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: 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. 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. 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