The role of the Fab-6 HS1 in the functioning of the iab-5 and iab-6 regulatory domains
Iampietro et al8 generated several deletions in the region between iab-5 and iab-6 that were expected to remove sequences critical for boundary function. One of these, Fab-62, removes ~ 8 kb including both HS1 and HS2. A second ~ 2 kb deletion, Fab-63, extends from a site in iab-5 ~ 1.1 kb from HS1 to a site within HS1 located just beyond the CTCF sites (Fig. 1B). Both deletions give a mixture of GOF/LOF phenotypes in A5 (PS10) and A6 (PS11). A third ~ 4,7 kb iab-6Δ7 deletion, that removes HS2, had no apparent phenotypic effects8. Based on these findings and those of Perez-Lluch et al41, it was proposed that HS1 is the core Fab-6 boundary while HS2 corresponds to the iab-6 PRE.
To more precisely map the sequences required for boundary function, we used CRISPR/Cas9 to generate a 1389 bp deletion, F61attP, that excises all of HS1 plus several hundred bp of surrounding DNA (Fig. 1 and S1). The CRISPR/Cas9 construct carries a dsRed reporter (3 × P3-DsRed) to select for deletion events. Also included is an attP site that can be used for boundary replacement experiments46. In the adult cuticle the HS1 deletion, F61attP, displays a strong GOF transformation of segment A5 towards A6. This transformation is most clearly evident in the ventral sternite. Instead of the characteristic quadrilateral shape (wt: Fig. 1), the A5 sternite has a banana shape like that normally observed in A6. It also differs from wt in that it lacks bristles. On the other hand, there are patches of depigmented tissue in the A5 tergite, which is indicative of a LOF transformation towards A4. Interestingly, we also observe weak LOF transformations in A6. These include small depigmented patches in the A6 tergite, and an occasional bristle in the A6 sternite (F61attP, Fig. 2). The depigmentation seen in the tergite would be consistent with an A6◊A4 LOF transformation.
A similar, though not identical mixture of GOF/LOF cuticle phenotypes in A5 and a weak LOF phenotype in A6 were observed by Iampietro et al8 in their Fab-63 deletion that removes part of iab-5 and most but not all of HS1 (Fig. 1A). One notable difference is that the GOF transformations of the sternite were much more modest in Fab-63 than in F61attP. Presumably these differences reflect the locations of the deletion breakpoints.
The Fab-6 HS1 cooperates with HS2 in blocking crosstalk between the iab-5 and iab-6 in vivo
To further define sequences important for Fab-6 function we generated three replacements. The largest was 1330 bp and contained nearly the entire sequence deleted in F61attP. The two other replacements were 873 bp and 529 bp (Fig. 2A). All three included the two CTCF sites. We introduced these replacements into F61attP using the φC31 attP/attB integration system46. To monitor blocking activity in the context of BX-C, the replacement included a minimal yellow reporter, mini-y (Fig. 2A). The reporter has a 340 bp yellow promoter linked to a yellow cDNA but lacks the wing, body and bristle enhancers of the endogenous yellow gene. As a result, mini-y expression depends upon enhancers in the neighborhood. The mini-y was placed relative to the test boundary sequences so that it is located in the iab-6 regulatory domain. To minimize any possible repressive effects of the iab-6 PRE a mCherry gene was placed in front of the reporter.
In order to recover insertion events and also to monitor blocking activity, we used a y1 genetic background. In flies carrying the null y1 allele, the tan gene is still expressed under the control of Abd-B and A5 and A6 have a light brown-yellow instead of black pigmentation47 (Fig. 2B, wt y1). When mini-y is introduced into the Fab1attP deletion without a boundary, reporter expression is driven by enhancers in the fused iab-5:iab-6 domain. As can be seen by the dark pigmentation in the A5 and A6 tergites, the enhancers drive mini-y expression in both segments. However, as is observed when expression of the endogenous y gene is driven by Abd-B in the starting F61attP platform, there is “tan” pigmentation along the posterior margin of A5 as well as patches elsewhere in the tergite without pigmentation indicative of a LOF transformation of A5 into A4 (Fig. 2B). In these cells, the fused domain is shut down and mini-y (like Abd-B) is not expressed. A different pattern of mini-y expression is evident when the 1330 bp HS1 sequence is included in the replacement. In this case, mini-y is expressed at high levels throughout the A6 tergite, while expression is (with the exception of variable number of small darkly pigmented dots) absent in A5. Thus, the 1330 bp fragment effectively blocks crosstalk between iab-5 and iab-6. This is also true for the two smaller replacements, 873 bp and 529 bp. Both eliminate mini-y expression in A5 as effectively as the larger fragment. The blocking activity of these replacements is also evident in the morphological phenotypes of A5 in both y1 and y+ flies. Like wt the A5 tergite is covered in trichome hairs, while the sternite has a quadrilateral shape with many bristles. These features indicate that even the smallest DNA sequence effectively blocks cross talk between iab-5 and iab-6. There was, however, one anomaly. In replacements carrying mini-y the A6 sternite has several bristles that are not seen in wt. However, this is likely due to promoter competition between mini-y and Abd-B for the iab-6 enhancers as it is not observed in the replacements after the mini-y is excised (Fig. 2B).
Fab-6 HS1 cannot substitute for Fab-7
In previous studies a deletion F7attP50 (Fig. 3A) that replaces the four Fab-7 nuclease hypersensitive regions with an attP site was generated 27. We used this platform to further assess the functional properties of Fab-6 HS1 (Fig. 3A). For this purpose, we introduced the 1330 bp and 873 bp Fab-6 fragments described above, and a 685 bp fragment that has the same proximal end as the 529 bp fragment.
The F7attP50 deletions results is a complete GOF transformation, and in males both A6 and A7 are absent. Surprisingly, all three Fab-6 HS1 fragments failed to rescue F7attP50 (Fig. 3B). In all three replacements, only a rudimentary A6th tergite is present, while there is no sternite. These findings indicate that Fab-6 HS1 is not sufficient to reconstitute a functional boundary in a different BX-C chromosomal context.
One plausible explanation is that the two deletions we have used to test Fab-6 HS1 boundary function are not equivalent. The Fab-7 deletion removes all of the nuclease hypersensitive regions including the HS3, which has both boundary and PRE activity. By contrast, the Fab-6 deletion only removes HS1 but not the HS2 PRE. If this explanation is correct, then it may be possible to reconstitute Fab-7 by combining DNA fragments that encompass Fab-6 HS1 and HS2.
To test this possibility, we generated a Fab-7 replacement F61330 + 3212 that includes both HS1 and HS2. Figure 3 shows that the A6 segment is almost fully wild type, indicating that F61330 + 3212 is an effective substitute for Fab-7. The tergite is fully formed and the trichome hairs are largely restricted to the anterior and lateral edges as in wt, while the sternite has the appropriate banana shape. However, there are two anomalies: there are patches of ectopic trichomes on the A6 tergite, while the sternite has several bristles. These weak LOF defects would suggest that the boundary bypass activity of F61330 + 3212 is not fully effective. Alternatively, since both HS1 and HS2 have PRE activity, the two together could sometimes silence iab-6.
We also tested a 2264 bp fragment that includes the 1339 bp HS1, but extends in the opposite direction towards iab-5. Unlike the F61330 + 3212, F62264 failed to substitute for Fab-7, suggesting that there are no additional sequences conferring insulator function on the centromere proximal side of Fab-6 HS1.
Combination of Fab-6 HS1 and Fab-7 HS3 substitutes for Fab-7
The finding that Fab-6 HS1 substitutes for Fab-7 when combined with HS2 suggests that that HS2 has both PRE and boundary activity like Fab-7 HS3.39 If this idea is correct, then Fab-6 HS1 might be able to substitute for Fab-7 when linked to Fab-7 HS3. To explore this idea, we combined four different Fab-6 HS1 fragments (Fig. 3C and Fig. 2S) with Fab-7 HS3. The largest was F61330, while the smallest was F6425 45. We also tested two intermediate Fab-6 fragments, F6744 and F61119 (Fig. 3).
The two larger combinations, F61330 + F7HS3 and F61119 + F7HS3, have similar activities. In both cases they rescue the GOF transformations of the F7attP50 deletion. The sternite has the appropriate banana shape, while the tergite is wt in size (Fig. 3C). However, as observed for F61330 + 3212, the sternite usually has a few bristles, while there are ectopic trichome patches on the tergite. Surprisingly, the two smaller combinations, F6744 + F7HS3 and F6425 + F7HS3, fail to fully rescue F7attP50. The GOF phenotypes are most pronounced in the F6425 + F7HS3 combination where both the sternites and tergites are significantly reduced in size. In the case of F6744 + F7HS3 the sternites are typically misshapen while the tegrites have nicks or are smaller than normal. As was the case for the larger F6 fragments, weak LOF phenotypes are also observed, likely due to minor defects in bypass activity.
Fab-6 HS1 + HS2 deletions have a more complete GOF phenotype
Our Fab-7 replacement experiments indicate that F61330 (HS1) must be combined either with the Fab-6 HS2 or Fab-7 HS3 to rescue the F7attP50 deletion. To further pursue the role of the Fab-6 HS2, we designed a CRISPR/Cas9 deletion, F61 + 2attP, which is 5995 bp and removes both HS1 and HS2.
As anticipated from studies on Fab-7, the phenotype of F61 + 2attP differs from F61attP. As shown in Fig. 1A, there is a nearly complete GOF transformation of A5 towards A6. The difference in the phenotypic effects of F61 + 2attP and F61attP are most clearly evident in the A5 tergite. While the A5 tergite in F61attP has patches of unpigmented cuticle indicative of an LOF transformation, the A5 tergite in F61 + 2attP is fully pigmented in > 90% of the males. Similarly, though the A5 sternite in F61attP has an A6-like banana shape, it also has several bristles, which are not present in the A6 sternite. In F61 + 2attP the GOF transformation is more complete as the sternite lacks bristles in 70–80% of the males.
Thus, as was observed for Fab-7 deletions which retain or remove HS333, removing both Fab-6 HS1 and HS2 results in much more complete GOF transformation than HS1 only. In this context, we would note that a strong GOF transformation of A5 was not observed by Iampietro al8 in the Fab-62 deletion (Fig. 1B) which removes both Fab-6 HS1 and HS2. Instead they observed a mixed GOF/LOF phenotype not altogether different from their smaller deletion, Fab-63. We suspect that the difference in phenotypes is that their deletion removes a larger segment from iab-5. At this point, it is not clear why the loss of iab-5 sequences enhances the LOF transformation of A5.
HS2 contributes to Fab-6 boundary activity
Next, we generated four F61 + 2attP replacements. The first replacement, F61330, has only HS1. The second, F61330 + 3212, has both HS1 and HS2, but lacks 730 bp between the HS1 and HS2. The third, F61330 + 930, has only HS1 and HS2. The fourth replacement was F62264 which contains the 1330 bp HS1 sequence, but extends into iab-5 (see above). These fragments were introduced into the mini-y replacement vector (Fig. 4).
Figure 4 shows that the two replacements that contain only HS1, F61330 and F62264 are unable to insulate mini-y and it is expressed in A5 and A6. Consistent with ineffective insulation, the sternite in A5 has a banana shape while the arrangement of the trichomes in the A5 tergite resembles that normally observed in A6 (they are restricted to the anterior and dorsal lateral margins). Both of these morphological features are indicative of a GOF transformation of A5 towards A6 and are observed with and without the mini-y reporter. It is worth noting that the GOF transformations are less complete when the mini-y reporter is present. Though the A5 sternite has a banana shape, it has some bristles while there are small patches of trichomes on the tergite. However, the ectopic bristles and trichomes disappear and the flies resemble the starting deletion, F61 + 2attP after excision of mini-y.
A different result is obtained when the replacement combined Fab-6 HS1 plus HS2. The pigmentation pattern in the A5 and A6 tergites of y1; F61330 + 3212 mini-y and y1; F61330 + 930 mini-y indicates the mini-y is (with the exception of a few small dots of pigmented cuticle in A5: see arrowhead) insulated from enhancers in iab-5. The morphology of the dorsal and ventral cuticle in A5 is also normal. The one possible exception is some gaps in the trichome field in the smaller F61330 + 930 replacement.
These findings suggest that iab-6 PRE, HS2, contributes to boundary function much like is observed for Fab-7 where HS3 functions both as the iab-7 PRE and as part of the Fab-7 boundary. If this suggestion is correct, then one would predict that Fab-7 HS3 would be able to substitute for Fab-6 HS2. To test this prediction, we combined Fab-6 HS1 with Fab-7 HS3 (F61330 + F7HS3). As shown in Fig. 4, the F61330 + F7HS3 combination also restores boundary function.