Tài liệu Báo cáo khoa học: Long-distance interactions between enhancers and promoters The case of the Abd-B domain of the Drosophila bithorax complex pdf

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

also that of abdominal segment 10 (for examples see
[1,2]). Here we focus on the transcriptional unit coding
for the class A transcript, and refer to it and its regula-
tory regions as the Abd-B domain. The expression pat-
tern of Abd-B is regulated by a set of large (over 10 kb),
autonomous cis-regulatory domains, iab-5, iab-6, iab-7
and iab-8 in segments A5, A6, A7 and A8, respectively
(reviewed in [3,4]). As illustrated in Fig. 1A, these
cis-regulatory domains are located downstream of the
Abd-B transcription unit, and, as is the case for the other
Keywords
Abd-B; chromatin structure; Drosophila;
homeotic genes; promoter targeting
Correspondence
H. Gyurkovics, Institute of Genetics,
Biological Research Center, Hungarian
Academy of Sciences, H-6726 Szeged,
Temesvari krt. 62, Hungary
Fax: +36 62 433503
Tel: +36 62 599687
E-mail:
(Received 21 February 2005, accepted
10 May 2005)
doi:10.1111/j.1742-4658.2005.04757.x
Abdominal-B (Abd-B) is a complex homeotic gene with a difficult task: one
transcript determines the identity of four different abdominal segments
throughout development in Drosophila. Although an increasing amount of
information is available about the structure and the functioning of the reg-
ulatory regions that determine the expression pattern of Abd-B, it is still
not clear how these regulatory regions can contact the distantly located

(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

the more posterior segments is not altered. As expected
from this loss-of-function phenotype, the Abd-B expres-
sion pattern normally seen in A7 is replaced by an
A6-like pattern [5].
Cis-regulatory regions contain a set of different func-
tional and structural elements (Fig. 1B) identified in
transgenic reporter constructs (for example see [6]).
Among them, ‘early enhancers’ drive segmentally
restricted gene expression patterns in blastoderm
embryos as a response to the action of gap and pair-
rule gene products. Another class of enhancers iden-
tified in cis-regulatory regions are ‘cell-specific
enhancers’, which turn on reporter genes in particular
cell types without any segmental specificity. Polycomb
and trithorax response elements (PREs ⁄ TREs) are
involved in generating and maintaining ‘closed’ or
‘open’ chromatin conformations, respectively, accord-
ing to the spatial activity pattern of the ‘early enhanc-
ers’. These alternative chromatin conformations will
eventually restrict the action of ‘cell-specific enhancers’
to segmental boundaries. Finally, boundary elements
flank the regulatory regions. Boundary elements can
block or greatly weaken the interactions of an enhancer
and a promoter if placed between them in transgenic
constructs, and can protect a reporter gene from the
effects of the neighboring chromatin (e.g. heterochro-
matinization) if the reporter is flanked by two of them.
The apparent function of the boundaries within BX-C
is to separate neighboring cis-regulatory regions, and to
Fig. 1. Schematic structure of the Abd-B

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

physical contact, while the intervening sequences loop
out. Although the looping model was formulated many
years ago, direct in vivo evidence for its validity has
been found only recently for the chicken beta-globin
gene cluster (reviewed in [7]). In this case, all sequences
necessary for the efficient transcription of one of the
genes in the cluster were found to be in close proxim-
ity, forming a ‘hub’, while inactive regions proved to
be pushed aside. The organization and functioning of
the Abd-B gene suggest that the looping model is also
applicable to the Abd-B regulatory unit. In A6, for
example, enhancers in iab-6 have to reach over the
entire inactive iab-7 region to act on the Abd-B pro-
moter (Fig. 2). However, the looping model raises the
question of how potentially promiscuous enhancers in
the cis-regulatory regions are able to avoid other pro-
moters, and to locate and physically approach their
proper target promoter in the viscous environment of
the nucleus.
Somatic pairing of chromosomes and
the regulation of Abd-B
A peculiarity of Drosophila is the fact that the homo-
logous chromosomes are tightly paired during the
interphase in almost all types of somatic cells (the
exceptions are cells in the early embryo), a situation
that occurs only exceptionally in most other eukaryo-
tes. Somatic pairing may affect long-distance regula-
tory interactions by interfering with loop formation. It
has been suggested that a gene may be regulated by
being switched between two states: in the case of un-

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

large distances (sometimes between different chromo-
somes). Two of these sequences are derived from the
Mcp [11], and the Fab-7 [12] regions. Both contain a
boundary and at least one PRE, and are able to medi-
ate long-distance regulatory interactions via the associ-
ation between homologous regions. In transgenic lines,
these sequences can interact with another copy inserted
somewhere else in the genome, or with their homolog-
ous sequence in the BX–C. These interactions between
distantly located copies usually result in silencing of
the reporter gene, or a gene next to the insertion site
of the transgene, although Mcp can also mediate posit-
ive regulatory interactions in exceptional cases [11].
However, these effects are observed only if at least one
copy of them is present in a transgenic insert, and the
significance of this high affinity pairing in the regula-
tion of the Abd-B is therefore unclear. Perhaps tight
homologous pairing between these sequences within
the BX-C plays a role in restricting the extent of loop-
ing-out domains.
Tethering elements
Deletion analysis of the Abd-B gene strongly suggests
the existence of a novel mechanism that tethers cis-
regulatory regions to the promoter-upstream region
[13]. It has been found that while Abd-B point muta-
tions do not complement the phenotype of an iab-7
deletion in A7, Abd-B alleles deleted for the promoter
region do complement iab-7 deletions in trans-hetero-
zygotes. The complementation is a result of the
action of the wild-type iab-7 on the wild-type Abd-B

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

iab cis-regulatory regions and the Abd-B gene when
they are separated by large-scale chromosomal rear-
rangements [17,18]. However, in contrast to previously
known cases of transvection in the BX-C, the tmr does
not require pairing with homologous sequences for its
effect. On the contrary, uninterrupted pairing of tmr
regions seems to prevent the trans–regulatory inter-
action between the Abd-B promoter and the iab
regions [13]. The unusual properties of the tmr led to
the hypothesis that it may be involved in a process
that normally targets the iab regions to the Abd-B pro-
moter. This assumption prompted a detailed analysis
of the tmr, which led to the identification of a set of
different cis-acting elements within it [19] (see Fig. 1C
for a detailed map of the region). Among these ele-
ments, a short sequence with highly intriguing pro-
perties has been suggested to play a major role in
directing distant enhancers to the Abd-B promoter.
This region is next to, or overlaps with the Fab)8
boundary, and is called promoter targeting sequence,
PTS [16]. It is important to note, however, that the
function of PTS is unlikely to be related to the tmr-
mediated trans regulation, as its deletion does not alter
the functioning of the tmr [16].
Promoter targeting sequence (PTS)
In transgenic assays, the PTS alone does not seem to
have a detectable function; it has to be placed next to
a boundary sequence for it to exhibit its intriguing
properties. Moreover, if a PTS+ boundary is placed
between two, divergently oriented reporter genes (5¢-to

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 is combined with a boundary: in Type I, the
enhancer is targeted to the proximal promoter, in Type
II, it is restricted to the distal promoter (Fig. 4, bot-
tom), and finally, in Type III, the boundary retains its
enhancer-blocking activity and no targeting occurs
(not illustrated). These three types of expression pat-
tern are stably maintained for many generations in the
different lines, and no reversion of the original choice
is observed [20]. A possible explanation for this unex-
pected stability of promoter targeting is that the partic-
ular chromosomal environment at the site of insertion
may determine the way the PTS functions in the trans-
gene. Recent experiments, however, ruled out this pos-
sibility, as even after mobilization and reinsertion of
the transgene into entirely different locations, the pro-
moter choice observed in the original strain was main-
tained in most cases [20]. These results strongly suggest
that the PTS and the promoter sequences are incorpor-
ated irreversibly into a protein-DNA complex in the
germ line cells, and the established contact is main-
tained through meiotic and mitotic cell divisions. For
the initial establishment of the PTS-promoter contact,
the presence of a boundary sequence is required: no
targeting is observed if the transgene does not contain
a boundary. However, the subsequent maintenance of
targeting does not require the presence of the bound-
ary, as its later removal has no effect on the pattern of
expression of the transgene. Thus, the specific task of
locating and contacting the target promoter by the
enhancers would be bypassed: a contact between 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

the Fab)7 boundary, but also a PTS, the function of
which is to overcome the enhancer-blocking effect of
the boundary. However, detailed analyses of the
Fab)7 boundary region [23,24], suggest that the mini-
mal boundary is slightly larger than the deletion used
in the swapping experiment [22]. Thus, it appears that
there is no space for a PTS-like sequence in the deleted
region. If this is the case, it follows that, although an
anti-insulator activity must be present in the iab-6
ci-regulatory region in order to overcome the enhan-
cer-blocking activity of the Fab)7 boundary, this
activity is unlike that of a generalized PTS, as it can
not bypass the su(Hw)orscs insulators, and therefore
appears to be adapted specifically to its normal part-
ner, Fab-7.
Trans-regulation-based experiments indicate that the
interaction between the PTS and the selected promoter
can not be as rigid as the transgenic results suggest.
As mentioned earlier, the homologous chromosomes
are tightly paired in the somatic tissues during the
Fig. 4. Schematic representation of transgenic constructs used to
assay PTS function. The PTS cannot overcome the enhancer-block-
ing effect of an adjoining insulator (brick patterned oval) when
placed 5¢ of two divergently transcribed reporter genes (top panel).
Each enhancer regulates (curved arrows) only the reporter gene
situated on the same side, relative to the boundary, of the con-
struct. However, when the combination of the PTS and the bound-
ary is placed 3¢ to one of the reporter genes, the enhancer is
targeted to one or the other promoter over the boundary (lower
panel). E1 and E2: enhancers; P1 and P2: promoters.

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
`

ve,
Switzerland), personal communication], arguing that
PTS is only one of the elements involved in targeting
iab-7 enhancers to the Abd-B promoter. The latter
assumption is indirectly supported by transvection
studies, indicating that a relatively large (larger than
7.6 kb) region just upstream of the Abd-B promoter is
involved in keeping the enhancers of different cis-regu-
latory regions near the promoter [13]. Deletion of these
sequences together with the promoter appears to free
the enhancers from a bond that tethers them to the
Abd-B gene in cis, and allows them to regulate the
expression of the Abd-B gene in trans (Fig. 3).
This observation suggests that promoter-upstream
sequences are critical for proper enhancer targeting in
the Abd-B domain. The observation that the larger the
deletion within this region, the stronger the resulting
trans regulation, indicates that the critical region is
built up from modules that function together (Fig. 3).
Such a complex system at the promoter suggests a
similarly complex counterpart at the side of the
enhancers, and is difficult to reconcile with a model
that attributes an exceptional role to a single PTS for
promoter targeting in Abd-B.
Taken together, the genetic studies suggest that in
its natural context the PTS region may function differ-
ently from that suggested by transgenic studies. Upon
incorporation into the transcriptionally inactive precur-
sor cells of the germ line, a protein complex may be
formed irreversibly on the transgenic DNA, this DNA-

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

studies would greatly benefit from a detailed know-
ledge of the looping structure of the Abd-B domain in
order to predict relevant regions. However, studies
similar to those on the chicken beta-globin gene cluster
are greatly hindered in the case of Abd-B by the fact
that the chromatin topology of the latter is likely to be
different in different segments. Hopefully, an increas-
ing number of reporter genes inserted within different
cis-regulatory regions, and advances in cell sorting
techniques, will overcome this problem in the near
future. Additional genetic studies may promote the
identification of the genes involved in the mediation of
long–range interactions between regulatory regions
and the Abd-B promoter.
Acknowledgements
We thank Welcome Bender, Francois Karch, Pe
´
ter Vil-
mos, Jo
´
zsef Miha
´
ly and Izabella Bajusz for their crit-
ical reading of the manuscript. H. G. is supported by
Long-distance interactions in Abd-B L. Sipos and H. Gyurkovics
3258 FEBS Journal 272 (2005) 3253–3259 ª 2005 FEBS
an NIH grant as a subcontractor and by the Hungar-
ian National Granting Agency, OTKA. L. S. is sup-
ported by an NIH FIRCA grant.
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