DNA FISH

In order to verify a physical interaction between the Abd-B-Gal4 BAC and the endogenous Abd-B locus, we performed two-color DNA FISH analysis. 15 kb probes were designed to the 51C region (the site of integrations of the Abd-B BAC) and a region in the Abd-B locus not included on the Abd-B-Gal4 BAC. We used these probes to stain adult, larval (ring cells) and embryonic salivary glands. While embryonic salivary glands could not be imaged, we were able to detect signals in the two remaining tissues. Unfortunately, in these cells we always detected two distinct signals in the nuclei. No dramatic increase in colocalization was ever detected between lines carrying the BAC and lines without the BAC. (Figure 4).

This lack of positive data, while disappointing, does not mean that no transvection exists.

Indeed, the genetic data strongly suggests that an interaction must exist between the Abd-B-Gal4 BAC transgene on the second chromosome and the endogenous Abd-B region on the third

29 chromosome. We simply could not visualize an interaction. There are a number of possible explanations for this problem.

Figure 4. Double labeled DNA fluorescent in situ hybridization. Abd-B locus in green, 51C locus in red. Images represent screen shots of 3D rendered confocal stacks by the software Imaris. A) is a control, a larval salivary gland nuclei where Abd-B expression is not detected with the reporter BAC in the background, no overlap between the two signal is expected and we didn’t detect any overlap. B) Ring cells of larval salivary glands, no overlap between the signals was detected. C) larval salivary gland duct cells, no overlap of the two signals detected. D) Adult salivary gland, no overlap of the two signals was detected. (videos are available on demand).

First and foremost among the possibilities stems from the fact that we do not know when the interaction will take place. We know from studies mentioned in the introduction that the transcriptional availability of the BX-C is generally set early in development and then maintained throughout later stages of development. Because of this maintenance of transcriptionally permissive chromatin, the transvection interaction might have occurred earlier in development in just a few cells to set up an open chromatin state that is then maintained until later stages. This change of chromatin state would lead to expression of Abd-B at earlier stages, as additional positive factors may be required to activate transcription. These factors might bind to elements within the BX-C that, under normal circumstances, would be packed into Polycomb silenced chromatin. The transient nature of such interactions has been suggested by other studies

30 examining chromatin interactions between BX-C elements. In these studies, the authors generally found that the two interacting loci did not show colocalization in the vast majority of cells (Bantignies, Grimaud et al. 2003).

Salivary gland morphological phenotype

While establishing stocks for an unrelated set of experiments, we noticed that flies carrying the Abd-B-Gal4::UAS-GFP reporter and different deletions in the Abd-B cis-regulatory region had markedly reduced adult salivary glands (Figure 5). This phenomenon was observed only in the newly established lines and was not seen in the stocks carrying the Abd-B-Gal::UAS-GFP reporter alone. Interestingly, after several generations, we noticed that this salivary gland morphological phenotype gradually weakened.

Figure 5. Collection of salivary gland phenotypes. The mutation in question is homozygous with the Abd-B-Gal4 UAS-GFP reporter in the background. The names of the mutants are written on the picture. WT is a wild type salivary gland. As can be seen WT is a long straight tube like organ with a squiggly end. In comparison most of the mutant salivary glands presented here are smaller with undetermined shape.

Based on these initial observations, we performed preliminary experiments to determine if different mutations in the Abd-B gene could disturb the morphology of the adult salivary gland.

These experiments made particular sense, given that we already observed ectopic Abd-B expression in the salivary glands and wondered if this ectopic expression led to the

31 morphological phenotypes. The experiment was designed as a simple F1 screen to test if heterozygosity for various mutations could modify the morphology of the adult salivary gland in the presence of a copy of the Abd-B-Gal4::UAS-GFP BAC. A numerical scale was established for scoring the phenotype based on the length of the gland and its morphology. This scale is presented in Table 1. From these crosses, it soon became evident that the BAC itself was also capable of modifying the morphology of the adult salivary glands, if outcrossed. This finding was consistent with the previous findings that the phenotype is suppressed over generations. As this suppression occurs over only a couple of generations, we hypothesize that this suppression is probably occurring at the level of chromatin structure and not an accumulation of suppressor explanation of the phenotype. Column one names the three body parts of the fly and subdivides them. Depending on the mutant the salivary gland length can reach all the way to the abdomen or be just the head. Column two shows the numbers 1 which removes the entire BX-C, Df D18, which removes the entire Abd-Bm coding region plus a little of the neighboring sequences, and Abd-B^D16 , which is an Abd-B point mutation that is a protein null. While outcrossed Abd-B-Gal4::UAS-GFP flies show mild defects in salivary gland

32 formation (scores between 4 and 5), crossing in either Df P9 or Df D18 mutations was able to suppress this mild effect (Figure 6). This effect was particularly noticeable if the Abd-B-Gal4::UAS-GFP came from male (see below). Thus, these results are consistent with the idea that reducing the level of Abd-B in the BX-C or changing the pairing interactions (as these deletions remove substantial portions of the Abd-B region), is able to decrease the salivary gland phenotype. The confusing result comes from the Abd-B^D16 allele. When Abd-B^D16 is crossed into the BAC background, an enhancement of the phenotype is seen. Like the other Abd-B mutations, the Abd-B^D16 allele should show reduced Abd-B levels. The difference between Abd-B^D16 and the other Abd-B alleles tested lies in the possibility that the other Abd-B alleles remove interaction motifs important for transvection. Relative to a wild type copy, however, D16 should differ only by the loss of one copy of Abd-B. Thus, if we believe that ectopic Abd-B expression causes the salivary gland phenotype, we must conclude that Abd-B^D16 has some peculiar characteristics (like a mutation in a regulatory element binding site) that we still do not understand.

Continuing this mutational analysis, we also examined the effects that other elements or proteins known to be involved in transvection could have on the salivary gland phenotype. For these experiments, we crossed in mutations removing BX-C domain boundaries (Mcp¹; Fab7¹), and mutations in known transvection-mediating molecules (zeste, Polycomb proteins (Pc, pcl and Asx) and boundary proteins (CTCF)). As before, the progeny of the cross were dissected and their salivary glands scored based on their length (Figure 6).

Interestingly, as implied above, the phenotypes change depending upon the parental origin of the BAC, with females often showing a weaker phenotype (more-wild-type) than males (Figure 7). One possible explanation for this is based on the different epigenetic inheritance from male and from female. As mentioned earlier, the quick suppression of the salivary gland

33 phenotype suggests that epigenetic/chromatin regulation may be involved in the manifestation of the salivary gland phenotype. In males, it is known that during spermatogenesis all the histones get replaced by protamines, thereby, removing all histone marks that regulate gene expression ((Daxinger and Whitelaw 2010) – review on transgenerational epigenetic inheritance). In

Figure 6. Chart showing salivary gland phenotype variation in different mutant backgrounds. The flies scored for their salivary gland phenotype were produced by crossing the stock Abd-B-Gal4 UAS-GFP reporter flies (striped bar marked with AGFP/Cy) with ten different mutants (Pcl,Asx; Pc3; Mcp; Z^op6; Abd-B^D16; CTCF⁰⁴⁶³; Fab-7¹; CTCF^p366; Abd-B^D18 and Abd-B^P9) and Oregon R as control (Green bars marked with AGFP/+). The left side of the chart, left of the stripped bar, are results where the origin of the BAC reporter is from the male while the right side are results where the origin of the BAC reporter is from the female. The scored mutations were heterozygous. About 30 flies (15 male and 15 females) were scored for their phenotype based on the salivary gland length per mutation per origin of the BAC reporter. Score of one was given for a severe phenotype while score of six is for normal salivary gland phenotype (see table 1 for more detailed explanation of the scoring of salivary glands).

females, however, histone marks have the possibility to be transmitted to the next generation.

Potentially, this is what we observe in our experiments. When the Abd-B-Gal4 BAC comes from

34 the female, there may be a certain level of epigenetic marks in place to deal with the problematic transgene. When the Abd-B-Gal4 BAC comes from the male, no such marks are present, and therefore we observe a stronger abnormal salivary gland phenotype.

Figure 7. Dependence of the adult salivary gland phenotype on the origin of the Abd-B-Gal4 BAC reporter chromosome. In picture A is an adult salivary gland coming from a cross (see above the picture A) where the male is the donor of the Abd-B-Gal4 BAC chromosome. In B the situation is reversed and the Abd-B-Gal4 BAC reporter chromosome is coming from the female. A clear difference can be seen in this example between the two salivary glands when the Abd-B-Gal4 BAC reporter chromosome is coming from the male we observe a much more severe phenotype than when the BAC reporter chromosome is coming from the female. Pictures are taken at the same magnification.

Luckily, in our experiments, the trends remain the same for both males and females. The Polycomb mutants seem to have the highest impact on the salivary gland phenotype with a maximum score of two for salivary gland length in males (Figure 6, orange and red bars). This enhancement could be due to the silencing effect of PcG proteins or on the modification of the long-range interactions. However, if Abd-B expression is important for the phenotype then interfering with the long range interactions would be expected to suppress the salivary gland phenotype instead of enhancing it, unless as we mentioned before, it’s the boundaries that mediate long-range interaction. Mutations in the Zeste gene also shows significant enhancement of the salivary gland phenotype irrespective of the origin of the reporter chromosome (Figure 6, yellow and light purple bars). As the Zeste protein is thought to be involved in transvection and

35 homologous chromosome pairing, (Bickel and Pirrotta 1990), perhaps one can imagine that disrupting the pairing interaction between the two homologues may somehow strengthen long-distance BAC interactions, and thus, leading to higher levels of Abd-B mis-regulation.

Deletion of individual insulator elements like Mcp¹ and Fab-7¹, as well as mutations for the insulator protein CTCF produces no significant difference from the control (Figure 6, light blue, grey, pink and purple bars). These results were not particularly surprising. The deletion of individual boundaries would only remove one of many such elements (our BAC contains four known boundary elements) and thus, might not be expected to cause a strong phenotype. The CTCF result can also be explained by redundancy or the fact that the mutation was only present as a heterozygote. CTCF is known to bind to most BX-C boundaries (with Fab-7 being the known exception). However, given the fact that many boundaries seem to contain multiple binding sites for multiple boundary proteins, it is likely that removal of a single copy of a single, boundary protein might not cause a dramatic effect. While it is possible that homozygous CTCF deletions might show a stronger rescue phenotype, our work on the Fab-7 boundary suggests that boundary proteins play highly redundant roles.

Secondary cell enhancer studies suggest Gal-4 toxicity

An alternative hypothesis to ectopic Abd-B causing the salivary gland phenotype is that the Gal-4 or GFP expressed from the BAC may cause the phenotype. Although high levels of Gal-4 and GFP have been shown to be toxic, this possibility was originally thought to be unlikely due to the fact that the BAC driver does not seem to express at extremely high levels (probably less than the endogenous Abd-B locus).

36

Figure 8. Orientation of the D5/DI enhancer fragments in relation to Gal4. Gal4 itself should always have the same location and orientation within the construct, next to the white gene, with its transcription going towards the white, however because of cloning issues for DIrsG4 (E) that was impossible. All constructs were integrated in the same integration site, 59D3 (VK00001) A) Represents the location of the enhancer fragment D5/DI within the iab-6 domain of the Abd-B cis-regulatory region. The blue line is the D5 fragment while the light blue section is the part that the DI fragment covers. The dashed arrow line represents the direction of transcription of the male specific abdominal (msa) transcript whose actual start site is somewhere in the DI fragment region. On the right of the enhancer in red is the Fab-7 boundary. On the right most area is the Abd-B transcription start with an arrow showing the direction of transcription. B) Construct where the orientation of the D5 (based on the direction of transcription of msa) in relation to Gal4 transcription is opposite. C) D5 is in the same orientation as Gal4. D) DI is basically the same as B with DI having the opposite orientation of Gal4. E) DI has the same orientation as Gal4.

Data we obtained from our secondary cell-specific drivers made us reconsider this assumption.

As mentioned earlier, we made multiple Gal-4 drivers based on the sequences deleted in the iab-6^cocu allele. One driver carries the 2.8 kb region deleted in the iab-6^cocu allele, while the other contains only the 1.1kb region that seems to be important for the iab-6^cocu phenotype. Although enhancers are generally thought to function in an orientation-independent manner, we decided, in this case, to isolate transformants from embryos injected with plasmids where the enhancers were placed in both orientations relative to the Gal-4 coding sequence. These drivers are called D5rsG4rs and D5G4rs for the the 2.8 kb enhancer and DIrsG4 and DIG4rs for the 1.1 kb enhancer. For convenience, we will refer to enhancer orientation based on the promoter for the male-specific abdominal (msa) transcript, located on both of these fragments. As expected, all constructs were able to drive expression in the secondary cells (Figure 8).

37 Surprisingly, however, we found that when heterozygous, these drivers behave slightly differently. It turns out that drivers in which the enhancer is oriented with the msa transcript going away from the Gal4 showed a secondary cell morphological phenotype similar to iab-6^cocu (Figure 9). Upon closer inspection, especially when looking at homozygous drivers, the morphological phenotype was far worse than the iab-6^cocu phenotype (Figure 10). As the secondary cell phenotype was observed in a line containing the UAS-GFP transgene and high levels of GFP are known to be toxic ((Haseloff and Amos 1995), we decided to test if the GFP marker was having an effect on the secondary cells. As seen in (Figure 11), the phenotypes persisted, even when the GFP marker was removed.

Figure 9. Secondary cells of accessory glands from flies heterozygous for the transgenic reporter construct crossed with to a uasGFP. From the pictures it can be seen that A and C have a severe secondary cell phenotype reminiscent of iab-6^cocu phenotype. B and D on the other hand have no secondary cell phenotype.

38

Figure 10. Secondary cells of accessory glands from flies homozygous for the transgenic reporter construct with a uasGFP recombined on the same chromosome.

From the pictures it can be seen that D5G4rs and DIG4rs have a very severe secondary cell phenotype. D5rsG4rs and DIrsG4 on the other hand have no or very mild secondary cell phenotype.

Next, we wondered if the drivers were somehow affecting the levels of Abd-B expression.

In as much as the phenotypes we observe with the drivers are reminiscent of the iab-6^cocu phenotype, this seemed like a distinct possibility. We, therefore, stained accessory glands from the driver lines for Abd-B protein. This analysis showed that Abd-B protein levels seemed relatively normal. While we cannot rule out a slight change in Abd-B protein levels, based on previous RNAi results, we know that the iab-6^cocu phenotype only occurs when Abd-B levels are substantially reduced.

Figure 11. GFP’s impact on secondary cell phenotype.

Pictures of secondary cells of accessory glands from flies homozygous for the transgenic reporter construct captured under a light microscope using Nomarski filter.

From the pictures it can be seen that even without GFP, D5G4rs and DIG4rs have a severe secondary cell phenotype. D5rsG4rs and DIrsG4 on the other hand have no secondary cell phenotype. Dashed lined circles mark individual secondary cells.

39 Based on these experiments, we conclude that changes in Abd-B expression are not the cause of this phenotype.

Thus, we are left with the hypothesis that Gal-4 expression might be the cause of the secondary cell phenotype with the drivers. It has been reported by the Lehmann lab (Liu and Lehmann 2008), that overexpression of Gal4 can have toxic effects on the cell. They show that Gal-4 can cause misexpression of many genes by a factor of 1.5X causing cell death (apoptosis or necrosis). As Gal4 is known to be temperature sensitive, we examined accessory glands from driver flies grown at 18º C (Duffy 2002). Consistent with Gal-4-based toxicity, lowering growth temperature suppressed the secondary cell phenotype (Figure 12).

Figure 12. Effect of temperature on the phenotype. Secondary cells from flies heterozygous for the transgenic Gal4 driver kept at 18°C. All constructs have normal looking secondary cells even D5G4rs and DIG4rs that at room temperature show drastic secondary cell phenotype.

40 Furthermore, lines with the same construct as D5 but without Gal4 show no secondary cell phenotype (Figure 13).

Figure 13. Effect of Gal4 on the secondary cell phenotype. In panels A and B are secondary cells from D5 and D5rs insertion of the same constructs as the driver lines but without Gal4 (see figure 8 for directionality information). There is no discernible secondary cell phenotype in any of them.

41 Conclusion

Based on these experiments, we can now re-examine our results regarding the salivary gland phenotype. Although the amount of Gal-4 does not seem (to our eyes) to be very high in lines carrying the BAC, it is still possible that it is sufficient to cause some problems. In that case, ectopic Abd-B expression could simply be benign and the salivary gland atrophy would be due to Gal-4 expression.

How, then, do we integrate all of this data into a coherent picture? The simplest answer is we probably can’t. Still, I would like to propose some ideas regarding our findings. First, we do believe that some sort of transvection-like phenomenon is occurring in these animals.

Regardless of phenotypic consequences, Abd-B is ectopically expressed when the BAC is present in these animals. While our original hypothesis was that an enhancer near the BAC integration

Regardless of phenotypic consequences, Abd-B is ectopically expressed when the BAC is present in these animals. While our original hypothesis was that an enhancer near the BAC integration