REVIEW ARTICLE
Long-distance interactions between enhancers and
promoters
The case of the Abd-B domain of the Drosophila bithorax complex
La
´
szlo
´
Sipos and Henrik Gyurkovics
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
Introduction
The normal development of eukaryotic organisms
requires a precise and coordinated control of gene
expression, both spatially and temporally. In the case
of genes with a highly complex expression pattern, this
is achieved through the action of a large set of enhanc-
ers, which are often located at a considerable distance
from the regulated gene. Accordingly, one of the key
questions involved in an understanding of complex
gene regulation is how distant enhancers communicate
with their target promoters. Despite its importance,
the available scientific data relating to this question are
still extremely scarce. In this respect, one of the best-
studied systems is the regulation of the homeotic
Abdominal-B (Abd-B) gene in Drosophila.
Abd-B, one of the three genes in the bithorax complex
(BX-C), determines the identity of the posterior-most
segments in the fly. One Abd-B transcript (class A tran-
script) is responsible for the proper identity of abdom-
inal segments 5–8, while three other transcripts are
required for the identity of abdominal segment 9 and

(several tens of kilobases away) promoter in the nucleus, what mechanism
restricts promiscuous enhancers to this specific interaction, and how differ-
ent regulatory regions replace one another at the same promoter in subse-
quent abdominal segments. Moreover, several of these regulatory regions
have to act over chromatin domain boundaries and extensive inactive chro-
matin domains, similarly to the situation found in the chicken beta-globin
cluster. In this minireview we survey mechanisms and factors that may be
involved in mediating specific interactions between the Abd-B promoter
and its regulatory regions.
Abbreviations
Abd-B, Abdominal-B gene; BX-C, bithorax complex; Pc-G, polycomb-group; PREs, polycomb response elements; PTS, promoter targeting
sequence; trx-G, trithorax-group; TREs, trithorax response elements; tmr, transvection-mediating region.
FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS 3253
BX-C cis-regulatory domains, their proximal-distal
order along the chromosome corresponds to the anter-
ior-posterior order of the segments they specify.
Cis-regulatory regions in the Abd-B domain are
sequentially activated on proceeding from anterior to
posterior segments. In A5, for example, only one of the
four Abd-B cis-regulatory regions, iab-5, is thought to
be active, while the other three are silenced. In A6, both
iab-5 and iab-6 are active, and iab-7 and iab-8 are
silenced, but only iab-6 drives the expression of Abd-B.
Similarly, although three different Abd-B cis-regulatory
domains are active in A7, the expression of Abd-B is
directed predominantly (or exclusively) by iab-7 in this
segment of wild-type animals (Fig. 2). However, if iab-7
is deleted, the expression of Abd-B is controlled by iab-6
in both A6 and A7, resulting in the transformation of
A7 into a duplicated copy of A6, while the identity of

domain. The proximal Abd-B promoter (d)
and insulator regions (brick-patterned ovals)
separating independent 3¢ cis-regulatory reg-
ions (iab-5 to iab-8) are shown (A). Each cis-
regulatory region is required for the proper
identity of one of the abdominal segments
from A5 to A8, indicated by vertical arrows.
(B) The generalized structure of a cis-regula-
tory region. (C) An enlargement of the
 10 kb tmr region with the known
cis-acting elements.
Fig. 2. Model of the regulation of the Abd-B
gene in abdominal segments A6 and A7.
Although the iab-5 cis-regulatory region is
also in an active conformation in A6, only
iab-6 is presumed to contact the Abd-B
promoter region (indicated by a series of
horizontal lines), while the inactive iab-7 and
iab-8 regions (thick dotted figures) loop out.
In the next abdominal segment, A7, iab-7
becomes activated and takes over the
regulation of Abd-B from iab-6.
Long-distance interactions in Abd-B L. Sipos and H. Gyurkovics
3254 FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS
provide them with the autonomy necessary for inde-
pendent functioning.
The looping model
The most widely accepted model of long–range regula-
tory interactions is the looping model, which postu-
lates that enhancers and distant promoters are in

interrupted pairing of homologous sequences (‘linearly
locked state’), the enhancers are locked away from the
promoter, while in the event of local unpairing, intra-
molecular looping is allowed to promote the inter-
actions between the enhancers and the promoter [8]. In
this context, it is interesting to note that the pairing of
BX-C occurs only after the tenth hour of embryonic
development [9], eight hours later than in the case of
the histone gene cluster [10]. This difference in the tim-
ing of somatic pairing perhaps reflects the difference
between the complexities of the regulation of the two
systems: a longer time is required for the formation of
the complex looping structure in the case of BX-C,
while a shorter time is sufficient for the establishment
of the much simpler regulatory interactions of the his-
tone cluster. However, the pairing of BX-C was found
to be a dynamic process, with the paired state never
exceeding 70% of the embryonic cells at a given time
[9]. This ‘breathing’ of the paired state might be
required for the reorganization of intramolecular inter-
actions and the correction of an inappropriate looping
structure in later stages of development.
If the uninterrupted pairing of homologs is consid-
ered to be an obstacle to loop formation, then there is
an intrinsic interest in well-defined sequences that can
counteract the forces of homologous pairing under
experimental conditions. Trough the use of different
approaches, such as transgenic assays, several short
sequences from the Abd-B have been shown to be able
to mediate regulatory interactions over exceedingly

in trans (Fig. 3). As this trans regulation is not detec-
ted when the somatic pairing of homolog chromo-
somes is disturbed by chromosomal rearrangements,
it represents a case of ‘transvection’. (The term
L. Sipos and H. Gyurkovics Long-distance interactions in Abd-B
FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS 3255
transvection was coined by Edward Lewis in 1954 to
designate the phenomenon when the expression of a
gene on one chromosome depends on the pairing
with its homologous region [14]) The degree of com-
plementation in A7 depends on the size of the pro-
moter deletion: the larger the deletion, the stronger
the trans regulation (Fig. 3), suggesting that the pro-
moter upstream region of the Abd-B gene consists of
numerous discrete elements that cooperate in locking
individual cis regulators to the Abd-B gene [13]. The
putative tethering region is extremely large (over
7.6 kb) as compared to that proposed for the white
gene (95 bp [15]), and it goes well beyond the region
necessary for the basal Abd-B promoter activity
(0.9 kb [16]). However, putative counterparts of the
tethering complex in the iab regulatory regions have
not been found to date.
Transvection-mediating region (tmr)
Another region from the Abd-B domain that has been
found to mediate long-distance interactions is called
the transvection-mediating region, tmr [17]. It is an
approximately 10 kb sequence immediately 3¢ of the
Abd-B transcription unit, and is responsible for a
weak, but extremely tenacious interaction between the

both), the boundary retains its enhancer-blocking
activity, and each promoter can be regulated only by
enhancers on the same side of the boundary (Fig. 4,
top). If, however, the PTS and boundary are placed
outside the region defined by the promoters of the two
reporter genes (3¢ to one of them, Fig. 4, bottom), the
PTS is able to overcome the enhancer blocking effect
of the boundary, and restricts the enhancer activity to
only one of the promoters in the transgene. Addition-
ally, the PTS is able to co-target different enhancers to
Fig. 3. Correlation between the size of 5¢ deletions in the Abd-B
transcription unit and the strength of trans regulation. Open circles
represent elements of the putative upstream tethering region, grad-
ual removal of which shifts the ratio between the strength (indica-
ted by the thickness of the curved arrows) of the cis and trans
interactions in favor of the latter. An increase in the trans inter-
action results in an increase in the level of the functional Abd-B
protein. Continuous lines represent the DNA of homologous chro-
mosomes, brick-patterned ovals symbolize boundaries, short verti-
cal lines indicate endpoints of deletions, black dots denotes the
site of the Abd-B promoter, and crossed lines indicate a point muta-
tion in the Abd-B gene.
Long-distance interactions in Abd-B L. Sipos and H. Gyurkovics
3256 FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS
the same promoter. All of these activities of the PTS
are independent of its orientation [16,19,20,21].
Surprisingly, however, the apparently random choice
between the two promoters is maintained not only
through mitoses, but also through meioses. Thus, three
types of transgenic strains can be obtained when the

PTS and a promoter (or a non-erasable covalent modi-
fication generated by the PTS in the promoter region),
inherited from previous generations, would provide a
pre-prepared and obligatory path for the enhancers to
the promoter.
However, a number of earlier observations raise the
possibility that the PTS might function differently in
transgenes and in its native context. For example, the
irreversibility of targeting, suggested by the transgenic
experiments, is difficult to reconcile with the fact that
the Abd-B promoter has to contact different sets of
enhancers in different segments, and also with the
observation that the identity of Abd-B-controlled seg-
ments can be changed in later stages of development,
implying that different sets of enhancers can replace
one another at the Abd-B promoter under certain con-
ditions, e.g. in some polycomb-group (Pc-G) or tritho-
rax-group (trx-G) mutant background. This problem
can be solved by assuming that each cis-regulatory unit
has its own PTS (presumably next to the relevant
boundary), and that these PTSs can effectively com-
pete for the same Abd-B promoter, perhaps in a hier-
archical manner. The results obtained by swapping
the Fab)7 boundary with heterologous boundary
sequences, such as su(Hw) and scs, are compatible
with this possibility [22]. If Fab-7 is replaced by either
of these boundaries, communication will be blocked
between the proximal enhancers and Abd-B. The simp-
lest interpretation of this result is that sequences
removed in the swapping experiments contain not only

L. Sipos and H. Gyurkovics Long-distance interactions in Abd-B
FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS 3257
interphase. In theory, this means that the cis inter-
actions between the enhancer and the promoter are
constantly challenged by the same promoter on the
homolog chromosome. For Abd-B, it has been revealed
that enhancers in the iab-7 cis-regulatory unit can
indeed regulate, albeit weakly, the Abd-B promoter in
trans even when the Abd-B promoter and the PTS
region are intact in both homologs (see Fig. 3), sug-
gesting that enhancer targeting is a dynamic process in
this case [13].
Is the PTS a unique or dominant player
in directing iab-7 enhancers to the
Abd-B promoter?
The genetic evidence suggests that it is not. For exam-
ple, even if the relevant promoter of Abd-B is deleted,
the iab-7 enhancers appear to remain partially ‘bound’
to some region in the vicinity of the promoter (see
below). More importantly, deletion of the PTS
sequences from an otherwise intact BX-C results in a
rather mild transformation of segment A7 toward A6
[J. Mihaly (Institute of Genetics, Biological Research
Center, Szeged, Hungary) and F. Karch (De
´
partement
de Zoologie et biologie animale, Universite
´
de Gene
`

protein complex differing in some fundamental way
from that formed within the BX-C. Conceivably, the
normal function of the PTS in iab-7 is to help enhanc-
ers bypass the enhancer-blocking activity of the Fab)8
boundary without compromising other functions, such
as preventing the ‘spreading’ of competing chromatin
structures between iab-7 and iab-8. This assumption is
compatible with the location of the PTS. The discovery
and characterization of the PTS in a transgenic con-
text, however, provided a strong case for the idea that
enhancer-promoter contacts do not need to be estab-
lished anew in each cell cycle; rather, they could be
maintained through many cell divisions if once it is
formed at an early stage of development. Moreover,
identification of transacting factors involved in promo-
ter targeting now seems feasible with the help of trans-
genic lines containing a PTS and different boundary
sequences.
Perspectives
The genetic evidence indicates that proper targeting of
the Abd-B promoter is most likely to be a result of a
hierarchical cooperation among a number of different
elements, analogous to the formation of enhanceo-
somes (reviewed in [25]), but on a much larger scale.
Cooperating elements may include PTS-like sequences,
boundaries, upstream tethering elements, PREs ⁄ TREs
and other, as yet unidentified components of the
Abd-B regulatory unit. A better understanding of the
promoter targeting in this system requires a careful
in situ analysis involving targeted mutagenesis. Such

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