Transcriptional interference is a very basic kind of gene regulation, by which the simple act of transcription from one promoter represses (in cis-) the expression of other, normally placed in its immediate vicinity, or with an overlapping transcriptional unit (Shearwin et al. 2005). The use of transcriptional interference by the cell as a mechanism of repression was initially described for the GAL10/GAL7 genes of the yeast Saccharomyces cerevisiae. These two genes, included in the galactose cluster, are transcribed from two promoters located in tandem. When the upstream gene, GAL10, is strongly transcribed, this expression can lead to silencing of the downstream GAL7.
According to this, the deletion of the UAS sites of GAL10, necessary for its expression, leads to an overexpression of GAL7. The repression of GAL7 seems to be due to an inefficient transcription termination of GAL10, which transcribes over the GAL7 promoter (Greger and Proudfoot 1998).
Since then, it has been proven that transcriptional interference is a more widespread repressive mechanism than initially thought. The molecular basis of this repression is clearly not uniform, and it has been widely studied on yeast (Gullerova and Proudfoot 2010). The mechanism of transcriptional interference depends on the relative position of both promoters. Following this criterion, three situations can be described:
− Overlapping divergent promoters: Both transcripts originate from the same area.
− Convergent promoters: The promoters produce antisense genes with overlapping regions.
− Tandem promoters: Both promoters drive co-directional transcription, and the upstream transcript may overlap with the downstream promoter or enhancers located in the vicinity.
The repression can occur via different mechanisms, being the most studied occlusion, promoter competition, RNApol collision and RNApol pausing.
42 Transcriptional interference by occlusion
Occlusion refers either to the transcription across enhancers, preventing their accessibility by transcription factors, or across promoters, preventing the access of the RNA polymerase to the target gene (Adhya and Gottesman 1982). This has been proposed to be the case in the interaction between GAL10 and GAL7 previously described (Greger and Proudfoot 1998), and also in the repression of Ubx by the bxd noncoding RNAs as described in (Petruk et al. 2006).
Promoter competition
A second case is the promoter competition, in which both promoters are located close to each other, and one of them interacts preferentially with an enhancer. This seems to happen between the beta- and epsilon- globulin genes in chickens (Choi and Engel 1988, Foley and Engel 1992). In the Antennapedia Complex of Drosophila, the AE1 enhancer, located between the genes fushi tarazu (ftz) and Sex combs reduced (Scr), interacts preferentially with the homeobox-containing gene ftz, over Scr (Ohtsuki et al. 1998). This mechanism is predominant in the cases of overlapping divergent promoters, like in the case of the permease gene aroP of Escherichia coli, whose expression from the main promoter, P1, is prevented by the transcription from the divergent noncoding promoter P3 (Wang et al. 1998).
RNA polymerase collision
It is the most likely situation in the case of transcriptional interference between convergent promoters. These cases are rare in natural circumstances. There seems to be evolutionary pressure to prevent the occurrence of simultaneous convergent transcription (Marin et al. 2004), as the RNA polymerase collisions can apparently lead to chromosome instability (Pannunzio and Lieber 2016).
43 Dislodgement of transcription factors
In the case of tandem promoters, occlusion and collision have been described as not very likely to happen First, the upstream promoter should be extremely active for it to effectively repress the target gene by occlusion of enhancers (Sneppen et al. 2005).
Second, the collision between an upstream elongating polymerase and a paused one seems to enhance transcription of the second one, rather than to repress it (Epshtein et al.
2003). The most likely situation would be the dislodgement of transcription factors from the assembling pre-initiation complex, especially in the case where the recruitment of this complex to this promoter is slow (Palmer et al. 2011).
Pausing of the elongating RNApol II
It is a special case of occlusion, in which the elongating RNA polymerase coming from a neighbor gene pauses over certain areas (enhancers or promoters) of another, prolonging the time during which they are inaccessible by transcription factors. This phenomenon must be distinguished from the pausing of the initiating RNApol II over the promoter of developmentally regulated genes. The pausing of the elongating RNApol causes the repression of the second gene, for as long as the promoter or enhancer stays occluded by the polymerase. This kind of transcriptional interference takes place between two convergent promoters of the λ phage, and it has been shown that the duration of the pause can be increased or reduced by different regulatory proteins (Palmer et al. 2009). This mechanism can, therefore, be finely tuned, increasing or decreasing the amount of repression over the target gene depending on the circumstances.
Chromatin modifications
They can occur independently or in addition to any of the mechanisms listed above. The transcription of the interfering RNA over certain areas can cause the recruitment of proteins with chromatin remodeling activity that would, in time, render the chromatin inaccessible to transcription factors. This has been proposed in eukaryotes as a mechanism for gene inactivation (Camblong et al. 2007, Saxena and Carninci 2011, Erokhin et al. 2013, Gill et al. 2019), and it has the advantage of not requiring continuous transcription of the interfering RNA for the permanent silencing of a gene.
44 Transcriptional interference in Hox clusters
The description of the regulatory interaction between bxd and Ubx and between the iab-8 ncRNA and abd-A has led to the interesting proposition that transcriptional interference could have been an ancestral way of regulation in Hox gene clusters. According to this model, posterior prevalence would be explained by the transcription of posterior Hox genes over the promoter of their immediate downstream neighbors. In this way, the expression of posterior genes would repress the anterior ones in cis-. This mechanism probably evolved later into a more refined interaction, in which Hox proteins acquired the ability to exert that repression in trans- (Gummalla et al. 2012).
Interestingly, the study of Ubx transcription in Arthropods has shed light over some interesting processes that could be derived from this ancient regulation, such as the production of a conserved antisense ncRNA that represses Ubx expression, or the polycistronic transcription between Ubx and Antp, repressive of Antp expression.
Antisense transcription of Hox genes
In 2006, (Brena et al. 2006) described the existence of antisense transcripts to the Hox gene Ubx of the myriapod Strigamia maritima, which are expressed in a complementary expression pattern to Ubx, beginning in a slightly more anterior position of the body.
They proposed that these RNAs repress Ubx expression by transcriptional interference or by the generation of double-stranded RNA. The production of these RNAs is conserved in different Arthropods, as described in (Janssen and Budd 2010). Interestingly, the expression of the Ubx antisense transcript resembles to the expression pattern of the bxd ncRNAs in Drosophila. Both RNAs begin to be expressed slightly before Ubx, and they are expressed in the posterior compartment of parasegments. These transcripts have been proposed to be functional repressors of Ubx by transcriptional interference, and the authors propose that they might have a common evolutionary origin.
45 Polycystronic Hox transcripts
The Ubx gene of the crustaceans Daphnia magna and Artemia franciscana shows a readthrough that invades the transcriptional unit of its immediate neighbor, Antp. This readthrough transcription produces a bicistronic chimeric RNA Ubx/Antp, in which the only ORF that is translated is the one of Ubx, following the posterior prevalence model (Shiga et al. 2006). Interestingly this is reminiscent of the observation that we make in this thesis between the iab-8 ncRNA and abd-A (see below). This bicistronic RNA has only been shown to exist in myriapods (Shiga et al. 2006) and several crustaceans (Janssen and Budd 2010), suggesting that it could be an evolutionary relic.
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OBJECTIVES
Previous studies appoint the iab-8 ncRNA as the sole repressive element for the Hox gene abd-A in PS13 of the embryonic central nervous system. This regulation is exerted via a double mechanism: the production of the mir-iab-8 microRNA from one of the intronic sequences of iab-8, and a second, cis-acting, repressive mechanism, proposed to be transcriptional interference.
Although the evidence provided by the research into abd-A regulation favors so far the model of transcriptional interference, it has been extremely difficult to rule out the influence of other factors in the regulation of abd-A. The reasons for this are quite diverse, but perhaps the main one is the mir-iab-8 microRNA. The unusually strong repressive action of this microRNA adds an additional layer of complexity to the study of abd-A repression. In addition, the sterility caused by its absence constitutes an important obstacle to any attempt to characterize the microRNA-independent repression of abd-A by studying abd-A expression in ΔmiR embryos.
In the light of all this, I set as the main objective of this thesis to build a genetic reporter system that, when inserted in the BX-C, would be repressed by transcriptional interference. In addition, I wanted to make this reporter immune to the repressive action of the mir-iab-8 microRNA.
The tissue-specificity of the regulation of abdominal-A was also of great interest to me.
Even though the iab-8 ncRNA is expressed in the ectoderm, its regulatory role over abd-A seems to be restricted to the embryonic CNS. This is not a surprise, given that in the ectoderm the expression of abd-A in PS13 is prevented by Abd-B. I wanted to verify if the ectopic expression of the iab-8 ncRNA could repress abd-A by transcriptional interference in other tissues.
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