Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA
*Author for correspondence (e-mail: hcai{at}arches.uga.edu)
Accepted July 24, 2001
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SUMMARY |
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Key words: Chromatin boundary, Enhancer specificity, Insulator, Promoter competition, suHw, Drosophila
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INTRODUCTION |
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Core promoter sequences have been shown to influence the ability of promoters to compete for a given enhancer (Merli et al., 1996; Ohtsuki et al., 1998). Specifically, the contribution of core sequences to the promoter competitiveness has been shown with two early Drosophila enhancers, AE1 and IAB5. The autoregulatory enhancer (AE1) of the fushi tarazu gene (ftz) directs the expression in seven transverse stripes during germ band extension in Drosophila embryogenesis. AE1 selectively activates ftz but not the neighboring homeotic gene Sex combs reduced (Scr), in spite of its intergenic position and comparable distance from both promoters (Hiromi et al., 1985; LeMotte et al., 1989; Ohtsuki et al., 1998; Pick et al., 1990; Schier and Gehring, 1993). The AE1 promoter specificity could be determined by the differences in the basal promoters of the two genes: whereas the ftz promoter contains a canonical TATA box, the Scr promoter contains no optimally defined core promoter motifs such as the TATA sequence, initiator (INI) or downstream promoter element (DPE) (Burke and Kadonaga, 1996; Kutach and Kadonaga, 2000; Smale, 1997). The infra-abdominal 5 (IAB5) enhancer interacts specifically with the Abdominal B gene (Abd-B) and directs a broad band of expression in the presumptive abdomen of gastrulating embryos. Both AE1 and IAB5 contain binding sites for the FTZ activator. In transgenic Drosophila embryos, AE1 or IAB5 placed between two divergently transcribed reporter genes preferentially activates transcription from the TATA containing evenskipped (eve) promoter, but not from the TATA-less white promoter (Fig. 1A, also see Construct 1 in Table 1) (Ohtsuki et al., 1998). The promoter specificity of these two enhancers is due to the competition from the TATA containing eve, rather than to incompatibility with white, as the enhancers can activate the white gene if the eve promoter is replaced with a TATA-less promoter such as that from the Transposase gene; or if the TATA region in the eve promoter is changed to the corresponding region from white (Fig. 1B) (Ohtsuki et al., 1998). The contribution of core sequences to promoter competitiveness is also supported by the observation that inserting an 8 bp TATA sequence into the white promoter enhances its competitiveness and results in its activation against eve (Ohtsuki et al., 1998). The competitive nature of promoter selection is further demonstrated by the observation that the AE1-white interaction can be restored by blocking the competing AE1-eve interaction with the suHw insulator (Fig. 1C) (Ohtsuki et al., 1998).
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The molecular basis of insulator action is not known. The best understood example is suHw, a 340 bp DNA element from the Drosophila retrotransposon gypsy. The suHw insulator causes mutations when transposed into regulatory regions of various genes. It also acts as a boundary by shielding chromosomal position effect when flanking a transgene (Hagstrom et al., 1996; Roseman et al., 1993; Sigrist and Pirrotta, 1997). Both the mutagenic effect and the boundary function of suHw are related to its ability to disrupt enhancer-promoter interactions. In fact, enhancer-blocking activity is observed in insulators either identified as enhancer modulators, or as chromatin boundaries (Cai and Levine, 1995; Chung et al., 1997; Kellum and Schedl, 1992; Milot et al., 1996; Scott and Geyer, 1995; Zhou et al., 1996). The insulator activity of suHw requires the function of two cellular proteins: SuHw, which directly interacts with the suHw DNA element, and Mod(mdg4), a chromosomal protein that interacts with the insulator element through SuHw (Cai and Levine, 1995; Gerasimova et al., 1995; Geyer and Corces, 1992; Parkhurst et al., 1988).
Previous studies have shown that insulators can function quantitatively and selectively. The strength and selectivity of their enhancer-blocking function depend on the qualitative and quantitative characteristics of the insulator and enhancers involved (Cai and Levine, 1995; Hagstrom et al., 1996; Scott et al., 1999; Zhou et al., 1996). In this study, we have investigated how promoter competitiveness and gene configuration affect insulator function. The suHw-mediated blockage of the AE1 enhancer was examined between two divergently transcribed fusion genes in transgenic embryos. We show that the properties of the core promoters that interacts with AE1 (interacting promoters) determine both their competitiveness for AE1 and their susceptibility to the suHw-mediated blockage. In addition, the suHw-mediated blockage of AE1 is also influenced by the neighboring promoters (competing promoters). Promoter-AE1 interactions strongly challenged by competing promoters are more susceptible to the suHw-mediated blockage. These observations provide evidence for a novel mechanism through which insulator function is modulated according to its regulatory contexts.
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MATERIALS AND METHODS |
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Construction of AE1 fusion promoter constructs
All P-transposons used in this report are derivatives of pCaSPeR containing the mini-white marker/reporter gene. Construction of the white-AE1-eve and white-AE1-suHw-eve transgenes has been described previously (Ohtsuki et al., 1998). For constructs containing ftz-Scr, ftz-white, ftz-eve, Scr-eve and Scr-white promoter pairs, BamHI-EcoRI fragments containing minimal promoter elements and a short coding region containing the first ATG codon were excised from pFEP (eve) (Ohtsuki et al., 1998), and pFpSu3 (ftz and Scr; S. Ohtsuki, personal communication). Pairwise combinations of purified promoter fragments were then ligated and cloned into the BamHI site in a pBluescript vector (KSEcoRI), resulting in dual-promoter subclones (KS-2Ps). EcoRI fragments containing AE1 and AE1-suHw, respectively, were inserted into the EcoRI site between the two promoters in each of the KS-2P plasmids. The position and orientation of the enhancer and insulator were determined by restriction digestions and DNA sequencing. BamHI fragments containing dual-promoter combinations with AE1 or AE1-suHw between the two promoters were then ligated into pCaSPeR based injection vectors containing divergently pointed CAT and lacZ reporter genes. The resulting constructs contain AE1 or AE1-suHw between divergently transcribed promoters fused in-frame with reporter genes. The transgenic constructs were characterized extensively by restriction digestion and some by sequencing analysis before used for the micro-injection procedure as described above.
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RESULTS |
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suHw function is influenced by the interacting and neighboring promoters
The differential blockage of AE1 by suHw in the above two pairs of transgenes could result from different interacting promoters (eve in Fig. 1C, and ftz in Fig. 2C), which directly participate in the interactions the insulator impedes. Previous studies have shown that the promoter preference of AE1 depends on the presence of the TATA core motif and that eve and ftz, both containing TATA motif, can compete comparably for AE1 (Ohtsuki et al., 1998). However, the sequences immediately flanking the TATA motif diverge significantly between the eve and ftz promoters, so it is possible that AE1-ftz interaction is stronger and not blockable by the suHw insulator. Alternatively, the differential blockage of AE1 by suHw may result from the different neighboring competing promoters in the two transgenes (white in Fig. 1C, Scr in Fig. 2D), which could influence the ftz-AE1 interaction through promoter competition, thereby altering the effectiveness of suHw.
To distinguish between these two possibilities, we examined the effectiveness of suHw in blocking the same ftz-AE1 interaction against different neighboring promoters. For the following constructs, we used the white and eve promoters, whose core sequences have been shown to affect their ability to compete for enhancers such as AE1 and IAB5 (Fig. 1) (Ohtsuki et al., 1998). The white promoter, which lacks TATA but contains an INI and a DPE, was first tested in place of the Scr promoter, which lacks any recognizable core promoter motif (Fig. 3A-D, Table 1 (Constructs 5, 6)). As in the white-eve promoter pairing, the TATA containing ftz promoter is preferentially activated by AE1 at the expense of the white promoter (Fig. 3A,B, compare with Fig. 1A; Table 1 (Construct 5)). However, when suHw was placed between the ftz promoter and AE1, their interaction is attenuated by the insulator, not completely blocked as seen with eve-AE1 (Fig. 3d, compare with Fig. 1C; Table 1 (Construct 6)) (Ohtsuki et al., 1998). Comparison of the suHw-mediated blockage of ftz-AE1 and eve-AE1 interactions, when opposed by the same white promoter, shows that the difference in the interacting promoters does affect the insulator effectiveness. It indicates that although eve and ftz both contain TATA elements, eve-AE1 interaction appears significantly weaker than ftz-AE1 interaction in terms of resistance to the suHw-mediated blockage. More interestingly, the suHw-mediated blockage of the same ftz-AE1 interaction is significantly enhanced when the competing promoter changed from Scr to white (Fig. 3C,D, compare with Fig. 2C,D; Table 1 (Constructs 4, 6)). This result confirms that the neighboring promoters, although out-competed by ftz and transcriptionally silent (Fig. 1A, Fig. 2A), indeed influence the dynamics of AE1-ftz interaction and therefore the effectiveness of suHw. It is also worth noting that the attenuated ftz-AE1 interaction is not accompanied by an increase in the white-AE1 interaction (Fig. 3C,D, Table 1 (Construct 6)), although such redirection was seen when the eve-AE1 interaction was completely blocked (Fig. 1C; Table 1 (Construct 2)) (Ohtsuki et al., 1998).
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Our previous analyses have demonstrated that promoter competition, which depends on the core promoter sequences, is important for enhancer-promoter specification (Ohtsuki et al., 1998). The use of the AE1 enhancer and the same set of promoters in the current study further suggests that promoter competition is the underlying mechanism through which neighboring genes modify the outcome of insulator function. These findings provide the first evidence for a mechanism through which the effectiveness of an insulator is determined by regulatory context of neighboring genome. Our results further suggest that the greater the difference between the two competing promoters, the more difficult it is to insulate the enhancer from the interacting promoter (usually the more competitive one). Conversely, a given insulator would be more effective modulate enhancer specificity among more comparable promoters.
Modulation of the insulator function by the neighboring genome is not limited to the cognate ftz-AE1 interaction or TATA promoters
The ability of suHw to block the ftz-AE1 interaction is sensitive to the influence of neighboring promoters, as shown by the above three pairs of transgenes containing dual promoters (Scr-ftz, white-ftz and eve-ftz). To analyze if the effect of the competing promoters is unique to the cognate ftz-AE1 interaction, we constructed transgenes containing the Scr-eve promoter pair to complete a second series of transgenes with eve as the interacting promoter (Scr-eve, white-eve and ftz-eve). As seen in Fig. 4A,B, AE1 preferentially activated eve but not the Scr promoter (also see Table 1 (Construct 9)). However, when suHw is inserted in between eve and AE1, the interaction between the two elements is partially impeded without stimulation in the Scr-AE1 interaction (Fig. 4C,D, Table 1 (Construct 10)). The partial blockage suggests that the difference between eve-Scr is smaller than that of ftz-Scr, but greater than that of eve-white, which is consistent with our observations from previous and current studies of competitive interactions among these promoters. These result indicate that the competing influences from neighboring genomic context can modulate insulators effectiveness in blocking both cognate and non-cognate interactions.
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DISCUSSION |
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Interacting promoters
In the current study we have further analyzed the suHw-mediated insulation as a function of the interacting promoters that differ in their core promoter sequences and in their ability to compete for AE1. Previous studies have shown that distinct cis elements in the core promoter such as a TATA, INI and DPE, and their associated trans-factors determine the ability of the promoter to interact and compete for regulatory enhancers (Merli et al., 1996; Ohtsuki et al., 1998). We found that the suHw-mediated blockage of AE1 depends on the promoter with which it interacts, as shown by the complete blockage of AE1 from white, but not from eve or ftz, when opposed by Scr; or the complete blockage of AE1 from eve, but not from ftz when opposed by white. Our results suggest that the ability of a promoter to compete for an enhancer correlates with the ability of their interaction to resist insulator blockage. These abilities may reflect a characteristic of the enhancer-promoter interaction that is distinct from the one reflected in transcriptional activation. Our results further indicate that even promoters with the same core motifs, such as the TATA sequence, may differ significantly in their interactions with a given enhancers, suggesting a role for sequences outside of core motifs to also contribute to enhancer specificity. Recent studies indicate that different TATA-binding proteins (TBP, TRFs) and/or TBP associated factors (TAFs) may interact with distinct TATA promoters to confer gene and tissue specificity (Buratowski, 1997; Holmes and Tjian, 2000). The transcription complexes assembled at the these basal promoters may be different and so are the interactions they forge with upstream regulatory proteins.
Competing promoters
A key finding from our study is that insulator function is affected by the balance of promoter competition among neighboring genes. The ftz-AE1 interaction becomes more susceptible to the suHw-mediated blockage when challenged by neighboring promoters such as eve, which has been shown in our previous study to be highly competitive for AE1, owing to the presence of the TATA sequence. The same ftz-AE1 interaction is less susceptible when opposed by non-competitive promoters such as white or Scr. Our results indicate that the neighboring promoters, although out-competed by ftz and apparently transcriptionally silent, can nonetheless alter the dynamics of the ftz-AE1 interaction and the effectiveness of suHw in blocking it. This property, which we showed with different types of promoters both at interacting and at competing positions, afford insulators with a greater regulatory flexibility according to the integrated input from its genomic context.
It is not known how an enhancer interacts with multiple competing promoters. Previous studies indicate that an enhancer may form a large complex that includes multiple promoters (Freidman et al., 1996), or it may alternate between separate enhancer-promoter complexes (the flip-flop model (Milot et al., 1996; Wijgerde et al., 1995). In our paired promoters configuration, the insulator function is not compatible with a complex formed between the enhancer and the interacting promoter, but is compatible or even synergistic with a complex formed between the enhancer and the competing promoter. This model predicts that a strong competing promoter enhances insulator function, which we observe.
A surprising observation from our data is that insulator function does not necessarily increase sharing of an enhancer among neighboring genes. For example, AE1 is not shared between promoters very different in their competitiveness (e.g. between eve and Scr, or ftz and white), even with the aid of an insulator. AE1 remains specific even when it is partially blocked. As the differences between the competing promoters reduce, the suHw-mediated insulation becomes complete, upon which the redirection of the AE1 interaction to the competing promoters occurs. The redirection occurs in an all or none fashion: no expression was detected from the competing promoters in cases of partial blockage. The reason for such an all or none switch is not clear. However, it is possible that the synergistic interaction between the formation of the complex among insulator components and the complex between the competing promoter and the enhancer could contribute to such abrupt transitions.
Selective insulation
Selective insulation (or differential blockage) has been documented for several insulator and boundary elements in Drosophila (Cai and Levine, 1995; Hagstrom et al., 1996; Muller, 2000; Scott et al., 1999). It is now our understanding that insulators, including those identified as boundary elements and those identified as enhancer blockers, are not impenetrable walls to transcriptional interactions. Rather they function with great flexibility depending on the regulatory context, including the characteristics of the interacting enhancer, promoter and competing interactions within the neighboring genome. The selectivity for any given insulator therefore reflect, in addition to the intrinsic characteristics of the insulator, but also the apparent effectiveness with which it respond to its cis and trans environment.
Integrated transcription regulation and genome organization
Promoter competition and insulator function are two important mechanisms that specify enhancer-promoter interaction in complex genetic loci. Our results demonstrate that these two mechanisms are interdependent, or may even be synergistic at times. The regulatory implication of such synergy is that enhancer specificity among different types of promoters may be determined mainly through promoter competition, and that insulators exert a greater influence among genes with more comparable promoters. Although our study focused on the effect of promoter competition, it is possible that other types of regulatory elements such as competing enhancers also exert similar influences on insulators through competition with promoters. The range and intensity of these influences may vary, but the regulatory interdependence could in principle relay along neighboring genes through cross-interactions between promoters, enhancers and insulators (see Fig. 6). The functional interdependence of regulatory elements in such genomic contexts imposes organizational restraints on closely linked gene groups (see Fig. 6). Change in the relative positioning or regulatory capacity of any one component could influence the outcome of neighboring interactions, and in turn, interactions further away, thereby linking a larger genomic region into one regulatory, organizational and evolutionary unit. The observations from transgenic Drosophila may reflect aspects of regulatory principles in authentic gene complexes and provide a molecular explanation for the highly conserved genetic organization of the Hox genes during evolution.
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ACKNOWLEDGMENTS |
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