The role of ELAV in transcriptional interference
A) Experimentally determined ELAV-binding sites in the target genes Nrg and ewg, and consensus sequence built from these sites
B) ELAV-binding plot of genes that undergo ELAV-mediated 3'UTR extension (red line) or other neural-specific transcripts (grey line). Reproduced from (Oktaba et al. 2014).
C) Comparison of the density of EBS between the first and terminal exon of the iab-8 ncRNA and abd-A.
95
In addition, I analyzed the results of the ChIP-seq against ELAV described in (Oktaba et al. 2014). In this paper, the authors provided a list of the genomic coordinates where they could detect ELAV binding in 6-8h embryos and in 10-12h embryos. I manually looked at the coordinates located between abd-A and Abd-B, and found ELAV binding in both developmental stages, all along the iab-8 ncRNA transcriptional unit. It is remarkable that the only exon in which ELAV binding is detected in embryos of 10-12h is exon 8, which supports the finding of potential ELAV-binding sites in it (Figure 27, arrows, and Appendix, Tables S1-S2). This time window also coincides with the embryonic stage in which the alternative isoforms of the iab-8 ncRNA are easily detected by in situ hybridization.
Figure 27 ELAV can be detected by ChIP binding to exon 8 of the iab-8 ncRNA Manual annotation of the genomic coordinates of the BX-C region between abd-A and Abd-B where ELAV binding has been detected by ChIP-seq in (Oktaba et al. 2014).
96
More recently, the identification of the iab-8 ncRNA as a potential ELAV target has been supported by a cell-specific transcriptome analysis (McCorkindale et al. 2019), in which the iab-8 ncRNA was appointed to be one of the noncoding RNAs enriched in elav+ cells (neurons) in comparison to repo+ cells (glia) of the embryonic CNS.
Taking into consideration the neural specificity of the intergenic splicing between the iab-8 ncRNA and abd-A, and the indications of the iab-8 ncRNA as a potential ELAV target, I decided to study the potential role of elav in this process.
ELAV is not necessary for the expression of the iab-8 ncRNA in the embryonic CNS, but the production of the neural-specific iab8/abdA isoform of this gene is ELAV-dependent
The alternatively spliced isoforms of the iab-8 ncRNA can be detected by in situ hybridization using the intergenic probe. I used this probe in embryos carrying the elav loss-of-function allele elavE476 (Simionato et al. 2007), kindly provided by C. Klämbt (Figure 28). To sort out the elav mutants, I placed the elavE476 chromosome over a GFP-marked balancer chromosome (FM7,act:GFP). I did the in situ hybridization in presence of a probe against the GFP gene present in the FM7,act:GFP balancer. I manually sorted the embryos that did not present ubiquitous staining, and I discarded the rest. This procedure was repeated for all the subsequent in situ hybridization experiments in elav embryos.
While in WT embryos I am able to detect strong and consistent readthrough transcription of the intergenic area between the iab-8 ncRNA and abd-A (28 A), this signal is strikingly reduced in elav mutant embryos (28 B). These embryos show a mix of phenotypes: while some are only mildly affected, in most of them the readthrough signal becomes barely visible.
97
Figure 28 The readthrough transcription of the iab-8 ncRNA is strongly reduced in elav mutants
Dissected central nervous systems of WT (A) and elavE476 embryos (B) stained by in situ hybridization with the intergenic probe.
The reduction in readthrough transcription is already a good indicator of the role of elav as a transcriptional regulator of the iab-8 ncRNA. However, it does not provide definitive evidence to support the hypothesis of ELAV being involved in the alternative splicing of this gene. Indeed, the reduction of the intergenic signal could also be caused by a downregulation of the iab-8 ncRNA expression in elavE476 mutants.
In order to discard this possibility, I studied the transcription of different exons of the iab-8 ncRNA in elav mutants: exons 1 and 2 (as reporters of the activity of the promoter) and exon 8 (to verify that the transcription of the iab-8 ncRNA does not terminate before this exon). The results of this study, and the comparison with the previous results using the intergenic probe, are illustrated in Figure 29:
98
Figure 29 elav loss-of-function does not affect the overall levels of transcription of the iab-8 ncRNA
Dissected central nervous systems of WT and elavE476 embryos stained by in situ hybridization with different probes along the transcriptional unit of the iab-8 ncRNA (see diagrams, left).
As shown in 29A and 29B, the expression of the universal isoform of the iab-8 ncRNA is not affected in elav mutant embryos. The iab-8 promoter drives transcription with similar strength in WT and elav embryos, and in both cases, the transcription reaches the genomic area that corresponds to exon 8 at a similar rate. However, transcription of the iab-8 ncRNA is more efficiently terminated at the end of exon 8 in elav mutants than in WT embryos, as shown in 29C. Therefore, I concluded that ELAV enhances the failure of termination of the iab-8 ncRNA in the CNS and its subsequent splicing into the abd-A locus.
99
The iab-8 ncRNA acts as a repressor of abd-A expression only in neurons
The alternative splicing of the iab-8 ncRNA generates an iab8/abdA chimeric RNA that lacks the first coding exons of abd-A and therefore is unable to produce ABD-A protein.
It is an attractive idea to think that this mechanism could be the responsible for the repression of abd-A by transcriptional interference (even more knowing that this repression only takes place in the central nervous system, and that in this tissue Abd-B mediated repression is absent).
I decided to study the expression of abd-A in the embryonic CNS, to determine the cell type in which the iab-8 ncRNA exerts its regulation. abd-A is a known regulator of programmed cell death in larval neuroblasts (Bello et al. 2003), so I hypothesized that the iab-8 ncRNA could be repressing its expression in this cell type in the embryo. If that were the case, I would expect to see ectopic abd-A expression in embryonic neuroblasts in Fab-864 mutants, which do not express the iab-8 ncRNA. I used the marker Dpn, expressed exclusively in neuroblasts and ganglion mother cells (GMCs), to look for abd-A expression in these cell types. However, the double immunostaining against DPN and ABD-A shows these two proteins expressed in cells located in different layers of the CNS, with most of the DPN+ cells on the ventral side and most of the ABD-A+ cells being localized in inner layers of the nerve cord. Even more, I was never able to detect ABD-A expression in embryonic neuroblasts (or GMCs), neither in WT of in Fab-864 homozygous embryos (Figure 30).
The other cell type in which the iab-8 ncRNA could be exerting negative regulation over abd-A is in the neurons. abd-A is expressed in postmitotic neurons of the embryo, where it has diverse and essential roles, giving them positional information and specifying their neuropeptidergic identity (Estacio-Gomez et al. 2013, Gabilondo et al. 2018). I studied by immunostaining the expression of ABD-A and ELAV in the embryonic CNS on WT embryos (Figure 31). Strikingly, I observed that all of the ABD-A expressing cells are ELAV positive. (31A). This observation not only confirms that ABD-A is expressed in neurons, but also rules out the possibility of it being expressed in any other cell type in the CNS, at least in this developmental stage.
100
This suggests that the iab-8 ncRNA acts as a cell-specific repressor for abd-A expression in the neurons of PS13, which supports the hypothesis of ELAV acting as a mediator of this repression.
Figure 30 abd-A is not expressed in embryonic neuroblasts or GMCs
Dissected central nervous systems of WT (A) and Fab-864 embryos (B), immunostained against DPN and ABD-A. The scale bar corresponds to 20 µm.
Figure 31 ABD-A is exclusively expressed in the neurons of the embryonic CNS Dissected central nervous system of a WT embryo immunostained against ELAV and ABD-A. The right picture shows a single confocal slice. The scale bar corresponds to 20 µm.
101
Effect of the loss of function of elav in the expression pattern of abdominal-A
In order to test if elav had a role in the repression of abd-A taking place in the CNS, I studied abd-A expression in embryos of the previously described elavE476 mutant line. I knew from previous studies (Thomsen et al. 2010) that the protein levels of abd-A are strongly reduced in elav mutants. Therefore, in order to study the potential de-repression of abd-A in this context, I observed its expression pattern by in situ hybridization using the abdA 3'UTR probe (Figure 32).
Figure 32 abd-A presents diverse degrees of de-repression in PS13 of the CNS in elav embryos
Dissected central nervous systems of WT (A) and elavE476 (B) embryos stained by in situ hybridization with the abdA 3'UTR probe.
Strikingly, I observed that while abd-A transcript is absent from PS13 of WT embryos (32A), elav mutants show a diverse degree of de-repression of abd-A in PS13 of the CNS (32B). This always correlates with a significant reduction in the amount of staining detected in PS14 (32B, arrow). As I have shown in the previous section, this absence of signal in PS14 is due to the nearly total absence of transcriptional readthrough of the iab-8 ncRNA in elav mutant embryos.
102
The repression of abd-A in PS13 of the embryonic CNS could be affected by an elav loss of function via two different mechanisms.
On one hand, the failure of termination of the iab-8 ncRNA, and its splicing into the abd-A transcriptional unit, which I had already proven to be mediated by ELAV, could be the cause of the repression exerted in cis- by the iab-8 ncRNA over abd-A in the CNS.
The absence of ELAV would have as a consequence a more efficient termination of the iab-8 ncRNA in exon 8 and, if this is the mechanistic cause of transcriptional interference, a failure in cis-acting repression.
On the other hand, elav mediates the elongation of the 3'UTR, adding two additional potential target sites for the mir-iab-8 microRNA. One interpretation to the de-repression observed in elav embryos would be the lack of this elongation and, therefore, of the additional mir-iab-8 target sites. It has been proposed that the 3'UTR extension, at least in the case of the Hox gene Ubx, modulates its sensitivity to the repression mediated by microRNAs (Thomsen et al. 2010). However, this does not seem likely, as show the studies made in (Tyler et al. 2008) on ectopic repression of abd-A by mir-iab-8 in the genital disc. In this organ (where ELAV is absent), the abd-A isoform does not have an elongated 3’UTR, and therefore it only presents 4 out of 6 target sites for mir-iab-8.
Despite of this, the GAL4-driven expression the microRNA produces the total repression of abd-A, suggesting that the 4 sites present in the universal isoform of abd-A are enough to mediate repression by mir-iab-8.
103
If transcriptional interference is failing in elav mutants, how is it possible that these embryos do not show a complete de-repression of abd-A transcript in PS13? This might be explainable by the capacity of the mir-iab-8 microRNA to promote rapid degradation of the abd-A transcript, in addition to prevent its translation. In effect, previous observations made in our laboratory (Gummalla 2011) indicated in ΔmiR mutants, which do not produce the mir-iab-8 microRNA, it was possible to detect a higher level of abd-A mRNA in PS13 of the CNS . I wanted to confirm this observation, so I stained ΔmiR embryos using my abdA 3'UTR probe (Appendix, Figure S1). Indeed, my probe against abd-A is able to detect high transcript levels in PS13 of the CNS of these embryos.
ELAV mediates transcriptional interference of the abdA:GFP transgene
To further evaluate the repressive efficiency of the iab-8 ncRNA in elav context without the interference of the mir-iab-8 microRNA, I introduced the elav mutation into my abdA:GFP strain
I crossed females heterozygous for the elavE476 allele with males carrying the abdA:GFP transgene (see Figure 33A for a detailed description of the cross). 50% of the males resulting from this cross are elav null- mutants, and they are distinguishable from the remaining males by the absence of the FM7,act:GFP balancer chromosome. However, 50% of the female progeny do not carry the FM7,act:GFP balancer neither, but they are only heterozygous for the elavE476 allele. This allele is recessive, and therefore, these heterozygous females should not show any phenotype.
To control for this, I did the reciprocal cross (see 33B), in which FM7,act:GFP males were crossed with abdA:GFP females. None of the embryos resulting from this cross would be elav mutants, and they would be heterozygous for the abdA:GFP construct, serving as a perfect control group for the experiment.
Again, the two sets of embryos were hybridized with probes against the abdA:GFP gene and against the GFP present in the FM7,act:GFP balancer.
104
Figure 33 Design of the experimental and control crosses for the study of abdA:GFP expression in elav mutant embryos
The experimental cross generates two clearly differentiated populations of embryos (Figure 34, A and B).
105
Figure 34 The repression by transcriptional interference of the abdA:GFP transgene is abolished in elav embryos
Dissected central nervous systems from embryos derived from the experimental cross (left) or the control cross (right) between the abdA:GFP line and the elavE476/FM7,act:GFP line, stained by in situ hybridization using the abdA:GFP probe.
The first population (34A) corresponds to the heterozygous elav/+ females, which are phenotypically similar to the male embryos laid by the reciprocal control cross (34C).
They present a very strong staining from PS7 to PS12, which corresponds to the
"domino" pattern characteristic of abd-A and described in previous chapters. In PS13-14, however, the signal becomes more diffuse. Knowing from previous experiments that the abdA:GFP transgene is not expressed in PS14, and only weakly expressed in PS13 (see Chapter III), I tentatively conclude that the strong signal detected in PS13-14 reflects the readthrough transcription of the iab-8 ncRNA, and not a de-repression of the abdA:GFP gene. This conclusion is confirmed by the staining observed in Fab-864,abdA/GFP embryos in elav+ background, where no signal is visible in PS14 (Figure 35, also Figure 19).
106
In the second population embryos derived from the experimental cross, however (34B), there is a different situation. These embryos show strong staining in PS13, in a similar level to PS12, and very weak or absent signal in PS14. In this case, the staining observed in PS13 cannot correspond to the readthrough transcription, as this signal would also be present, with the same strength, in PS14. Furthermore, I had already proven that in elav embryos the iab-8 readthrough transcript is strongly reduced (Figure 28). Finally, as the mir-iab-8 microRNA does not have a repressive effect over the abdA:GFP transgene, this strong de-repression cannot be caused by a potential effect over the microRNA-mediated repression caused by the loss-of-function of elav. As a final argument, this staining is reminiscent of the abdA:GFP expression pattern that can be observed by in situ hybridization against abdA:GFP in Fab-864,abdA:GFP recombinant embryos (Figure 35, also Figure 19).
Therefore, I can conclude that the signal detected in PS13 of the CNS in elavE476 embryos corresponds to transcription coming from the abdA:GFP promoter, reflecting a failure of transcriptional interference in absence of ELAV.
Figure 35 The de-repression of abdA:GFP in elav background is similar to the one caused by the absence of transcriptional interference in Fab-864,abdA:GFP embryos Dissected central nervous systems from elavE476, abdAGFP / + or Fab-864, abdA:GFP embryos stained by in situ hybridization with the abdA:GFP probe.
107