At the beginning of this PhD, I first attempted to find the Fab-6 boundary element using the classical approach based on transgenic assays. At the time, this seemed like a reasonable approach. Genetic data generated in the lab allowed us to identified a region likely to contain the Fab-6 boundary (Mihaly et al., 2006). However, this data was based on inference from large deficiencies generated by imprecise excisions of a P-element located at the Fab-7 boundary. This tool could not be used to make smaller deletions affecting only the Fab-6 region of the BX-C. As no P-elements are known to exist near Fab-6, we concluded that studying Fab-6 genetically would be quite difficult.
The transgenic approach seemed well suited to our needs. Boundaries had previously been shown to behave as insulators in the enhancer-blocking assay and the Fab-8 boundary had been isolated in the lab using this method. Therefore, we decided to dissect the area of the BX-C implicated in Fab-6 boundary activity using the enhancer-blocking assay. Three overlapping DNA fragments covering this area were tested. And although all evidence suggested that Fab-6 should be located within one of these fragments (or multiple fragments), we failed to isolate a Fab-6 enhancer-blocker. At the time, we were disappointing by this result, because we believed that we our choices of DNA fragments was judicious, and that among them should have contained a boundary element. Discussion of these results led us to question the enhancer-blocking assay. Could a boundary exist that was not an insulator?
The real “in vivo” function of a boundary in the bithorax complex is to keep domains autonomous. Strictly interrupting communication between enhancers of the BX-C and their target gene is not their primary function. We know that the replacement of the Fab-7 boundary by other enhancer-blocking elements, like the Drosophila Su(Hw) or scs element, cause a disruption of enhancer-promoter interactions in the BX-C, and hence, lead to a loss-of-function phenotype (Hogga et al., 2001). This means that enhancer-blocking activity, by itself, is not what makes a boundary a boundary. So mechanistically, how do boundaries function?
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At the time, the idea of elements with insulator activity mediating long-distance interactions was gaining popularity. The labs of Pavel Georgiev and Hai-Ni Cai showed that placing two Su(Hw) insulators in between an enhancer and a promoter in the enhancer-blocking assay behaved as if no Su(Hw) insulators were present in the construct (Cai and Shen, 2001; Muravyova et al., 2001). In essence, the two Su(Hw) elements cancelled each other. Further characterization of this phenomenon, and staining experiments from the lab of Victor Corces, led to a model in which Su(Hw) insulators interact to form chromatin loops genome to make loops with, how many appropriate sites might exist for BX-C boundaries? It is possible that for BX-C boundaries (and especially Fab-6), there might not be many. Indeed, for both Fab-7 and Fab-8 the enhancer-blocking assay suffers from position effect. We concluded then, that to continue to work on boundaries, a more biological assay might be needed. We turned to what we felt was the most biologically relevant assay of all by studying boundaries in their native environment.
To study boundaries in situ, we turned to the method of gene conversion. Gene conversion is a form of homologous recombination that allows one to make specific mutations in a region of interest. The technique is quite powerful but suffers from some limitations.
First, it requires a method for creating a double-stranded break near the region to be mutated.
This is often supplied by a P-element transposon, but can also be made by rare-cutting endonucleases. Second, the site of the double-stranded break limits where you can make a mutation. Conversion rate drops dramatically as one moves further away from the site of the double-stranded break. And third, each conversion mutants take some time to make. In our lab, we have found that gene conversion at Fab-7 works best from a transgenic source of donor DNA (the DNA whose mutated sequence will be copied into the site of the double-stranded break). This means that to make a gene conversion in our hands takes almost half a year to complete. Because of this time restriction, and the amount of work required to produce one gene conversion line, the choice of what mutations to make is important.
Rather than attempting to localize potential binding sites, we decided to try a broader approach and ask what are the similarities and differences between BX-C boundaries. As P-element insertions exist next to both Fab-7 and Fab-8, we decided to replace each of these
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elements with a copy of the other. We did these reciprocal experiments because we wanted to know if there was a regulation of boundaries along the A/P axis. The results of these experiments are presented in chapter 3 and published in the journal Development (Iampietro et al., 2008). Basically, we showed that the Fab-7 boundary can substitute for the Fab-8 boundary, but not visa versa. This substitution works even though these two elements show little in common at the level of DNA sequence. Even the CTCF factor, shown to be important for Fab-8 function in transgenic assays, seems to be dispensable, given the right context. This substitution suggests that there might be a commonality to the mechanism by which boundaries function in the BX-C. This commonality might not require the same exact proteins to function, but might require a certain type of activity. Recruitment to the Abd-B promoter or a specialized chromatin structures (as alluded to by the H3.3 replacement studies) might account for this activity.
This kind of work could not have been performed in transgenic assays. Because of position effect, even insulator strength could not really be compared until recently (and even this may be questionable). Furthermore, this work, combined with the work of others, conclusively demonstrates that BX-C boundaries are not equivalent to other enhancer-blocking elements.
Although we obtained interesting results from these experiments, we did not see the gene conversion technique as a way to address more detailed analysis of the Fab-7 element.
This is mostly because of the amount of time it takes to create and characterize a gene conversion mutant. It is clear that gene conversion could be used to address questions like the importance of potential protein binding sites at Fab-7 (for example, we could have mutated mutational analysis was called for to better understand what elements like Fab-7 are made up of. Therefore, it was imperative that make a system to streamline the mutagenesis process and drive down our labor costs.
Fortunately, at the time, a number of technologies were becoming available to us in the lab. First, the lab of Kent Golic had recently published papers describing two new ways of creating site-specific mutations in Drosophila through homologous recombination (Gong and
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Golic, 2003; Rong and Golic, 2000). This meant that even regions away from P-elements were now open to precise genetic investigation. Second, our lab was working on the improvement of a new system to introduce DNA into specific places in the genome. This technology, based on the recombination system of the bacteriophage φC31, allows one to quickly integrate specific pieces of DNA into pre-prepared locations in the genome. For this technology to work, requires only a 255 bp attP site in the genome and a ~280bp attB site on the DNA element to integrate. By combining these two methods, we thought we finally had the tools to address the specific questions regarding Fab-7 and the rest of the BX-C.
The decision of where to concentrate our efforts was made based on the following reasons: First, we decided to focus on an iab domain because, as the initial development of the system would take a long time, we wanted to work on a large area with many elements to dissect using the same tool. Second, we wanted to stay within the Abd-B region of the BX-C because of our lab’s experience. Third, we wanted to study an area with easily visible characteristics in both sexes. And fourth, because the first step in our scheme would involve the creation of a deletion of the domain, we wanted a domain without known sterility problems. We thus chose to work on the iab-6 domain.
The tools we generated are described in chapter four. Basically, we started by replacing the iab-6 region with an attP site, using homologous recombination. This deletion, iab5-6CI, was chosen because it would not only delete iab-6 but also the Fab-6 boundary.
Overall, it took ∼11 months to finish making this chromosome (without counting all the cloning required to make a new homologous recombination vector, py25, marked with yellow (as white is silenced in the BX-C). This is quite a long time. But afterwards, using this tool, we were able to generate and characterize 14 mutants in less than 5 months. These 14 mutants allowed us to identify a minimal initiator element in the iab-6 domain and also define the location of the Fab-6 boundary. But what is more important is that with this tool, we can now generate as many iab-6 and Fab-6 mutants as we want, introducing only ∼370bp of foreign sequences (att-P and loxP sites). Using this methodology, we have found that the limiting step is not the isolation of mutations, but the characterization of mutations after isolation and the design of new mutations to test.
We have already started to create precise mutations in the iab-6 initiator to determine the individual elements important to its function. Bioinformatic and genome-wide mapping experiments have already pointed out potential binding sites for gap and maternal gene products. Constructs with mutated Krüppel, Caudal and Hunchback sites have already been made and the Krüppel binding site mutants have already been integrated. And this is only the
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beginning. As the deletions mutants are analyzed more precisely (on a cellular level), we hope to identify new enhancers and other regulatory elements important for iab-6 function.
In the lab, there are new areas of research being started using the iab-6 tools I created.
Continuing on our investigations of the initiator, we would like to understand how the initiator controls the activation of the domain. One of the most interesting models comes from the intergenic transcripts present in the BX-C. Within the BX-C there are early intergenic transcripts covering each domain. Moreover, the location of these transcripts along the A/P axis seems to correspond the area in which each domain should be active (Bae et al., 2002;
Lipshitz et al., 1987; Sanchez-Herrero and Akam, 1989). For example, the intergenic transcript that corresponds to iab-6 sequence is expressed from approximately PS11 where iab-6 should be active. This remarkable correspondence of the location of the transcript on the A/P axis, and the domain transcribed suggests causal relationship between the two events.
However, which event causes the other remains to be determined. What is clear is that the presence of these intergenic transcripts precludes the expression of the homeotic genes. Also, work from our lab, as well as from the labs of Welcome Bender and Renato Paro suggest that transcription through a PRE could inactivate its silencing activity (Bender and Fitzgerald, 2002; Hogga and Karch, 2002; Schmitt et al., 2005). Together, these results present the possibility that the role of initiators is to start intergenic transcription across the domain to inactivate PREs and hence, to activate the domain.
To investigate this further, we are now following two lines of investigation. First, we are looking at the intergenic transcripts in some of my iab-6 mutants. If deletion of the initiator arrests the production of iab-6 intergenic transcripts, then this would confirm the link between the two phenomena. Meanwhile, the study of the other deletion mutants would aid in localizing the promoters responsible for these transcripts. Second, we are now using the iab-6 integration tools to introduce transcriptional terminators next to the iab-6 initiator. We have chosen to place the terminators near the iab-6 initiator simply as a first guess, and a way to test if the terminators work to stop intergenic transcription (see Chapter 4). If our hypothesis is correct, stopping the iab-6 intergenic transcripts should result in a loss of iab-6 activity.
A second set of experiments that should prove interesting is investigating the evolutionary conservation of cis-regulatory domains. Although the entire genome sequences of 12 Drosophila species have already been sequenced and aligned to the sequence of D.
melanogaster, bioinformatics has had difficulty in actually finding analogous regulatory modules. For example, recently the group of Michael Eisen compared the even-skipped locus from D. melanogaster and six species of scavenger flies (Sepsidae) (Hare et al., 2008). At the
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DNA level, they detect little sequence similarity over the cis-regulatory region. Yet in transgenic Drosophila, the sepsid and Drosophila enhancers drive nearly identical expression patterns. Only on closer examination of sequences did they find a small number of short (20–
30 bp) sequences nearly perfectly conserved among the species. Using our integration tools, we could easily perform similar experiments by experimentally testing for conserved functions. The only difference would be that we would look for these changes at the endogenous Drosophila locus. Using our iab-6 system, it would be particularly interesting to look at evolutionarily divergent traits in A6 like pigmentation, the presence of a Muscle of Lawrence, and trichome pattern (see figure 5.1) (Gailey et al., 1997; Kopp et al., 2000).
Figure 5.1: Drosophila 12 Genomes Consortium (Clark et al., 2007)
And lastly, the facility and speed of generating precise mutants using the integration system has inspired us to perform similar experiments in other domains. This can be done using the same homologous recombination method I used (currently being performed around the Abd-B coding region), but we have also come up with another method to step from one domain to another. This method will allow us to move laterally from iab-6, limited only by the length of DNA we can inject (for now, this is 100kb).
Figure 5.2 explains this method. First, an integration plasmid is created. This plasmid should recreate all of the wild-type sequences surrounding the mutation you want to create, starting from iab-6. In place of the sequence you want to focus on, an attB site and an FRT
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flanked cassette containing a selectable marker and all of the plasmid backbone sequence are placed. The wild-type sequence on the distal side of the deletion you want to create (relative to iab-6) should be ~2.5 kb and contain an I-SceI cut site at its distal end. Finally, a second attB is placed between the 5’ and 3’ ends of this construct. By integrating this fragment into the attP site in iab-6 and selecting for integration occurring through the second attB site, we can obtain a chromosome with an attB site and an FRT cassette in the place of a new deletion.
However, this will not be a true deletion, as the sequence deleted on the plasmid will still be duplicated in the genome. By crossing the line to an I-SceI expressing line, a recombination event can take place between the integrated sequence and the chromosomal region homologous to the DNA at the site of the double-stranded break. The Flip recombinase can then be used to remove the marker gene and the plasmid sequence. What we would be left with is a situation exactly analogous to the iab-6 construct, but with an attB in place of the attP, and an FRT in place of the loxP. Note, however, that an attL and a loxP will be left next to iab-6 and future integration plasmids should contain an attP (instead of attB) and an FRT (instead of a loxP).
Closing thoughts
My work in Geneva has come full circle. In 2003, I started my work by look for the Fab-6 boundary using a classic transgenic approach, but with little success. After that frustrating experience, I would not have believed that six years later, I would have returned to Fab-6 and find it by a completely different method! Although my most important contributions to BX-C research may be my studies on the initiator, the finding of Fab-6 is still a point of great personal satisfaction.
Through the course of my six years of PhD training, I have had the opportunity to use (and slightly improve) some of the most powerful genetic tools currently available in Drosophila. Although my work has focused on the BX-C and has resulted in the discovery of interesting things, I believe my work’s greatest impact will be in the methods applied. I have shown that any locus in the fly can be specifically and efficiently targeted for systematic mutagenesis. By targeting endogenous loci, we keep the natural chromatin environment and study the actual function of the gene. I have shown that studies previously performed in transgenes can now be easily performed in situ.
Drosophila has the reputation of being a historic and classic model organism to study genetics. But with all of the tools available to Drosophila researchers, it is still an ideal scientific organism to perform modern genetic analysis. Its strengths lie in its speed and the
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fact that its powerful genetics allow scientists to look at phenotypes in vivo. I, therefore, hope that my work has helped to expand on these strengths and keep Drosophila at the forefront of modern genetics. Indeed, with the techniques available in Drosophila, it would be difficult for me to work on another model organism.
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Figure 5.2: Diagram of a possible ne w domain deletion using the iab-5,6CI deletion.
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