Introduction:

While experiments performed using lacZ reporter gene constructs are extremely useful in identifying regulatory elements with precise tissue or spatial specificity, they remain very limited in their ability to decipher large and complex regulatory regions, such as those of the BX-C. A typical example of these limitations can be seen in chapter two, where I describe my attempts to localize the Fab-6 boundary using transgenic reporter constructs. Boundaries in the BX-C have all been identified on the basis of their deletion phenotypes. They were subsequently found to behave as insulators in constructs designed to detect enhancer-blocking. In the case of Fab-6, the position of the boundary was inferred on the basis of three large deletions. Given the genetic mapping data and our success in localizing Fab-8 in the lab using the enhancer-blocking assay, it seemed reasonable for us to try to narrow down the position of Fab-6 using the enhancer-blocking assay. Although our results were not completely conclusive, we, nevertheless, failed to pinpoint an Fab-6 insulator activity using this assay. Based on these experiences, it became clear to us, that the best way to identify relevant elements in the BX-C would be to create deletions and mutations in the actual BX-C, and to look for homeotic transformations.

When I began my work in the Karch laboratory, homologous recombination in the fly had just been reported. Outside of the Golic laboratory, very few people had even tried using homologous recombination to create mutations. I was fortunate enough to have two of these people working right down the hall from the Karch lab, in the lab of Pierre Spierer (Seum et al., 2002; Seum et al., 2007). The possibility to perform homologous recombination within the BX-C would, in principle, be the technique of choice to identify elements like the Fab-6 boundary within the large cis-regulatory region. However, homologous recombination is time

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consuming, with each new mutation requiring upwards of 6 months of labor-intensive work.

This made the technique to too cumbersome to perform systematic and sequential mutagenesis. Gene conversion is another powerful method in Drosophila to perform site-directed mutagenesis (as seen in Chapter three). But it is limited by the location of P-elements to create the double-stranded chromosomal break. Therefore, for finding P-elements like Fab-6, where no P-element has been recovered in the vicinity, gene conversion could not be easily used.

In 2004, a new technique was reported using the bacteriophage φC31 recombinase to mediate unidirectional site-specific integration in Drosophila (Groth et al., 2004). The φC31 recombinase is an enzyme that mediates recombination between two distinct DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB.

Because the two recombination sites differ, upon recombination, the two recombined sites become hybrid sites that are not substrates for the same enzyme. This means that φC31 recombination is a unidirectional reaction, allowing integration without subsequent excision (as would happen with the Cre or Flp recombinases). This ability to mediate recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale, provides a very powerful tool for the genetic manipulation of large genomes.

In the framework of the “NCCR Frontiers in Genetics” program, our laboratory, in collaboration with the laboratory of Konrad Basler at the University of Zurich, helped to further develop and improve the method of φC31 recombinase-mediated integration (Bischof et al., 2007). With these improved methods, the φC31 integrase method is not only as fast as P-element transformation, but also extremely efficient.

Given expertise in the lab on homologous recombination and φC31 integration, we decided to combine the two techniques to create a powerful and quick method to introduce mutations within targeted regions of the BX-C. In a first step, we would use homologous recombination to replace a region of the BX-C with an attP integration site. Then, we would simply integrate, into this site, DNA fragments containing various amounts of the replaced sequence and look for phenotypes. For these experiments, we chose to focus on the iab-6 cis-regulatory domain.

73 Results and Discussion:

Creation of an attP integration site in the BX-C

Our reasons for choosing iab-6 are many. First, we wanted to choose a region that controls Abd-B expression, as it is the area of the BX-C that our lab focuses on. Second, we wanted to choose a large region that would control many phenotypes. As the longest part of the procedure is the homologous recombination, we wanted to delete a sequence large enough to be used for the study of many elements. Also, we wanted to take advantage of the lack of size limitation to integration (whose limitations seem to be dictated by the injection procedure and not the integration itself). Third, as we would have to create a large deletion line into which we would inject, we wanted our deletion line to be homozygous viable and fertile, for ease of manipulation (deletions of iab-5, 7, and 8 would be sterile in at least one sex). Fourth, we wanted to be able to screen for easily visible phenotypes in males and females. And lastly, we also wanted to be able to use this tool to identify the Fab-6 boundary.

The large iab-6 deletion we planned leaves intact the Fab-7 boundary. Because the Fab-7 area is extensively studied, the position of the distal breakpoint for the iab-6 deletion was easy to determine, in between the PTS-6 element (Chen et al., 2005) and the Fab-7 boundary. The determination of the proximal breakpoint of the iab-6 deletion was more difficult. We wanted to delete the putative Fab-6 boundary together with the whole iab-6 domain in order to precisely determine its position genetically. As our previous work failed to identify the Fab-6 boundary, we decided to enlarge our initial search area from what we had attempted in the past. On the other hand, we wanted to avoid deleting important elements in the iab-5 domain. In 1993, Busturia and Bienz identified the IAB5 initiator fragment (~1kb SalI fragment, coordinates 3R: 12704134-12705146, Busturia and Bienz, 1993) that is thought to control the expression of the enhancers in the iab-5 domain. We therefore extended the proximal border of our deletion from the Prox fragment (see chapter two) until just before the IAB5 initiator element. Overall, this makes our deletion 19.3kb long and extends from Fab-7 to the IAB5 initiator.

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Figure 4.1 Landmarks in the iab-6 region. IAB-5 initiator (12704133-12705035), CTCF binding site (12708438-12708574), HS1 (12707732-12708886), HS2 (12711833-12712760), PTS-6 (12724133-12724331), Fab-7 (12724552-12725060), 19,3kb deletion (12705250-12724572).

To create this deletion, we decided to use the “ends-out” homologous recombination technique developed by Gong and Golic (Gong and Golic, 2003). To this end, we modified the original “ends-out” donor plasmid to contain the attP insertion site and a yellow reporter gene (see HR annex). Two reasons dictated our choice of using yellow+ as marker gene to screen for the homologous recombination event. First, white is a cell autonomous gene and cannot be used in the BX-C because of the tight repression of the BX-C in the eye (which silences white+ transgenes). And second, using a body pigmentation marker such as yellow, allows us to screen for specific integration events by looking at the pattern of pigmentation.

Indeed, following the homologous recombination event, the yellow reporter gene will be inserted in the BX-C next to the iab-5 cis-regulatory domain. We, therefore, expected that the adult male flies would exhibit pigmentation in only the A5 and A6 segments, while the other segments of the fly would remain yellow. Figure 4.2 shows the result of such a recombination event. After a long and difficult procedure, we finally recovered the 19.3kb deletion that we called the iab-5,6CI deletion. The entire homologous recombination procedure and its verification are described in the annex (HR and southern annexes). Once we were sure of the integrity of our homologous recombination event, we deleted the yellow marker gene by cre/loxP-mediated excision so that the yellow marker could be used to screen for future integrations (see HR annex).

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Figure 4.2: Pigmentation pattern of used to find homologous recombinants

Phenotype of the 19.3kb deletion: iab-5,6CI

Although we were aiming at deleting only iab-6, homozygous iab-5,6CI males have both their A5 and A6 segments transformed into A4, indicating that iab-5 is also affected.

This is visible by the lack of complete tergite pigmentation on the 5th and 6th tergites. The transformation of A6 towards a more-anterior segment is also visible on the ventral side of the male cuticle, where we observe that the 6th sternite, normally devoid of bristles and displaying a crescent shape, develops like an A4 or A5 sternite, with bristles and a trapezoidal shape.

Based on this phenotype, we named the mutation iab-5,6CI (see Fig4.3) As mentioned above, we were careful to leave the IAB5 initiator intact when we designed our targeting construct.

In agreement with this, we find that only some A5 phenotypes are affected. For example, in the embryonic CNS, the PS10/A5 Abd-B expression pattern is normal, indicating that in embryos, iab-5 is still active (Fig A4.2) (Figures labeled with an “A” indicates that they are annex figures). Also, while true iab-5 mutants are sterile, iab-5,6CI is fertile. Therefore, we believe that this deletion removes a cell type-specific enhancer that is involved in the

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specification of the adult A5 cuticle. This cell-type-specific enhancer would map between the IAB5 initiator and the Fab-6 boundary.

At the other side of this deletion, we have kept the integrity of the Fab-7 boundary. In agreement with the expected role of the boundary, the iab-7 domain remains fully autonomous and functional, and homozygous males have a “normal” A7 segment. It should be emphasized here that adult males have only six abdominal segments because the 7th larval abdominal segment does not contribute to the formation of the adult cuticle during metamorphosis. Adult males mutant for iab-7 harbor a 7th abdominal segment and mutations affecting Fab-7 function cause a reduction in the 6th abdominal segments through the ectopic activation of iab-7 (see Fig A4.26).

Rescue of the iab-5,6CI deletion

The iab-5,6CI mutant contains a 255 bp attP insertion site. We constructed a stock carrying the φC31 integrase on the X chromosome, and the iab-5,6CI deletion over the TM6b balancer chromosome. This stock was used for all of the integration experiments to follow.

By having created a large deletion, we had to be able to clone and mutate large DNA fragments for re-integration. Unfortunately, classical cloning procedures with restriction enzymes become quite difficult when working with large fragments. Therefore, we turned to the method of recombineering, a method based on homologous recombination in E. coli, to make our large constructs. Using specific bacterial strains where the recombination proteins are deleted and replaced by heat-shock inducible λ phage recombination proteins, we can induce homologous recombination on a plasmid by electroporating in linearized DNA fragments containing only two regions of 50bp of homology to the region to be mutated (Lee et al., 2001; Liu et al., 2003). By using this procedure, we were able to create a large number of mutant fragments for re-integration in very little time (see Fig. 4.5).

In order to test the validity of our overall approach, we first decided to re-integrate the exact 19.3 kb DNA fragment deleted in iab-5,6CI and test for rescue. Once again a removable yellow gene was used to score for integration. We were extremely pleased to recover integration events in the progeny of about 40% of the embryos that survived the injection procedure. This high frequency came as a relief to us as we did not know if integration would work in the BX-C. The BX-C is subject to strong repression by the Pc-G machinery and we worried that the integrase would not be able to access the attP platform in the germline (a

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detailed protocol for the generation of the plasmids and the injections is presented in the annex).

Figure 4.3: Adult male cuticle preparations. (A) Wild type, (B) iab-5,6CI, (C) iab-6rescue.

Lines having the 19.3 kb fragment re-integrated show complete rescue of the iab-5,6 phenotype (see Fig 4.3 and Fig. A4.3). The homozygous adult males rescued for the deletion, named iab-6rescue, show a normal A5 segment (with a hairy sternite and a black tergite), and a normal A6 segment (with no bristles on the sternite and a black tergite). Another criteria of distinction between A5 and A6 is the distribution of the trichomes (peculiar small bristles) on the tergites (Kopp et al., 2000). In wild-type males, the trichomes cover the entire A5 tergite, whereas on the A6 tergite, they only cover the ventral and anterior side of the tergite (see Fig A4.1. Confirming the complete rescue, the trichome distribution on the A5 and A6 tergites of male iab-6rescue flies are completely wild-type. It should be noted that these phenotypes and all of the following phenotypes are reported from flies in which the yellow marker has been removed. When the yellow gene is still in place, we observe a small “enhancer-trapping effect” on the Abd-B promoter causing a very slight iab-6 phenotype (data not show).

Known elements within the iab-6 domain

During his Ph.D. work in our laboratory, Stephane Barges performed a molecular analysis to find new Abd-B regulatory elements. In this analysis, different sequences from the iab-4 to the iab-8 cis-regulatory domains were inserted upstream of a Ubx-lacZ reporter gene in the Casper Ubx-lacZ P-element vector (Qian et al., 1991). From the resulting transgenic lines, lacZ immuno-staining was performed in an attempt to discover novel regulatory elements. The figure below summarizes some of the findings of Dr. Barges in the iab-6 region. The 11.9 kb LO fragment does not drive any beta-galactosidase pattern in embryos,

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meaning the absence of visible embryonic enhancers in this fragment. Interestingly, the OU fragment directs a very specific lacZ pattern during early embryogenesis in PS11 and PS13.

In as much as iab-6 is responsible for the specification of PS11/A6, the OU fragment harbors the expected characteristics of an initiator for the iab-6 domain. The activity of the initiator was further narrowed down to the OT fragment and was given the name “IAB6 initiator” by Mihaly et al (2006). It should be noted that the OU and OT fragments may also contain a Polycomb-Response Elements (PRE), as the initial anterior border of expression of the lacZ gene in PS11 is maintained during later stages of embryogenesis. Finally, the U fragment was shown to direct lacZ expression in a tissue-specific pattern within the visceral mesoderm.

Therefore, altogether, these transgenes give us an indication of the location of the iab-6 initiator and a visceral mesoderm enhancer.

Figure 4.4: Summary of fragments pre viously tested in transgenic reporter assays.

Coordinates of the fragments on the 3R chromosomes: LO (12704594-12716498), OU (12713611-12721426), OT (12716498-12719280), TP (12719280-12720605), U (12720660-12725048).

More recent studies highlight the existence of putative regulatory elements within iab-6. First, Holohan et al. (2007) have localized by ChIP-on-chip methodologies, the distribution of CTCF binding sites within the BX-C (see also introduction). Very interestingly, the CTCF binding sites correspond to the sites of known boundaries (with the exception of Fab-7) as well as to the sites of putative boundaries, as inferred by the enhancer trap lines (Bender and Hudson, 2000; Maeda and Karch, 2006). A CTCF site is found in iab-6 within the region where Fab-6 is expected to localize. Second, Perez-Lluch et al. (2008) have localized DNAse hypersensitive sites within the proximal region of iab-6 and showed that two such hypersensitive behave as PREs in the miniwhite assay (Perez-Lluch et al., 2008). One of these

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hypersensitive sites corresponds to the PRE previously identified by the Schedl lab (personal communication, see chapter two).

In-silico analysis of iab-6

In order to get more information about the 19.3kb sequence that we deleted, we used the available bioinformatic tools to predict putative regulatory elements. Both multi-species alignments and binding site predictions were used in these studies. In particular, we used DNA sequence analysis tools like MOTIFs search and AliBaba2.1 (and some others available on the web site: gene-regulation.com). All these programs use data on transcription factor binding sites provided by the TRANSFAC database. Other particularly useful tools were Stubb and Fly-ahab, which detect potential clusters of binding sites for the transcription factors(s) (Rajewsky et al., 2002; Schroeder et al., 2004; Sinha et al., 2006). We got a really interesting result with the statistical algorithm, Ahab. Using this program we were able to detect a potential module (module Ahab_12391, 3R: 12717635-12718561) localized in the 2.8kb OT sequence previously identified in transgene assay as the IAB6 initiator (Mihaly et al., 2006) (See in silico annex).

Recently, new bioinformatics tools have become available that support the Ahab results. Whole genome ChIP-on-chip experiments with six maternal and gap gene transcription factors (Bicoid, Giant, Caudal, Krüppel, Hunchback, Knirps) verify the binding of Caudal, Giant, Hunchback and Krüppel to the initiator region (Li et al., 2008). These data are available on the UCSC Genome Browser together with the data obtained with antibodies for the RNA PolII polymerase and the transcription factor Zeste (Li et al., 2008; Moses et al., 2006). Altogether, these bioinformatics tools point to ~1kb DNA fragment bound in vivo by PolII, Caudal, Giant, Hunchback and Krüppel as the IAB6 initiator (Li et al., 2008) (see in silico annex).

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Figure 4.5: Deletions created in this study. A. Provides the position of the different elements thus far discovered in the iab-6 region. B. Presents very approximately, the position of the various deletion created through re-integration into the initial iab-5,6CI deletion. The exact position of the breakpoints of each deletion is provided on the left side. The deletions in grey have not yet been injected.

The IAB6 initiator is essential for the activity of the iab-6 cis-regulatory domain.

Using the bioinformatics data, we decided to genetically identify the iab-6 initiator by creating small deletions around the ∼ 1kb Ahab_12391 module. Our working model predicts that the activity of each cis-regulatory domain depends on the initiator element that, in response to a particular combination of the gap and pair-rule gene products, activates the regulatory domain in the appropriate parasegment. As Stéphane Barges identified an element with the expected activity of the IAB6 initiator on a 2.8kb fragment in a Ubx-lacZ reporter construct (Mihaly et al., 2006), we first decided to generate an iab-6 integration construct

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lacking the 2.8kb OT fragment (named 2.8XN in Mihaly et al. 2006). The resulting mutant is has been named, iab-61. Figure A4.4 shows the abdominal cuticle of an iab-61 homozygous male. In these flies, A6 is completely transformed into A5, as shown by the presence of a bristled 6th sternite, as well as by the trichome distribution on the 6th tergite. It is important to note that the other abdominal segments remain unaffected in iab-61 flies. The complete transformation of A6 into A5 is corroborated by the embryonic Abd-B expression pattern in the CNS, in which the PS11/A6-specific pattern is replaced by the pattern normally found in PS10/A5 (Fig A4.4). The complete homeotic transformation of A6 into A5, confirms our hypothesis that the initiator fragment is indispensable for the activity of iab-6.

In as much as the 2.8kb OT fragment had not been further dissected in the reporter gene assay, we used the previously described bioinformatics methods to further narrow down the critical portion of the IAB6 initiator. Among the three cis-regulatory modules detected by the Ahab algorithm in the 19.3kb long iab-6 domain, module Ahab_12391 falls within the 2.8kb initiator fragment. This 927bp module contains four binding sites for the Krüppel protein, two binding sites for Hunchback, and three binding sites for Caudal. We, therefore,

In as much as the 2.8kb OT fragment had not been further dissected in the reporter gene assay, we used the previously described bioinformatics methods to further narrow down the critical portion of the IAB6 initiator. Among the three cis-regulatory modules detected by the Ahab algorithm in the 19.3kb long iab-6 domain, module Ahab_12391 falls within the 2.8kb initiator fragment. This 927bp module contains four binding sites for the Krüppel protein, two binding sites for Hunchback, and three binding sites for Caudal. We, therefore,

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