bxdPRE silencing activity depends on the chromosomal context
In previous studies, PREs were found to repress expression of reporter genes in only about half of the transgene insertion sites [38,39,40,41, 58, 59]. To better understand the context-dependent factors that impact PRE activity, we used the ΦC31 site-specific integration system [60] to generate independent Drosophila transgenic lines. For this purpose, we selected five attP sites that had previously been shown to provide a context in which the expression of a white reporter is not subject to obvious repression or activation by the surrounding chromatin neighborhood (Additional file 1: Table S1). Three attP sites are on the 2nd chromosome (22A, 51C, and 58A), while two are on the 3rd chromosome (68E and 96E). As a PRE, we selected a well-characterized 656-bp bxdPRE element from the Ubx regulatory region [30, 31, 61, 62] (bxd construct, Fig. 1a). To evaluate the silencing activity of the bxdPRE as a reporter, we used a white gene with its eye tissue-specific enhancer (E). The bxdPRE was flanked by ~1 kb “neutral” spacers derived from coding regions of the eGFP and RFP genes and by terminators of transcription (SV40 terminator upstream and yellow gene terminator downstream) to reduce the influence of potential transcription from surrounding genomic sequences. To better assess the effects of the PRE, we also inserted a control construct at each site, which has all of these components except for bxdPRE (E-w construct, Fig. 1a). The transgene constructs were inserted in w- attP lines lacking a functional white gene. The silencing activity of the bxdPRE was assessed by the reduction in eye pigmentation as pigmentation is known to be directly correlated with the level of white gene transcription [62, 63].
Shown in Fig. 1b, c are the eye color phenotypes of hemizygous and homozygous flies for each of the five insertion sites. In the five lines carrying the E-w control transgene, there is no evidence of repression in either hemizygotes or homozygotes (Fig. 1b). In contrast, different degrees of repression are observed in 4 of the 5 lines with the bxd transgene (Fig. 1c). Line 22A is a classic example of PSS. It shows little or no evidence of silencing as a hemizygote, while as a homozygote there is a strong and almost uniform reduction in pigmentation. Though PSS is observed for line 58A, it differs from line 22A in that only a small sector of the eye shows a significant loss of pigmentation as a homozygote. Unlike 22A and 58A, weak silencing is observed in line 51С as a hemizygote. However, PSS is also evident as silencing is clearly enhanced when the flies are homozygous for the insert. For line 68E, eye pigmentation is greatly reduced in both hemizygous and homozygous flies. Finally, the bxdPRE is unable to induce silencing at 96E insertion site, suggesting that this chromatin environment renders the PRE inactive. In all cases, flies within a given line have similar eye pigmentation phenotypes. Since PSS is thought to be dependent upon homolog pairing, the different properties of the five insertion sites could be due to differences in the strength of local homolog pairing. To investigate this possibility, we took advantage of a recent genome-wide study that measured interaction frequencies between homologs at the embryo stage [64]. However, analysis of the available Hi-C data did not show any significant correlations (Additional file 2).
Silencing is expected to be accompanied by the association of PcG proteins with the bxdPRE. To confirm that this is the case, we isolated chromatin from adult heads of homozygous bxdPRE transgene flies and performed chromatin immunoprecipitation (X-ChIP) with antibodies against Ph, which is a core component of the PRC1 complex (Fig. 1d). To compare Ph association in different lines, we calculated the extent of enrichment relative to an internal positive control. For this purpose, we used primers to a sequence immediately adjacent to the endogenous bxdPRE (hereafter referred to as bxdPRE-Genome) that is known to be enriched in PcG/TrxG proteins. Figure 1c shows that Ph is associated with the transgenic bxdPRE in the four lines that show silencing of the white gene. Moreover, the extent of association correlates well with the level of repression observed in each line. Consistent with the lack of silencing of white in the 96E insertion, Ph is not found to be associated with its bxdPRE sequence.
Multimerized sites for Su(Hw) boundary induce bxdPRE silencing at 96E
PREs are often located near other transcriptional regulatory elements. As noted above, the PREs in the four Abd-B regulatory domains are positioned close to the boundary elements for each domain. Another example is one of the PREs for the even-skipped (eve) gene that is located next to the distal boundary of the eve locus homie [32]. These observations led us to wonder whether boundary elements might be able to augment the silencing activities of PREs.
To explore this possibility, we selected the 96E attP since the bxdPRE is unable to silence white at this insertion site. As the test boundary, we used an artificial element consisting of multimerized binding sites for the polydactyl zinc finger protein Su(Hw) rather than an endogenous boundary. Endogenous boundaries contain binding sites for many different proteins, and some are known to be required for PRE activity. For example, the GAF protein is implicated not only in insulation but also in PcG-dependent silencing [65].
The Su(Hw) protein is responsible for the boundary activity of the insulator element associated with the gypsy transposon [66, 67]. The gypsy transposon has 12 binding sites for the Su(Hw) protein [68, 69]; however, previous studies have shown that a multimer consisting of only four copies of the third Su(Hw) binding site from the gypsy insulator is sufficient for boundary activity in transgene reporter assays [70] and in the context of BX-C [71]. This 4xSu(Hw) multimer was placed on the distal side of the bxdPRE (Fig. 1a: Su-bxd construct) so that the PRE is between it and the white gene. In this position, the 4xSu(Hw) multimer would not be able to insulate white from PRE-dependent silencing [54, 57, 72]. To assess the effects of the multimer alone, we inserted a control transgene containing 4xSu(Hw) but not the PRE (Fig. 1a: Su construct). Figure 1e shows that combining 4xSu(Hw) with the bxdPRE has a dramatic effect on white expression. Silencing of white is evident in hemizygotes, while strong PSS is observed when the transgene is homozygous. In contrast, the 4xSu(Hw) multimer alone has no effect on eye pigmentation either as a hemizygote or a homozygote. Thus, the presence of the 4xSu(Hw) multimer can induce the establishment of silencing by the bxdPRE in a chromosomal location that is not conducive to PcG-dependent silencing.
Su(Hw) binding facilitates recruitment of PcG/TrxG and PRE DNA-binding proteins to the bxdPRE at 96E
As shown above, Ph is recruited to bxdPRE insertions that are able to repress white expression but is not found associated with the bxdPRE at 96E. If the addition of the 4xSu(Hw) multimer induces PcG-dependent repression, it should also facilitate the recruitment of Ph to the 96E bxdPRE. To test this prediction, we used ChIP to examine Ph association at 5 sites in the Su-bxd and bxd transgenes: (1) the distal end of the spacer sequence upstream of bxdPRE, (2) bxdPRE, (3) the distal end of the spacer sequence downstream of bxdPRE, (4) the white transcription unit, and (5) the white promoter (Fig. 2a). As a negative control, we used the Ras64B coding region, while the bxdPRE-Genome region was used as a positive internal control.
As would be predicted from the activation of silencing, there is a substantial increase in Ph association with the bxdPRE in the Su-bxd transgene as compared to the bxd transgene (Fig. 2b). While Ph is enriched at the bxdPRE, its association with the four other sites in the Su-bxd transgene is essentially the same as in the bxd transgene or the negative Ras64B control. This result is consistent with previous ChIP experiments in which we found that Ph (as well as several other PcG proteins: see below) associates with the bxdPRE element in the transgene construct, but not with other sequences even though white expression is silenced [62]. Like silencing, the recruitment of Ph requires a combination of the bxdPRE and the 4xSu(Hw) multimer as Ph is not associated with the control transgene carrying only the 4xSu(Hw) multimer (Additional file 1: Figure S1b). Consistent with the idea that the presence of Su(Hw) protein bound to its target sites is responsible for the acquisition of silencing activity and the recruitment of Ph, we find that Su(Hw) associates with the 4x multimer not only in the Su transgene but also the Su-bxd transgene (Additional file 1: Figure S1c).
Biochemical and genetic studies have shown that, like other PREs, the silencing activity of the bxdPRE depends upon several DNA-binding proteins that help recruit the PRC1 and PRC2 complexes. Thus, one explanation for the inability of the bxdPRE element alone to silence white at 96E is that these DNA-binding proteins are unable to associate with the bxdPRE. In this model, these proteins would be able to access their recognition sequences in the PRE when the 4xSu(Hw) multimer is included in the transgene, but not when it is absent. The DNA-binding proteins known or thought to be important for bxdPRE silencing include Pho, which binds PREs together with its partner Sfmbt (the PhoRC complex) as well as the Combgap DNA-binding protein. An alternative model is that the bxdPRE at 96E is unable to silence because it is occupied by TrxG proteins and these factors block association of Ph and other PcG proteins and/or their function. In this case, the presence of the 4xSu(Hw) multimer could shift the balance to favor of the recruitment of PcG complexes.
To test these two models, we used antibodies against Pho, Sfmbt, Combgap, and two TrxG proteins, Trx and CBP, for ChIP experiments. In the bxd transgene, we observe only background levels of Pho, Sfmbt, and Combgap in ChIPs for the PRE and other sequences in the transgene (Fig. 2c–e). In contrast, all three of these proteins are detected at the bxdPRE in the Su-bxd transgene. These results are consistent with the predictions of the first model. We infer from these finding that the presence of the 4xSu(Hw) multimer induces the association of key DNA-binding proteins with the bxdPRE.
The second model predicts that TrxG proteins will be associated with the transgene containing the bxdPRE alone, but will be displaced by PcG proteins when the 4xSu(Hw) multimer is present. However, like the PcG proteins, the Trx and CBP are recruited to the bxdPRE only when the 4xSu(Hw) multimer is present (Fig. 2f, g). Thus, the boundary induces the association of PRE DNA-binding proteins as well as both PcG and TrxG factors to the bxdPRE. It remains to be determined whether this is actual co-occupancy or whether it reflects instead a heterogeneity in the population such that some PREs are occupied by PcG proteins while others are occupied by TrxG proteins.
The Su(Hw) multimer augments bxdPRE silencing activity at different chromosomal sites
Genome-wide ChIPs indicate there is a minor Su(Hw) peak that overlaps the attP site at 96E, while there are two larger peaks on either side of the attP ~5 kb and ~10 kb away (Additional file 3, data from [73, 74]). This raises the possibility that the 4xSu(Hw) multimer is able to rescue the silencing activity of the bxdPRE at this particular attP site only because of the endogenous Su(Hw) protein. For this reason, we asked whether the 4xSu(Hw) multimer is able to augment bxdPRE silencing at the four other attP sites which do not have peaks for Su(Hw) nearby (Additional file 3). To control for the effects of the Su(Hw) binding sites on transcriptional activity, the Su transgene was inserted at these other attP sites as well. Shown in Fig. 3 is a comparison of the silencing activity of the bxdPRE with and without the 4xSu(Hw) multimer (Fig. 3a,b) and 4xSu(Hw) multimer alone (Fig. 3c). For inserts at 22A, 51C, and 58A, silencing of white in hemizygotes is enhanced when 4xSu(Hw) is included next to bxdPRE in the transgene (Fig. 3b). While there is no obvious effect on the silencing of inserts at 68E (Fig. 3b), the bxdPRE already strongly silences on its own as either a hemizygote or homozygote (Fig. 3a). In homozygotes, the 22A insertion containing 4xSu(Hw) and bxdPRE is lethal, while the PSS observed for the 58A insert is clearly stronger when the 4xSu(Hw) multimer is present (Fig. 3b). Although there is no obvious difference between hemizygotes and homozygotes for the Su-bxd transgene inserted at 51C, silencing is substantially greater when the 4xSu(Hw) multimer is present (Fig. 3b). For the control construct, the Su(Hw) multimer alone, there is no evidence for silencing in any of these lines either in hemi- or homozygotes (Fig. 3c). Taken together, these findings indicate that the 4xSu(Hw) multimer can augment the silencing activity of the bxdPRE in different chromosomal environments.
Binding sites for architectural proteins CTCF and Pita can induce bxdPRE silencing
Next, we wondered whether induction of PRE repressing activity is unique to Su(Hw) or whether other polydactyl zinc finger proteins that have chromosome architectural functions are able to induce PRE silencing. To test this, we linked the bxdPRE to either a Drosophila 4xCTCF multimer or a 5xPita multimer. Like 4xSu(Hw), both of these multimers have insulating activity in BX-C boundary replacement experiments [71, 75,76,77]. Figure 4 shows that combining either 4xCTCF or 5xPita with the bxdPRE induces silencing activity in hemizygotes and PSS in homozygotes. Control experiments show that white expression is not affected when the multimers are included in the transgene alone. Thus, multimerized sites for three different chromosomal architectural proteins can induce the silencing activity of the bxdPRE.
Su(Hw) architectural protein induces enPRE silencing activity
We also used the same strategy to test the silencing activity of another well-defined PRE, the 181 bp enPRE (PSE2) from the engrailed locus, in different chromosomal environments. In previous P-element transgene experiments, this element was shown to repress white expression as a hemizygote and/or homozygote and maintain the parasegmental expression pattern of a Ubx reporter [33, 38, 46, 59, 78,79,80,81,82]. Surprisingly, however, repression of white by the enPRE in hemizygote flies is weak or nonexistent at all five attP insertion sites and there is no evidence of PSS when the inserts are homozygous (en construct, Fig. 5a, b) Moreover, Ph binding is not detected at the enPRE in homozygous insertions of this construct (Fig. 5c).
Since the lack of silencing was unexpected, we tested whether silencing activity could be induced by linking the 4xSu(Hw) multimer to the enPRE (Su-en construct, Fig. 5a). For this purpose, we chose the 68E attP integration site as it was most permissive for bxdPRE silencing. Figure 5b shows that the eye pigmentation of hemizygous Su-en flies is similar to that observed for en flies; however, silencing is observed in flies homozygous for the 68E insert. This is opposite of that observed for the en transgene at 68E where the eye color in homozygotes becomes darker not lighter. ChIP experiments provide further evidence that 4xSu(Hw) is able to activate PcG-dependent silencing. While Ph and Sfmbt are not associated with the enPRE in the en transgene, both are recruited to the enPRE when it is linked to 4xSu(Hw) (Fig. 5c).
Increasing the distance between the bxdPRE and the boundary element disrupts silencing in hemizygotes
How do the architectural proteins help establish PRE activity? Two different mechanisms could be in play. The architectural proteins could facilitate the establishment of PcG silencing by locally displacing nucleosomes and/or by recruiting chromatin remodeling complexes so that key DNA-binding factors and PcG complexes are able to assemble on the PRE. Alternatively, the architectural proteins could enhance PRE activity by helping target the transgene from an active chromosomal compartment to a PcG-silenced chromosomal compartment [83]. Since the effects of nucleosome displacement and chromatin remodeling are expected to be limited to closely linked sequences, the former model predicts that the impact of architectural proteins on the establishment of PcG silencing will decrease as the distance between the multimers and the bxdPRE is increased. In the latter model, relatively small changes in distance (<10 kb) should have little or no effect on the ability of the boundary to target the transgene to a PcG-silenced compartment.
To test these two models, we increased the distance between the 4xSu(Hw) or 4xCTCF multimers and the bxdPRE by 1 kb and 3 kb (Fig. 6a). For the distance of 1 kb, we inserted each multimer upstream of the left eGFP coding sequence spacer (the Su-1kb-bxd and CTCF-1kb-bxd constructs). To adjust the distance between the multimers and the PRE to 3 kb, we introduced an additional 2 kb spacer derived from the Escherichia coli LacZ coding sequence (the Su-3kb-bxd and CTCF-3kb-bxd constructs). A 1-kb distance would be sufficient for approximately five nucleosomes, while 3 kb would correspond to about fifteen nucleosomes. All of the constructs were then introduced into the 96E attP site. The results of this analysis are shown in Fig. 6. In hemizygotes (P/+) increasing the distance between the multimer and the bxdPRE adversely impacts silencing activity. For the 4xSu(Hw) multimer, a distance of 1 kb is sufficient to substantially reduce silencing (compare with bxdPRE alone). In the case of the 4xCTCF multimer, the disruption of silencing is greater when it is located 3 kb away from the bxdPRE than it is at a 1-kb distance; however, even at 1 kb, silencing is reduced compared to the control CTCF-bxd construct. These findings argue in favor of the first model, namely that the 4xSu(Hw) and 4xCTCF multimers act in cis to facilitate the assembly of silencing complexes on the PRE (Fig. 6c).
Boundaries located at a distance from the bxdPRE can induce PSS
While silencing activity in hemizygotes is significantly compromised when the multimers are moved away from the bxdPRE, this is not true for PSS. As shown in Fig. 6 (P/P), the bxdPRE represses white expression even when the boundary multimers are located 3 kb away. A plausible explanation for this result is that pairing of the 4xSu(Hw) or 4xCTCF multimers in trans would tend to stabilize pairing interactions between the PREs on each homolog, and this interaction facilitates the PSS-dependent assembly of functional silencing complexes.
Boundary pairing in Drosophila depends upon specific interactions between proteins associated with each element [56, 84,85,86]. A classic example of specificity comes from the boundary bypass assay. In this assay, an upstream regulatory element (enhancer or silencer) is separated from a reporter gene by a spacer DNA that is flanked by two boundary elements (endogenous or artificial). If the boundaries flanking the spacer DNA can pair with other, the upstream regulatory element is brought into close proximity with the reporter and can either activate (enhancer) or repress (silencer) its expression [48, 55, 57, 87, 88]. This is what is found when the spacer DNA is flanked by either Su(Hw) or CTCF multimers [81]. On other hand, bypass is not observed when the spacer DNA is flanked by a heterologous combination of Su(Hw) multimers and CTCF multimers [89]. Thus, if boundary-boundary interactions between homologs are required to induce PRE repression, then PSS should not be observed in the two sets of mixed pairs: Su-1kb-bxd trans to CTCF-1kb-bxd or Su-3kb-bxd trans to CTCF-3kb-bxd. Figure 6b shows that this prediction is correct: silencing depends on boundary pairing in trans and is not observed in heterologous combinations. A possible model is shown in Fig. 6d.
Binding sites of architectural proteins leads to local decrease of histone H3 enrichment
The finding that the boundary element must be closely linked to the bxdPRE for silencing in hemizygotes and enhanced PSS in homozygotes suggests that the boundary has a local effect on chromatin structure that enables PcG factors to gain access to the PRE and assemble functional silencing complexes. If this suggestion is correct, then the chromatin organization of the bxdPRE should be altered when it is closely linked to a boundary element. To test this prediction, we analyzed histone H3 association with six unique sequences (1n-6n) located at different distances upstream and downstream of the BamH1 site used to insert test DNAs (Fig. 7a). The distance between the midpoints of the 3n and 4n sequences and the BamH1 site are 75 bp and 83 bp, respectively. The midpoints of 2n and 5n are 187 bp and 233 bp from the BamH1 site, respectively, while those for 1n and 3n are 301 and 365 bp.
In the control E-w transgene, histone H3 association as measure by ChIP is nearly the same in all regions tested (1n-6n) and is equivalent to that observed for the control genomic sequence in the coding region of the Ras64B gene (ras) (Fig. 7b). The inclusion of the bxdPRE in the transgene (bxd) has no apparent effect on histone H3 occupancy and the profile across the six sequences is similar to that of the E-w control. A different result is obtained for Su, CTCF, or Pita transgenes containing the multimerized binding sites for the boundary proteins. In all three cases, there is a reduction in histone H3 occupancy in the sequences located immediately next to the multimers (Fig. 7b). This finding indicates that the multimerized sites for these three chromosomal architectural proteins generate a region that is depleted in nucleosomes. This effect is local and does not extend to sequences located more distant (1n, 2n, 5n, and 6n) from the multimerized sites.
We next examined the Su-bxd, CTCF-bxd, and Pita-bxd transgenes (Fig. 7b). As was observed for the transgenes containing only the multimerized binding sites, histone association with the two sequences, 3n and 4n, that immediately flank the multimer-bxdPRE combination is reduced. In each case, the reduction is approximately the same as that observed for the corresponding multimer alone. Importantly, this is true for 4n, which is separated from the multimers by the 650 bp bxdPRE. These results provide strong support for the idea that closely linked boundary elements can induce alterations in the association of nucleosomes with the PRE. Interestingly, this is not the only alteration in nucleosome association evident in these transgenes. In all three of the multimer-bxdPRE combinations, we found that histone H3 association with the more distant sequences (1n, 2n, 5n, and 6n) is enhanced relative to the various control transgenes (E-w, bxd and Su, CTCF, and Pita). Since enhanced association is not observed with the inactive bxdPRE or with any of the multimers alone, it seems likely that this effect is due to the recruitment of functional PcG complexes to the bxdPRE. It remains to be determined whether this alteration in histone H3 association reflects an increase in nucleosome density in the flanking DNA regions or an increase in the extent of compaction.
Interplay between PREs and boundary contributes to silencing
The studies in the previous sections indicate that boundaries can enhance PRE silencing by two different mechanisms. One takes place in cis and requires a close linkage of the boundary and PRE. This mechanism locally alters the pattern of histone association and facilitates the recruitment of factors critical for PcG repression. The other takes place in trans and is mediated by boundary:boundary pairing interactions. In this second mechanism, boundaries appear to provide a “spot weld” that holds the homologs in close proximity. This facilitates PRE:PRE interactions and results in PSS even when the boundary multimers are separated from the PREs. We undertook several additional experiments to further explore this “pairing” mechanism.
Boundary pairing can induce silencing in trans: When 4xSu(Hw) or 4xCTCF is placed next to the bxdPRE, the PRE can assemble a functional silencing complex and repress white in hemizygotes. However, the bxdPRE would not be expected to efficiently silence white in trans unless the homologs are tightly paired in the immediate neighborhood. To test this expectation, we generated trans-heterozygotes between the starting transgene E-w (the eye enhancer—white gene control construct which has all of the elements in the bxd construct except bxdPRE) and either Su-bxd or CTCF-bxd (Fig. 8a). While the bxdPRE silences the white gene in cis (Fig. 8a-II, Su-bxd/+ and CTCF-bxd/+ - compare with E-w/+ in Fig. 8a-I), it does not efficiently silence the white gene in trans (Fig.8a-III, Su-bxd/E-w, and CTCF-bxd/E-w: compare with E-w/+ and E-w/E-w in Fig. 8a-I). In these two combinations, the eye color phenotype is indistinguishable from that observed in E-w /+ or E-w/E-w. A different result is obtained when both transgenes have a copy of the same multimer (Su-bxd/Su or CTCF-bxd/CTCF, Fig. 8a-IV—compare with Su-bxd/E-w and CTCF-bxd/E-w, Fig.8a-III). While silencing is not as effective as when the two transgenes not only have identical multimers but also a copy of the bxdPRE (see Fig. 8a-V, Su-bxd/Su-bxd, CTCF-bxd/CTCF-bxd), the level of white expression is clearly reduced compared to that observed when the multimer is not present in the E-w transgene. This finding indicates that boundary:boundary pairing interactions can promote trans silencing by a PRE. To confirm that pairing interactions between the boundaries in the two transgenes provide a “spot weld” that facilitates trans silencing activity, we tested heterologous multimer combinations that do not pair with each other. As shown in Fig. 8a-VI, silencing is not observed with the Su-bxd/CTCF combination, nor is it observed with the converse CTCF-bxd/Su combination.
Boundary pairing is not necessary when both PREs are active: Placing multimerized binding sites for zinc finger architectural proteins next to the bxdPRE induces the assembly of functional silencing complexes in hemizygous flies and enhances PSS in homozygous flies. If the PREs on both homologs are activated by multimerized binding sites, then boundary:boundary pairing interactions would not be expected to be required for generating the synergistic trans interactions between PREs on each homolog that are responsible for PSS. To test this prediction, we generated trans combinations of bxdPRE transgenes that have closely linked multimerized Su(Hw), CTCF, or Pita-binding sites. Figure 9a-II shows that the silencing of white in the three trans-heterologous combinations of multimerized binding sites is close to that observed when both transgenes have multimerized binding sites for the same protein.
An inactive PRE can be transactivated by an active PRE: The bxdPRE insertion in the 22A attP site is a classic example of PSS. Little or no silencing is observed in hemizygotes, while silencing is quite efficient in homozygotes. The PSS phenomenon suggests that PRE:PRE interactions in trans can synergizes, promoting the assembly of functional PcG silencing complexes on both PREs. To test this idea for 96E, we generated trans combinations between the inactive PRE in the bxd transgene and transgenes in which bxdPRE is activated by closely linked 4xSu(Hw), 4xCTCF, or 5xPita sites. Consistent with prediction, the white reporter in the homolog carrying the bxdPRE (only) transgene is repressed when the other homolog has the boundary-bxdPRE (compare eyes in Fig. 9a-III with those in Fig. 9a-IV). However, silencing is not equivalent to that observed when the bxdPREs on both homologs are activated by multimerized binding sites (Fig. 9a-I).