Looking for the Fab-6 boundary using an Enhancer-Blocking Assay

6 This is a simplification of the actual phenotype. The actual phenotype also shows evidence of cells that revert to PS10 identity in PS11, as if both the iab-6 and iab-7 become silenced in these cells. The current model to

Because of the function of boundaries in keeping domains autonomous is in some ways similar to the function of insulators (in blocking position effect and enhancer-blocking), all of the identified BX-C boundaries have been tested in the insulator enhancer-blocking assay. When tested in this assay, all of the known boundaries (Mcp, Fab-7 and Fab-8) have been shown to behave as enhancer-blockers. In fact, the Fab-8 boundary was first identified using this transgenic approach.

At the time this work was performed, only three BX-C boundaries had been discovered: Mcp, Fab-7 and Fab-8 (Barges et al., 2000; Gyurkovics et al., 1990; Mihaly et al., 1997; Mihaly et al., 1998). And although a number of genetic works had been published to describe their activity, we still did not know much about the molecular mechanisms by which they function. This was partly due to the fact that their sequences yielded little hint as to what proteins might bind them. The only common protein binding sites were the GAGAG binding sites for the GAGA factor⁷. In order to have more sequences from which to draw similarities from, and to further our characterization of the cis-regulatory domains of the Abd-B portion of the BX-C, we decided to attempt to precisely identify the Fab-6 boundary predicted to separate the iab-5 and iab-6 domains. Because of our success in using the transgenic enhancer-blocking assay to identify Fab-8, we chose use the same method in our search for the Fab-6 boundary.

Results:

Analysis of deletion mutants in the Abd-B cis-regulatory region provided a preliminary map location for Fab-6 (Mihaly et al., 2006). Adults homozygous for the iab-6,7^IH deletion display a LOF phenotype, in which the abdominal segments A6 and A7 are transformed towards A5. In the embryonic CNS, however, the iab-6,7^IH deletion looks strictly iab-6,

explain this phenomenon is that there is a competition for the control of the domain between the IAB-6 initiator and the IAB-7 initiator. If the iab-7 initiator wins, the fused domain becomes silenced in PS11 where the iab-7 domain is normally silenced. This model is supported by the differences between Fab-7 mutant class 1 and 2 alleles which differ in the removal of a Polycomb response element from iab-7 (Mihaly et al., 1997).

7 We wanted to present in the introduction the different arguments, which at the time, led us to think in a certain way. When we started this project, dCTCF had been shown to play a role in the enhancer blocking activity of the Fab-8 boundary in transgene assay (Moon et al., 2005). It is in only 2007, that dCTCF was found to bind almost every frontier of each BX-C domains, and particularly the Fab-6 region (Holohan et al., 2007). Therefore we will discuss about dCTCF later in the discussion.

indicating that CNS specific iab-7 enhancers are still present in the iab-6,7ÎH deletion. In neither the adult nor the embryo does iab-6,7ÎH affect the identity of the abdominal segment A5 (PS10). Given that boundary deletions cause a fusion of two domains, one would expect an A5 to A7 transformation (at least in the CNS, in this case) if the Fab-6 boundary were deleted in the iab-6,7ÎH mutation. Therefore, we conclude that the iab-6,7ÎH deletion leave intact the Fab-6 boundary element (Mihaly et al., 2006). Cloning of the deletion breakpoints indicate that iab-6,7ÎH removes 26 kb of DNA with a centromere proximal breakpoint providing one limit on Fab-6 location (see figure 2.1; coordinates of the deletion on 3R chromosome: 12712604-12738899).

Figure 2.1: Diagram of the iab-6 cis-regulatory region. Only the proximal breakpoints of the deletions are indicated in this figure (black arrows). Coordinates of the fragments on the 3R chromosome (Genome assembly: FB2009_04, released April 27, 2009): iab-5 initiator (12704133-12705035), PRE (12708620-12708099), Prox (12706583-12708764), Dist (12708090-12710440), D-ext (12710315-12712575).

Further localization can be achieved through the analysis of the Fab-6,7¹ and Fab-6,7² deletions. Both the Fab-6,7¹ and Fab-6,7² deletions exhibit a striking dominant GOF phenotype, in which, the 5th and 6th male abdominal segments are largely reduced in size.

This phenotype corresponds to a transformation of A5 and A6 towards A7, the phenotype expected of a deletion removing iab-6 and both the Fab-6 and Fab-7 boundaries. Therefore, we believe that the two Fab-6,7 deletions must delete, at least partially, the putative Fab-6 boundary element separating the iab-5 and iab-6 domains (Mihaly et al., 2006). Molecular analysis of the breakpoints of these two deletions indicate that the Fab-6,7¹ and Fab-6,7² deletions share the same distal breakpoint (relative to the centromere; between the Fab-7 boundary and the iab-7 PRE (Fab-6,7¹ :12708067-12725372, Fab-6,7² : 12706476-12725365)), but differ slightly on their centromere proximal side (see Fig 2.1). However, as

Proximal Distal

both mutations generate the same phenotype, and seem to disable 6, we can say that Fab-6 must be located somewhere distal to the Fab-6,7² proximal breakpoint at 12706476.

Therefore, based on the data from the Fab-6,7 deletions and the iab-6,7^IH deletion, we conclude that the Fab-6 must be located within the ~6 kb area region between the proximal breakpoints of Fab-6,7² and iab-6,7^IH.

Analysis of the sequence of this ~6 kb region points to specific regions as candidates for the Fab-6 boundary. First, all the BX-C boundaries discovered thus far, are adjacent to PREs. Within this ~6 kb region, the group of Paul Schedl identified a hypersensitive site that behaves as a Polycomb Response Element (PRE) (personnal communication)⁸. If Fab-6 follows the same pattern as other BX-C boundaries, it should lie just proximal to this PRE. A second feature of all BX-C boundaries is the presence of GAGA factor binding sites. Within the Fab-7 boundary, the GAGA sites have been shown to be important for its enhancer-blocking activity (Schweinsberg et al., 2004). Examination of the ~6 kb sequence reveals the presence of four GAGA factor binding sites clustered in an area ~1.5 kb distal to the PRE, implicating this area as a candidate Fab-6 boundary.

Based on the location of the PRE and GAGA factor binding sites, we choose to split the ~6 kb Fab-6 region into three overlapping DNA fragments of ~2.2kb in length. We named these fragments, Prox, Dist and D-ext (see Fig 2.1). The two fragments Prox and Dist are largely overlapping. The common sequence, shared by these two DNA fragments, is the PRE identified in the Schedl laboratory. The D-ext fragment, on the other hand, contains the region where the four putative GAGA factor binding sites were identified. Each of these fragments was cloned into a transgene vector to test for enhancer-blocking activity (Hagstrom et al., 1996).

For our transgenic assay, we used the same P-element enhancer-blocking assay vector used to study the Fab-7 and Fab-8 boundaries (Barges et al., 2000)(see Fig. 2.2). This vector contains two enhancers from the fushi tarazu (ftz) gene to activate gene expression of a lacZ reporter: the upstream element (UPS) that activates expression in the even-numbered parasegments of germband-extended embryos (in early embryogenesis), and the neuronal element (NE) that activates expression in the central nervous system (CNS) of germ-band retracted embryos (in late embryogenesis). To test for insulator activity, putative insulator

8 Recently, other hyper-sensitive sites have been identified in the sequence near the Fab-6 boundary (Perez-Lluch et al., 2008). Since these results were not published when we designed our experiments, they will be presented and commented upon only in the discussion.

fragments are placed in between the enhancers and the promoter and tested for their ability to block the ftz enhancer driven expression of lacZ (see figure 2.2).

Figure 2.2: Diagram of the enhancer-blocking assays. A. Represents the P-element construct initially injected while B. represents the corresponding control line recovered after excision of the DNA fragment using the Cre recombinase. If the DNA fragment contained an enhancer-blocking activity, the LacZ staining should be darker in the control compared to the experimental line. The picture on the right shows germ-band extended embryos carrying a transgenic construct in which either Fab-7, Fab-8 or a lambda DNA fragment control were inserted in the same vector (Barges et al. 2000).

In our experiments, the three DNA fragments were inserted, in both orientations, in between the enhancers and the Hsp70 promoter. In addition, two LoxP recombination sites flanked each DNA fragment, in order to control for position effect and to obtain control lines for each P-element insertion. (See Fig. 2.2). Thus, we generated six different transgenes, containing the Prox, Dist or D-ext DNA fragment, in the two orientations. Each of these transgenes was injected into pre-cellular blastoderm Drosophila embryos and multiple lines were established. For each line, a corresponding control line was generated by crossing individual lines to the line expressing the CRE recombinase. After homozygous lines were established, X-gal staining was performed in parallel on homozygous experimental and control embryos.

The transgenic lines created from the Prox, Dist and D-ext fragments placed into the enhancer-blocking vector in the orientation in which the proximal side of each fragment is adjacent to the ftz enhancers, are called P1, Di1 and De1, respectively. Meanwhile, the P2, Di2 and De2 lines correspond to transgenes in which the prox, dist and d-ext fragments are inserted into the vector in the opposite orientation.

Due to problems of sterility, lethality and position effect, we were not able analyze all transgenic insertions. For example, we were never able to isolate a transformant for the P2 construct (after two series of injections) and although we isolated four transformants for the Di1 construct, none showed were homozygous viable and fertile and showed visible LacZ staining. In all, however, we were able to test at least one transgenic line for each putative insulator fragment in at least one orientation. A summary of the lines established can be found in Table 2.1

Construct Transformant Chromosome Homozygous Staining Control

P1 P1.1 II Viable √ OK control line could be recovered after excision with the Cre recombinase.

Overall, we found that all fragments tested showed a large degree of variability in staining intensity within a single experiment. Given this, it was difficult to discriminate subtle effects. Still, some general impressions could be drawn. For the P1 construct, we obtained two inserts that were both viable and fertile as homozygotes in both experimental and control forms. X-gal staining of these lines and their controls, provides conflicting results. Line P1.1 shows slightly lighter staining than its corresponding control line P1.1-Control (Fig. 2.3).

However, line P1.2 shows the exact opposite result, with the P1.2-Control showing lighter staining than P1.2 (Fig. 2.4). Therefore, based on these results, we cannot conclude that the Prox fragment (in one orientation) contains an enhancer-blocking activity.

The results for the Di2 lines (Di2.1 and Di2.2) are more consistent. Both Di2 lines and their controls display the same level of staining intensity (staining for line Di2.1 is seen in Fig 2.5), and therefore, indicate that fragment Dist (in this orientation) does not contain an enhancer-blocking activity.

Likewise, the D-ext fragment does not seem to have an enhancer-blocking activity. X-Gal staining of the two De1 fly lines (De1.1 and De1.2; see Fig 2.6 and 2.7) showed no marked decrease in staining intensity relative to their controls (De1.1 may even be more intense). In the opposite orientation, we also did not observe a decrease in straining in De2 lines relative to controls with De2.2, in fact they showed a strong increase in staining relative to its control (Fig 2.8 and 2.9). Thus, we conclude that the fragment D-ext (in both orientations) probably contains no enhancer-blocking activity.

Discussion:

Overall, our results were quite disappointing with only one line showing signs of a reduction in lacZ staining relative to its control line. This was for the P1.1 line. And, for this same Prox fragment, a second insert yielded the exact opposite result, showing a slight increase in lacZ expression. From our previous experiences using this vector, we knew that this assay was subject to minor position effects (Barges et al., 2000). This is the reason we flanked each fragment with loxP sites. However, we did not expect to find such dramatic inversions in the staining patterns between experimental and control lines. Given that this was the first time control lines had been generated using the same insertion sites, our expectations may not have been realistic. Obviously, we still lack the critical number of transformant lines to reach a definitive conclusion for this study. However, given the variability in the staining results and the confusing/negative results we obtained, we did not feel this assay would be useful in finding Fab-6 within this region of DNA.

But why might these experiments have failed? One possibility is that we chose the wrong region to investigate. However, all previous data pointed to this region as containing the Fab-6 boundary. Even the finding of a PRE and GAGA binding sites hinted that this area might contain the Fab-6 boundary. We broke up this region with these facts in mind. Our first hypothesis was that the Fab-6 boundary was probably located around the PRE, previously identified by the group of P.Schedl. The two DNA fragments Prox and Dist were designed based on this assumption. The D-ext fragment is located ∼1.5kb distal to this PRE and contains the four potential GAGA protein binding sites (Schweinsberg et al., 2004). Indeed, with the data we now know, our region of study was correct. The Drosophila homologue of the vertebrate insulator protein, CTCF, has been shown to bind in vivo to almost all of the putative and known boundaries of the BX-C (with the only noticeable exception being the Fab-7 boundary). Examining the identified binding sites for dCTCF in the BX-C, we find a strong peak of dCTCF binding within the hypersensitive site of the Schedl group that contains the PRE (Holohan et al., 2007) (see Fig. 2.10).

Figure 2.10: Map of the iab-6 DNA region.

A second possible reason for the failure of our experiments is that the breakpoints of our fragments somehow destroy Fab-6 enhancer-blocking function. Once again, we do not think this is the case. All identified boundary elements in the BX-C have been shown to be contained within ~1 kb of DNA with their associated PRE. Based on the dCTCF sites and the location of the PRE, the two most likely candidates for containing Fab-6 are the Prox and Dist fragments. These two fragments are each about 2,2 kb long and overlap by more than 600 bps.

They also both contain the PRE and the dCTCF binding region. Therefore, if Fab-6 is similar to other boundaries, it is highly unlikely that the breakpoints each destroy enhancer-blocking

Proximal Distal

activity. Of course, if the Fab-6 boundary is markedly different from the other three BX-C boundaries, both in length and in location relative to the PRE, we could be mistaken. We might then be able to recover enhancer-blocking activity by using larger fragments or fragments with different breakpoints. However, results published during the writing of this chapter seem to indicate differently.

Earlier this year, Smith et al. showed that a 2kb sequence containing the CTCF binding sites can behave as an enhancer-blocker in a transgenic assays, similar to the one we performed (Smith et al., 2009). The fragment tested in Smith et al. seems to roughly correspond to the Prox fragment, but, exact verification of this cannot be made as they do not report the sequence coordinates. However, it must be noted that they describe their Fab-6 DNA fragment as a weak enhancer-blocking element compared to other BX-C boundaries.

Given the variability we observed in our assay and in the staining in general, we doubt that we would feel comfortable determining weak enhancer blocking activity. This weak activity and perhaps subtle differences between our assays might account for our inability in identifying Fab-6 using the enhancer-blocking assay.

If we assume that Fab-6 has weak enhancer-blocking activity, then why is it different from other boundary elements, and does this difference reflect its function in the BX-C? It seems clear that enhancer-blocking is probably not the function of boundaries in the BX-C.

Indeed, the very idea of enhancer-blockers in the BX-C is almost paradoxical, as many enhancers are separated from their target promoter by multiple boundary elements. Then what property of boundaries is the enhancer-blocking activity representing? We know that BX-C boundaries each function to keep neighboring domains autonomous. Recent experiments suggest that the mechanism of boundary function is through the promoting of long-distance interactions. Work from the Cavalli laboratory has shown that a P-element containing the Fab-7 boundary located on the X-chromosome localizes in close proximity to the endogenous BX-C within the nucleus (Bantignies et al., 2003). Furthermore, work from our lab has shown that the Fab-7 boundary associates with a region near the Abd-B promoter, and that this association is mediated by the Fab-7 boundary itself (Fig 2.11)(Cleard et al., 2006). These data suggest that boundaries function through mediating long-distance chromatin interactions and that insulation may be a side-effect of this function. Indeed, this may be true for other DNA elements characterized as insulators like the Su(Hw) insulator, which also seems to promote long-distance interactions. Based on this, it seems possible that boundaries might

exist that may not have this insulator ability, or at least may behave differently in the enhancer-blocking assay. Perhaps, Fab-6 is one of these cases.

Fig 2.11 The Boundary tethering model. In this cartoon, the boundaries are represented by red circles, the inactive regulatory regions are covered by green circles (representing Polycomb silencing) and active regulatory regions are depicted by black lines (Maeda and Karch, 2007).

Three situations are represented here, when all the Abd-B cis-regulatory domains are inactivated (in A4 segment) or in segments more posterior when iab-5 and iab-6 are activated (in A5 and A6).

48 Experimental procedures

The pCfhL enhancer-blocking assay plasmid used in these experiments is described in (Aoki et al., 2008; Hagstrom et al., 1996) and is represented below.

The three DNA fragments were generated by PCR (prox 2182bp, dist 2351bp and d-ext 2261bp) by using the following oligos containing a KpnI restriction site on their 5’ ends:

Fab6 prox s:

The resulting PCR products were then cloned in between the two LoxP recombination sites in the pH7-loxP vector using the unique KpnI restriction site. NotI fragments containing the two LoxP sites and the inserted fragments were then excised and subcloned into the unique NotI restriction site present in the pCfhL vector. Clones with the NotI fragment in each orientation were isolated.

Transformants were generated by injecting each of the resulting plasmids into w¹¹¹⁸ flies using standard dechorionated embryo injection protocols.

49 Figures

Figure 2.3: Proximal fragment

All the embryos were stained the same day under the same conditions, in two batches, one for the X-gal coloration of the P1.1-Control (panel A, B and C) , the other one for the coloration of the line P1.1 containing the Prox DNA fragment (D, E and F). A,B, E, F and G show germ-band extended embryos. In the middle of panel C, a late embryo at the germ-band retracted stage, shows the X-gal coloration in the central nervous system (CNS).

P1.1-Control P1.1

Figure 2.4: Proximal fragment

All the embryos were stained the same day under the same conditions, in two batches, one for the X-gal coloration of the P1.2-Control (panel A and B), the other one for the coloration of the line P1.2 containing the Prox DNA fragment (C and D). Panel A shows a weak coloration in the CNS of two control embryos, and B a weak coloration in seven stripes, in an germ-band

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