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

Long-distance interactions between enhancers andpromoters The case of the Abd-B domain of the Drosophila bithorax complex La´szlo´ Sipos and Henrik Gyurkovics Institute of Genetics, Biol

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-Btranscription 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: Henrik@brc.hu

(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.

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.

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 pairtim-ing 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

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.

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) or scs 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 adjoinenhancer-block-ing 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.

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

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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