencer elements act to repress
transcription in a sub-set of cells where the promoter might otherwise be
activated by an enhancer (Fig.
1A, promoter B) (Drewell et
al., 2000). Neither of these mechanisms prevents the enhancer from
interacting with other promoters. Promoter competition is responsible for the
preferred interaction of an enhancer with a specific promoter when linked to
two or more promoters. In this way the distal `strong' promoter
(Fig. 1A, promoter D)
competitively recruits the enhancer in preference to the proximal `weak'
promoter (Fig. 1A, promoter C).
This is thought to occur via selective recognition of distinct core promoter
elements by the shared enhancer (Butler and
Kadonaga, 2002). However, at the bithorax complex (BX-C) in
Drosophila none of these mechanisms can explain enhancer-promoter
specificity. We present evidence of a fourth mechanism, promoter tethering,
capable of regulating enhancer-promoter specificity.
The BX-C contains over 300 kb of genomic DNA but codes for only three
homeotic transcription factors which pattern the thorax and abdomen:
Ultrabithorax (Ubx), abdominal-A (abd-A)
and Abdominal-B (Abd-B)
(Lewis, 1978;
Martin et al., 1995;
Sanchez-Herrero et al., 1985).
In the case of abd-A and Abd-B, the cis-regulatory DNA
required for accurate spatial and temporal expression during embryonic
development lies in an organized array of genetically defined domains:
infraabdominal (iab) regions, iab2 to iab8
(Fig. 1B)
(Akbari et al., 2006;
Celniker et al., 1990;
Maeda and Karch, 2006;
Sanchez-Herrero, 1991). Each
iab region is thought to contain an enhancer capable of directing
expression in the corresponding abdominal parasegment
(Hogga et al., 2001;
Karch et al., 1985;
Mihaly et al., 2006). For
example, the IAB5 enhancer is located 55 kb 3' of the Abd-B
promoter and 48 kb 5' of the abd-A promoter
(Fig. 1B) but preferentially
directs expression only of Abd-B in presumptive abdominal segment 5
(parasegment 10) (Busturia and Bienz,
1993; Ohtsuki et al.,
1998). By contrast, the IAB2 enhancer is located 18 kb 3' of
the abd-A transcription start site
(Fig. 1B) and interacts only
with the abd-A promoter, directing expression in presumptive
abdominal segment 2 (parasegment 7)
(Shimell et al., 2000).
Disruption of IAB5-Abd-B or IAB2-abd-A interactions disrupts
normal embryonic development and results in homeotic segment transformations
(Busturia and Bienz, 1993;
Karch et al., 1985;
Shimell et al., 2000).
A critical question is how the IAB5 enhancer is selectively recruited only
to the Abd-B promoter. IAB5 has been shown to have a preference for
TATA-containing promoters on synthetic transgenes with exogenous promoters
(Ohtsuki et al., 1998).
However, at the endogenous locus neither of the Abd-B or
abd-A promoters contains a TATA box. We also examined whether the
Abd-B or abd-A promoters contained the other known core
promoter elements: initiator (Inr) or downstream promoter element (DPE) (for a
review, see Butler and Kadonaga,
2002). Neither promoter has 100% matching sequence for the weakly
defined consensus sites for these elements
(Fig. 1C). A weakly defined
consensus site allows for more variation in the nucleotides present at any
particular position within the site. Any variation from a weakly defined site
is indicative that the site is not present. It is, therefore, unlikely that a
promoter competition model accounts for the specific enhancer-promoter
interactions in the BX-C. In order for long-range activation of Abd-B
expression to occur, IAB5 has to bypass at least two known insulator
sequences, Fab-7 and Fab-8, that have enhancer-blocking
activity (Fig. 1B)
(Barges et al., 2000;
Hagstrom et al., 1996;
Zhou et al., 1999;
Zhou et al., 1996). One
characterized mechanism for IAB5 to bypass these insulator elements involves a
novel class of regulatory elements known as the promoter targeting sequences
(PTS). Two PTS elements have been characterized; PTS-7, which is located
adjacent to Fab-8, and PTS-6, which is located adjacent to
Fab-7 (Chen et al.,
2005). The PTS elements are thought to function as
anti-insulators, facilitating the correct promoter-enhancer interactions. In
addition, earlier studies suggested that genomic regions 5' of the
Abd-B promoter may be capable of recruiting enhancers in trans
(Sipos et al., 1998). In this
study we examined whether this 5' upstream region was able to direct
IAB5 to Abd-B in cis. In vivo analysis of transgenes identified a 255
bp DNA element that facilitates IAB5-Abd-B interactions. This
regulatory DNA is located 40 bp 5' of the Abd-B transcription
start site and permits IAB5 to activate a distal Abd-B-CAT
reporter gene in preference to a more proximal abd-A-lacZ
reporter. This element is also sufficient to direct IAB5 to an ectopic
promoter in competition assays. Deletion of this element results in the
redirection of enhancer-promoter interactions on transgenes. We suggest that
the 255 bp cis-regulatory sequence is a promoter-tethering element (PTE)
capable of selectively recruiting enhancers from the iab regions,
including the IAB5 enhancer, to the Abd-B promoter at the endogenous
locus.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was
isolated from genomic DNA as a 1522 bp PCR fragment (48,905-50,427 in
EMBL:DM31961), which includes 294 bp of 5' flanking sequence and 1228 bp
of 3' sequence. The abd-A promoter region was isolated as a 957
bp PCR fragment (152,853-153,810) which includes 538 bp of 5' flanking
sequence and 419 bp of 3' sequence. The IAB5 enhancer
(Ohtsuki et al., 1998) was
isolated as a 1026 bp PCR fragment (104,011-105,037). The IAB2 enhancer
(Shimell et al., 2000) was
isolated as a 1969 bp PCR fragment (171,019-172,988). The PTE was isolated as
a 255 bp PCR fragment that extends from -294 bp to -40 bp relative to the
Abd-B transcription start site. The Fab-8 insulator element was
isolated as a 743 bp PCR fragment (63,627-64,370) and specifically designed to
exclude the adjacent PTS7 element. The
Abd-B
PTE region was isolated as
a 1268 bp PCR fragment (49,159-50,427).
Construction of P-element transgenes
The P-transformation vector used in this study is a modified pCaSpeR and
contains divergently transcribed white, CAT and lacZ
reporter genes (Ohtsuki et al.,
1998). Genomic promoter regions from the Bithorax complex were PCR
amplified using conventional methods and cloned as
AscI-BamHI fragments in a modified pBluescript at the
5' end of either the lacZ or CAT reporter genes
(Calhoun et al., 2002). The
abd-A-lacZ fusion gene was isolated as an
AscI-XbaI fragment and used to replace the
AscI-XbaI lacZ fragment in the pCaSpeR vector. The
Abd-B-CAT fusion gene was isolated as an
AscI-NotI fragment and used to replace the
AscI-NotI CAT fragment in pCaSpeR. The IAB5
enhancer was isolated as a PstI fragment and cloned into the pCaSpeR
vector in the unique PstI site 3' of lacZ. The IAB2
enhancer was isolated as a NotI fragment and cloned into the unique
NotI site 3' of CAT. The Fab-8 insulator
element was isolated as an AscI fragment and cloned into the unique
AscI site between the Abd-A-lacZ and Abd-B-CAT
fusion genes. The previously described 1.6 kb spacer from bacteriophage
alone was sub-cloned into the AscI site in pCaSpeR
(Calhoun et al., 2002). The PTE
element was isolated as an AscI fragment and cloned into the unique
AscI site 5' of eve-lacZ and its orientation was
determined by sequencing. The eve-lacZ gene used was as previously
described (Ohtsuki et al.,
1998). The Abd-BPTE
promoter was isolated as an AscI-NotI fragment and used to
replace the AscI-NotI CAT fragment in pCaSpeR.
|
). Multiple transgenic lines were generated for each construct
and at least three independent lines were analyzed by in situ hybridization.
Embryos were collected, fixed and hybridized with digoxigenin-labeled
lacZ or CAT probe as previously described
(Bae et al., 2002).
Bioinformatic analysis
Levels of sequence conservation were calculated using VISTA
(Frazer et al., 2004) for the
PTE (chr3R:12,760,005-12,760,259), PTE 5' (chr3R:12,760,259-12,760,513),
PTE 3' (chr3R:12,759,751-12,760,005), BX-C (chr3R:
12,470,945-12,809,178) and iab5-iab8 (chr3R:
12,695,347-12,755,125) regions of the D. melanogaster genome (April
2004) using the following parameters: calc window, 24 bp; cons window, 24 bp;
cons identity, 80%. Absolute levels of conservation were obtained by comparing
the number of perfectly aligned base pairs to the total length of the
promoter-tethering element in D. melanogaster.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Ohtsuki et al., 1998). To
identify the cis-regulatory elements responsible for this specificity, an
Abd-B promoter was attached to a CAT reporter gene. The
Abd-B promoter region is 1.5 kb in length and includes 294 bp of
5' flanking sequence and approximately 1.2 kb of 3' sequence. On a
reporter transgene the IAB5 enhancer is able to direct strong CAT
expression from the Abd-B promoter in the characteristic three
posterior abdominal stripes in blastoderm-stage embryos
(Fig. 2A). One possible
mechanism by which promoter-enhancer specificity is established at the BX-C
could be the existence of promoter-proximal elements, which inhibit enhancers
from interacting with specific promoters. For example, IAB5 may be prevented
from interacting with the abd-A promoter by a negative regulatory
element close to the abd-A transcription start site. However, this
does not appear to be the case, as IAB5 is able to interact strongly with a 1
kb abd-A promoter-lacZ reporter gene when located close to
the abd-A promoter and in the absence of any other competing
promoters (Fig. 2B). If the
IAB2 enhancer is also added to this transgenic construct, a much broader band
of lacZ expression expanding towards the anterior of the
blastoderm-stage embryo is seen (Fig.
2C), indicating that IAB2 and IAB5 are both able to drive
expression from the abd-A promoter. On transgenic constructs, IAB2
directs expression from the abd-A promoter predominantly in
parasegments 7 and 9 (presumptive abdominal segments 2 and 4) and more weakly
in parasegments 11 and 13 (segments 6 and 8), as previously described for a
Ubx-lacZ reporter gene
(Shimell et al., 2000).
However, on transgenes the Abd-B promoter is also capable of
interacting with the IAB2 enhancer. When the IAB2 and IAB5 enhancers are
present on the same construct, bands of CAT expression are detected
extending from presumptive abdominal segment 2 towards the posterior of the
blastoderm-stage embryo (Fig.
2D). The promoters from the BX-C, therefore, appear to be
responsive to both of the enhancers tested, indicating that the promoter
regions themselves are not able to inhibit enhancer-promoter interactions. It is a formal possibility that promoter-enhancer specificity at the BX-C is established as a result of the endogenous enhancers interacting with promoters in an orientation-dependent manner. This is unlikely to be a general rule as the enhancers in the IAB3 and IAB4 regions must activate the abd-A promoter in the 3' direction, while IAB5 and IAB2 both interact with their respective target promoters in the 5' direction (see Fig. 1B). To test whether the enhancer activities are directional we also reversed the orientation of IAB2 and IAB5 relative to their target promoters on the transgenic constructs. This failed to disrupt enhancer-driven expression of the reporter genes (data not shown). Therefore, the enhancers from the BX-C appear to be orientation independent and promiscuous, able to activate any promoter. In addition, the homeotic gene promoters do not appear to harbor inhibitory regulatory elements capable of repressing activation from either enhancer. As a result, when only a single promoter is present, both IAB2 and IAB5 will drive expression strongly from either abd-A-lacZ or Abd-B-CAT.
|
Anti-insulator activity in the Abd-B promoter
In order to activate Abd-B expression at the endogenous gene
complex, IAB5 has to bypass at least two known insulator sequences,
Fab-7 and Fab-8, that have enhancer-blocking activity
(Fig. 1B)
(Hagstrom et al., 1996;
Zhou et al., 1999;
Zhou et al., 1996). These
insulator elements have previously been shown to disrupt promoter-enhancer
interactions when placed between a promoter and enhancer on transgenes
(Barges et al., 2000;
Zhou et al., 1999;
Zhou et al., 1996;
Zhou and Levine, 1999). To
further characterize the IAB5-Abd-B interaction, we therefore needed
to test the interaction in the presence of an insulator from the BX-C. The
2-B-Fab-8-A-5 construct was created in which the previously characterized
Fab-8 insulator element (Zhou and
Levine, 1999) was placed between the homeotic promoter elements.
The Fab-8 insulator sequence was specifically designed to exclude the
adjacent PTS7 element. The addition of the Fab-8 insulator sequence
did not disrupt the IAB5-Abd-B interaction, as strong CAT expression
was detected in posterior stripes in blastoderm-stage embryos
(Fig. 4A) whereas no
abd-A-lacZ expression was detected
(Fig. 4B). To confirm that the
CAT expression in these embryos was not solely an IAB2-driven pattern, we
generated transgenic lines carrying the B-Fab-8-A-5 transgene, from which the
IAB2 enhancer was removed. In these embryos the IAB5 enhancer was recruited to
the Abd-B promoter, as strong IAB5-driven expression was detected for
the Abd-B-CAT gene (Fig.
4C) and no abd-A-lacZ expression was observed
(Fig. 4D). To ensure that
altering the enhancer-promoter spacing would not modulate the
IAB5-Abd-B interaction, we created the 2-B-1.6-A-5 construct.
To generate this construct, a 1.6 kb lambda DNA fragment
(Calhoun et al., 2002) was
inserted between the promoter regions on the 2-B-A-5 construct
(Fig. 3). The addition of the
spacer had no effect on the IAB5-Abd-B interaction, as strong
CAT expression in a composite IAB2-IAB5-driven pattern was observed
(Fig. 4E) whereas no
lacZ expression was detected (Fig.
4F). These results indicate that the Abd-B promoter may
contain a tethering activity that allows the IAB5 enhancer to drive expression
of Abd-B-CAT across the Fab-8 insulator. This anti-insulator
activity is important in the context of the endogenous BX-C, as IAB5 must
bypass at least two insulators to interact with the Abd-B
promoter.
|
|
) close to the transcriptional start site but leaving the
promoter region capable of activating transcription. This 255 bp sequence also
overlaps with the 5' region previously shown to be functionally
important in regulating promoter-enhancer communication at the BX-C in trans
(Sipos et al., 1998).
|
PTE-A-5
construct resulted in re-direction of IAB5-driven expression from the
Abd-B promoter (Fig.
5C) to the abd-A promoter directing lacZ
expression (Fig. 5D) in
transgenic embryos. We therefore named the 5' 255 bp sequence the
promoter-tethering element (PTE). In a few transgenic lines carrying the
BPTE-A-5 construct very weak
IAB5-driven expression of the
BPTE-CAT reporter gene was
observed (data not shown), probably because of position effects. This
experiment indicates that the Abd-B promoter is functional in this
configuration, but only at certain integration points in the genome. It is
possible that the lack of expression of the CAT reporter gene on
these constructs could simply be a result of a non-functional Abd-B
promoter, caused by the truncation of the promoter. To verify that the
Abd-BPTE promoter was still
functional in a non-competitive situation, IAB2 was inserted 1 kb 3' of
the Abd-BPTE promoter to
generate the 2-BPTE-A-5 construct. This
resulted in IAB2-driven activation of the
Abd-BPTE promoter
(Fig. 5G), indicating that this
promoter is still functional, whereas IAB5 was solely recruited to the
abd-A promoter. (Fig.
5H). The level of expression detected from the truncated
Abd-BPTE promoter was
comparable to other transgenic lines analyzed in this study, suggesting the
promoter is fully active. In addition, these embryos clearly demonstrate the
different expression patterns driven by the two IAB enhancers
(Fig. 5G,H). The deletion of
the PTE, therefore, confirms that this cis-regulatory sequence is necessary
for the recruitment of IAB5 to the Abd-B promoter on transgenes.
PTE can regulate ectopic enhancer-promoter interactions
To further analyze the functional activity of the PTE in
Drosophila embryos, the W-5-EZ construct was created in which the
IAB5 enhancer was positioned between the mini-white reporter gene and
an even-skipped-lacZ fusion reporter gene. As previously described,
in this configuration the IAB5 enhancer has a strong preference for the
TATA-box-containing even-skipped (eve) promoter in
transgenic blastoderm-stage embryos and only weakly activates white
(Fig. 6A,B)
(Ohtsuki et al., 1998).
Insertion of the 255 bp PTE adjacent to the eve-lacZ gene on the
W-5-PTE-EZ transgenic construct resulted in redirection of the IAB5
enhancer-driven expression solely to the eve promoter as
lacZ was expressed in the three characteristic posterior abdominal
stripes (Fig. 6D), whereas no
white expression was detected
(Fig. 6C). The expression
patterns from these constructs demonstrate that the PTE from 5' of
Abd-B will regulate ectopic promoter-enhancer interactions. The
regulatory switch induced by the juxtapositioning of the PTE with the
eve promoter indicates that the tethering of IAB5 is not
promoter-specific. This fits with the regulatory logic required at the
endogenous locus as a PTE in a gene complex will only be required to recruit
specific enhancers to the promoter at which it is located.
Bioinformatic analysis of the PTE sequence in different Drosophila species
Our transgenic studies indicate that the PTE is a functional element in the
BX-C. One potential model for the tethering function of this regulatory
sequence is that it may contain binding sites for trans factors that directly
interact with the enhancers in the BX-C to bring them into close proximity
with the Abd-B promoter. A prediction of this model is that the
putative binding sites in the PTE should be conserved in different
Drosophila species. As a result it should be possible to identify a
high level of conservation for short stretches of sequence within the PTE that
contain binding sites. To test this we carried out bioinformatic studies
across seven different Drosophila species
(Fig. 7A). The PTE sequence as
a whole does not demonstrate a significantly higher level of conservation in
the different species when compared to other sequences from the BX-C,
including the IAB5 enhancer with known regulatory activity
(Fig. 7B). However, within the
PTE there are two short sequences that are more highly conserved: a 24-mer and
a 27-mer (Fig. 7B,C). It is
therefore possible that these short sequences represent conserved protein
binding sites and that the trans factors involved in the functional activity
of the PTE interact specifically with these conserved sequences (see
Discussion).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
At the endogenous BX-C, the IAB5 enhancer specifically directs expression from
the Abd-B promoter in cis, although it is located slightly closer to
the abd-A promoter and is separated from the target promoter by at
least two known insulator sequences. The present study provides evidence that
a novel promoter-tethering element (PTE), located -40 to -294 bp relative to
the Abd-B transcription start site, is responsible for mediating
these regulatory interactions and consequently plays a critical role in
controlling promoter-enhancer communication at the BX-C.
Functional properties of the PTE
A 1.5 kb Abd-B promoter specifically recruits the IAB5 enhancer on
reporter transgenes. The tethering activity contained in the Abd-B
promoter region is able to interact with IAB5 over a long distance (>5 kb)
and is capable of facilitating the bypass of an intervening promoter
(abd-A) from the BX-C (see Figs
3 and
4). Deletion of a 255 bp
sequence located in the 5' region of the Abd-B promoter reveals
the existence of a novel cis-element responsible for tethering of the IAB5
enhancer to the promoter. Removal of this PTE sequence from the Abd-B
promoter is sufficient to redirect the IAB5 enhancer to the adjacent
abd-A promoter on transgenes (see
Fig. 5). In addition, fusion of
the PTE sequence to an ectopic promoter results in complete recruitment of the
IAB5 enhancer to the promoter (Fig.
6).
|
). By contrast, the IAB8 enhancer may not require a tethering
element to activate the Abd-B promoter, possibly due to the close
proximity of this enhancer to the Abd-B promoter and the fact that
there are no intervening insulator elements (see
Fig. 1B).
Previous studies demonstrated that a deletion 5' of the
Abd-B promoter region, which included the PTE, resulted in reduced
IAB enhancer-Abd-B promoter interactions in trans
(Sipos et al., 1998). As
larger deletions were made, the interactions between the IAB7 enhancer and the
target Abd-B promoter became increasingly weaker. One explanation for
this observation could be the existence of additional elements in the extended
5' promoter sequence which may play a role in the tethering of the IAB
enhancers to the Abd-B gene at the endogenous BX-C. Our bioinformatic
studies across different Drosophila species support this idea. The
neighboring sequence 5' of the PTE is highly conserved; suggesting that
part of this upstream region may also contain sequences that aid in the
tethering activity (Fig. 7). In
this case, the critical in vivo function of the PTE may be supported by, as
yet unidentified, additional cis-regulatory sequences capable of facilitating
promoter-enhancer tethering. Transgenic constructs containing a PTE sequence
extended to include part of this 5' region will be important in
determining the function of this region.
The PTE may also function in conjunction with a different class of
anti-insulator elements at the BX-C, the promoter targeting sequences (PTS),
which are known to facilitate promoter-enhancer interactions
(Chen et al., 2005). Future
transgenic and genetic experimental approaches will help to unravel the
combinatorial regulatory activities of these complex cis-elements. However, it
is clear that the promoter-enhancer interactions facilitated by the PTE are
relatively strong, as neither spacer DNA nor endogenous insulator elements
were capable of disrupting these interactions in our transgenic assays. These
results provide insight into the regulatory requirements at the endogenous
locus. It seems likely that these strong interactions are necessary for the
IAB5 enhancer element to bypass the two known insulator elements to activate
the Abd-B promoter approximately 55 kb away in cis.
The precise molecular mechanism by which the tethering element functions is
not clear. It is possible that common trans factors may bind to both the IAB5
enhancer and PTE and establish protein-protein interactions, although the
Abd-B PTE and IAB5 enhancer do not share any extensive sequence
homology. There is, however, a precedent for this type of interaction as Sp1
has been shown to form DNA loops between binding sites proximal to promoter
sequences and distant binding sites to mediate an increased concentration of
activator protein at the promoter
(Mastrangelo et al., 1991).
Bioinformatic analysis reveals two separate short sequences within the PTE
that are highly conserved in different Drosophila species, when
compared to the other sequences in the PTE
(Fig. 7). It is possible that
these sequences harbor binding sites critical for the recruitment of the trans
factors involved in the molecular function of the PTE. As previously proposed,
it is possible that a mechanism involving the IAB enhancers looping to
interact with the PTE and drive expression from the target Abd-B
promoter could be facilitated by specific chromatin structures in the BX-C
(Akbari et al., 2006;
Sipos and Gyurkovics, 2005). A
similar spatial nuclear organization has also been suggested as a global
regulator of developmental gene expression in higher eukaryotes
(de Laat and Grosveld, 2003).
More recent studies have demonstrated that there are indeed physical
interactions between distant regulatory regions with the BX-C
(Cleard et al., 2006),
although the details remain to be fully elucidated. This model would explain
the necessity of a functional PTE in the BX-C, as the disruption of this
element would prevent the formation of the chromatin loop structures essential
for promoter-enhancer communication, leaving the Abd-B target
promoter inactive.
Implications for regulatory specificity in gene complexes
Promoter-tethering elements represent a precise mechanism for regulating
specific enhancer-promoter interactions in gene complexes. Other mechanisms of
cis-regulation may be less flexible. Recent studies have identified enhancers
in the Drosophila genome capable of interacting only with distinct
sub-sets of promoters. Some enhancers will only interact with DPE-containing
promoters, whereas others only interact with TATA-containing promoters
(Butler and Kadonaga, 2001). At
the BX-C this may not be a feasible method for regulating enhancer-promoter
interactions as the addition of a TATA box to one promoter, for example, may
result in recruitment of all the enhancers in the complex. However, the PTE
may function similarly to promoter competition in some respects, as the
specific IAB5-Abd-B interaction it mediates appears to predominantly
prevent the enhancer from activating other promoters.
The existence of a tethering element capable of specifically recruiting the
distal T1 enhancer to the Scr gene promoter at the antennapedia
Hox gene complex in Drosophila
(Calhoun et al., 2002) suggests
that promoter-tethering elements may represent a common mechanism for
regulating enhancer-promoter interactions at complex loci. The ability of the
IAB5 enhancer to activate Abd-B across insulator DNAs provides an
intriguing model for tethering activities at other gene complexes. An example
is the well characterized insulator at the mouse H19 imprinting
control region which separates 5' enhancers from the H19
promoter (Bell and Felsenfeld,
2000; Drewell et al.,
2002b; Hark et al.,
2000; Szabo et al.,
2000). It is possible that a tethering element is required to
selectively recruit these 5' enhancers to the target promoter. The
identification of an enhancer-containing global control region at the mouse
Hoxd complex (Spitz et al.,
2003) raises the possibility that promoter tethering over long
distances may also be required at mammalian Hox genes.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akbari, O. S., Bousum, A., Bae, E. and Drewell, R. A. (2006). Unraveling cis-regulatory mechanisms at the abdominal-A and Abdominal-B genes in the Drosophila bithorax complex. Dev. Biol. 293,294 -304.[CrossRef][Medline]
Bae, E., Calhoun, V. C., Levine, M., Lewis, E. B. and Drewell,
R. A. (2002). Characterization of the intergenic RNA profile
at abdominal-A and Abdominal-B in the Drosophila bithorax complex.
Proc. Natl. Acad. Sci. USA
99,16847
-16852.
Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, 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.[Abstract]
Bell, A. C. and Felsenfeld, G. (2000). Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405,482 -485.[CrossRef][Medline]
Busturia, A. and Bienz, M. (1993). Silencers in abdominal-B, a homeotic Drosophila gene. EMBO J. 12,1415 -1425.[Medline]
Butler, J. E. and Kadonaga, J. T. (2001).
Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.
Genes Dev. 15,2515
-2519.
Butler, J. E. and Kadonaga, J. T. (2002). The
RNA polymerase II core promoter: a key component in the regulation of gene
expression. Genes Dev.
16,2583
-2592.
Calhoun, V. C., Stathopoulos, A. and Levine, M.
(2002). Promoter-proximal tethering elements regulate
enhancer-promoter specificity in the Drosophila Antennapedia complex.
Proc. Natl. Acad. Sci. USA
99,9243
-9247.
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.[Medline]
Chen, Q., Lin, L., Smith, S., Lin, Q. and Zhou, J. (2005). Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation. Dev. Biol. 286,629 .[CrossRef][Medline]
Cleard, F., Moshkin, Y., Karch, F. and Maeda, R. K. (2006). Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification. Nat. Genet. 38,931 -935.[CrossRef][Medline]
Crosby, M. A., Goodman, J. L., Strelets, V. B., Zhang, P., Gelbart, W. M. and The FlyBase Consortium (2007). FlyBase: genomes by the dozen. Nucleic Acids Res. 35,D486 -D491.[CrossRef][Medline]
de Laat, W. and Grosveld, F. (2003). Spatial organization of gene expression: the active chromatin hub. Chromosome Res. 11,447 .[CrossRef][Medline]
Drewell, R. A., Brenton, J. D., Ainscough, J. F., Barton, S. C., Hilton, K. J., Arney, K. L., Dandolo, L. and Surani, M. A. (2000). Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch. Development 127,3419 -3428.[Abstract]
Drewell, R. A., Arney, K. L., Arima, T., Barton, S. C., Brenton,
J. D. and Surani, M. A. (2002b). Novel conserved elements
upstream of the H19 gene are transcribed and act as mesodermal enhancers.
Development 129,1205
-1213.
Drewell, R. A., Bae, E., Burr, J. and Lewis, E. B.
(2002a). Transcription defines the embryonic domains of
cis-regulatory activity at the Drosophila bithorax complex. Proc.
Natl. Acad. Sci. USA 99,16853
-16858.
Frazer, K. A., Pachter, L., Poliakov, A., Rubin, E. M. and
Dubchak, I. (2004). VISTA: computational tools for
comparative genomics. Nucleic Acids Res.
32,W273
-W279.
Geyer, P. K. (1997). The role of insulator elements in defining domains of gene expression. Curr. Opin. Genet. Dev. 7,242 -248.[CrossRef][Medline]
Hagstrom, K., Muller, M. and Schedl, P. (1996).
Fab-7 functions as a chromatin domain boundary to ensure proper segment
specification by the Drosophila bithorax complex. Genes
Dev. 10,3202
-3215.
Hark, A. T., Schoenherr, C. J., Katz, D. J., Ingram, R. S., Levorse, J. M. and Tilghman, S. M. (2000). CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405,486 -489.[CrossRef][Medline]
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.[CrossRef][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]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[CrossRef][Medline]
Maeda, R. K. and Karch, F. (2006). The ABC of
the BX-C: the bithorax complex explained. Development
133,1413
-1422.
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.
Mastrangelo, I. A., Courey, A. J., Wall, J. S., Jackson, S. P.
and Hough, P. V. (1991). DNA looping and Sp1 multimer links:
a mechanism for transcriptional synergism and enhancement. Proc.
Natl. Acad. Sci. USA 88,5670
-5674.
Mihaly, J., Barges, S., Sipos, L., Maeda, R., Cleard, F., Hogga,
I., Bender, W., Gyurkovics, H. and Karch, F. (2006).
Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex.
Development 133,2983
-2993.
Ohtsuki, S., Levine, M. and Cai, H. N. (1998).
Different core promoters possess distinct regulatory activities in the
Drosophila embryo. Genes Dev.
12,547
-556.
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., Vernos, I., Marco, R. and Morata, G. (1985). Genetic organization of Drosophila bithorax complex. Nature 313,108 -113.[CrossRef][Medline]
Shimell, M. J., Peterson, A. J., Burr, J., Simon, J. A. and O'Connor, M. B. (2000). Functional analysis of repressor binding sites in the iab-2 regulatory region of the abdominal-A homeotic gene. Dev. Biol. 218,38 -52.[CrossRef][Medline]
Sipos, L. and Gyurkovics, H. (2005). Long-distance interactions between enhancers and promoters. The case of the Abd-B domain of the Drosophila bithorax complex. FEBS J. 272,3253 -3259.[CrossRef][Medline]
Sipos, L., Mihaly, J., Karch, F., Schedl, P., Gausz, J. and
Gyurkovics, H. (1998). Transvection in the Drosophila Abd-B
domain: extensive upstream sequences are involved in anchoring distant
cis-regulatory regions to the promoter. Genetics
149,1031
-1050.
Small, S., Blair, A. and Levine, M. (1992). Regulation of even-skipped stripe 2 in the Drosophila embryo. EMBO J. 11,4047 -4057.[Medline]
Spitz, F., Gonzalez, F. and Duboule, D. (2003). A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 113,405 -417.[CrossRef][Medline]
Szabo, P. E., Tang, S.-H. E., Rentsendorj, A., Pfeifer, G. P. and Mann, J. R. (2000). Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function. Curr. Biol. 10,607 -610.[CrossRef][Medline]
West, A. G., Gaszner, M. and Felsenfeld, G.
(2002). Insulators: many functions, many mechanisms.
Genes Dev. 16,271
-288.
Zhou, J. and Levine, M. (1999). A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo. Cell 99, 567.[CrossRef][Medline]
Zhou, J., Barolo, S., Szymanski, P. and Levine, M.
(1996). The Fab-7 element of the bithorax complex attenuates
enhancer-promoter interactions in the Drosophila embryo. Genes
Dev. 10,3195
-3201.
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.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  O. Kyrchanova, S. Toshchakov, Y. Podstreshnaya, A. Parshikov, and P. Georgiev Functional Interaction between the Fab-7 and Fab-8 Boundaries and the Upstream Promoter Region in the Drosophila Abd-B Gene Mol. Cell. Biol., June 15, 2008; 28(12): 4188 - 4195. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
100 kb in length. The iab regions that control
expression of the two homeotic genes are indicated: IAB2-IAB8 (shown with
respect to the corresponding embryonic parasegment). IAB2, IAB3 and IAB4
(shown in blue) regulate expression of abd-A. IAB5, IAB6, IAB7 and
IAB8 (shown in green) direct Abd-B expression. The insulator DNAs
that separate the different iab regions are marked by red ellipses.
Characterized enhancers within the iab regions are shown as orange
rectangles. The IAB5 enhancer is located 55 kb 3' of the Abd-B
promoter and 48 kb 5' of the abd-A promoter, but only interacts
with Abd-B over the intervening insulator sequences. The IAB2
enhancer is located 18 kb 3' of the abd-A promoter and directs
expression specifically from abd-A. (C) Core promoter
sequences at abd-A and Abd-B. The consensus sites for
initiator (INR) and downstream promoter elements (DPE) in Drosophila
are shown (Butler and Kadonaga,
2002
