It is already well established that DNA can undergo a variety of conformational transitions, adopting structures other than the canonical B form. Among these, G-quadruplexes (G4s) are tetra-helical structures that originate at guanine-rich genomic sites, including telomeres and gene promoters. For many years, G4s were thought to constitute obstacles to replicational and transcriptional machineries and therefore they were considered as interesting targets for the selective downregulation of oncogene expression. However, recent findings suggested an active role of G4s in promoting gene expression. Therefore, a clear understanding of the physiological role of G4s is lacking. This work aims to investigate the effect B-DNA to G-quadruplex transition at the proximal promoter of the oncogene c-KIT. Here, three G4 forming motifs have been characterized, namely KIT1, KIT2 and KIT*. They are all closely located in the region spanning the first 160 bp upstream of the transcription start site (TSS). Both KIT1 and KIT* adopt a unique well-defined G-quadruplex topology, while KIT2 is more polymorphic. Indeed, KIT2 can adopt multiple G4 topologies, some being kinetically favored and others being thermodynamically stable. The close proximity between KIT2 and KIT* allows the two G4s to interact through stacking interactions between the external tetrads, arising a further level of structural complexity. In this study, point mutations were introduced at these three G4 forming motifs, to disrupt G-quadruplex folding. This resulted in an increase of the promoter activity that was proved to be mostly dependent on the increased recruitment of the transcription factor SP1 at the region harboring KIT2 and KIT* G-quadruplexes. Therefore, the B-DNA to G-quadruplex transition at KIT2 and KIT* seemed to prevent SP1 recruitment at this site and thus appeared as a feature that can be exploited to reach the downregulation of the oncogene expression. This promoted the search for ligands that could stabilize KIT2 and KIT* G4s. Given that transcription factors bind to their consensus sites on a timescale of seconds, the kinetically favored G4 topology adopted by KIT2, whose folding occurs within few seconds, was selected as preferential target. The perylene derivative K20 was proved to efficiently bind to this arrangement and promote the conformational selection of this topology, against the more thermodynamically stable one. The same ligand also displayed a good binding profile towards KIT* G4. Therefore, K20 can be used as a probe to further validate the effect of G4 folding at KIT2 and KIT* on SP1 displacement and consequent downregulation of c-KIT, with a particular focus on the kinetically favored conformation adopted by KIT2. Eventually, the latest work herein presented aimed to provide a wider perspective on the KIT2KIT* mediated control of c-KIT expression, by searching for proteins that can interact with this motif when folded into G4. Surprisingly, the intermediate filament protein vimentin displayed high affinity for the G4 motif comprising both KIT2 and KIT*, with no binding to the isolated G4 units. This pointed to a distinct role of the higher-order G4 arrangement adopted by KIT2KIT*. Although the biological implications of vimentin recruitment at this site are still to be addressed, this work provides an opposite perspective about the role exerted by G4 structures at the promoter of c-KIT. Indeed, not only they can prevent the binding of the transcription factor SP1, thus leading to a reduction of c-KIT expression, but also, they can trigger the onset of distinct physiological processes that rely on vimentin recruitment at this site.
It is already well established that DNA can undergo a variety of conformational transitions, adopting structures other than the canonical B form. Among these, G-quadruplexes (G4s) are tetra-helical structures that originate at guanine-rich genomic sites, including telomeres and gene promoters. For many years, G4s were thought to constitute obstacles to replicational and transcriptional machineries and therefore they were considered as interesting targets for the selective downregulation of oncogene expression. However, recent findings suggested an active role of G4s in promoting gene expression. Therefore, a clear understanding of the physiological role of G4s is lacking. This work aims to investigate the effect B-DNA to G-quadruplex transition at the proximal promoter of the oncogene c-KIT. Here, three G4 forming motifs have been characterized, namely KIT1, KIT2 and KIT*. They are all closely located in the region spanning the first 160 bp upstream of the transcription start site (TSS). Both KIT1 and KIT* adopt a unique well-defined G-quadruplex topology, while KIT2 is more polymorphic. Indeed, KIT2 can adopt multiple G4 topologies, some being kinetically favored and others being thermodynamically stable. The close proximity between KIT2 and KIT* allows the two G4s to interact through stacking interactions between the external tetrads, arising a further level of structural complexity. In this study, point mutations were introduced at these three G4 forming motifs, to disrupt G-quadruplex folding. This resulted in an increase of the promoter activity that was proved to be mostly dependent on the increased recruitment of the transcription factor SP1 at the region harboring KIT2 and KIT* G-quadruplexes. Therefore, the B-DNA to G-quadruplex transition at KIT2 and KIT* seemed to prevent SP1 recruitment at this site and thus appeared as a feature that can be exploited to reach the downregulation of the oncogene expression. This promoted the search for ligands that could stabilize KIT2 and KIT* G4s. Given that transcription factors bind to their consensus sites on a timescale of seconds, the kinetically favored G4 topology adopted by KIT2, whose folding occurs within few seconds, was selected as preferential target. The perylene derivative K20 was proved to efficiently bind to this arrangement and promote the conformational selection of this topology, against the more thermodynamically stable one. The same ligand also displayed a good binding profile towards KIT* G4. Therefore, K20 can be used as a probe to further validate the effect of G4 folding at KIT2 and KIT* on SP1 displacement and consequent downregulation of c-KIT, with a particular focus on the kinetically favored conformation adopted by KIT2. Eventually, the latest work herein presented aimed to provide a wider perspective on the KIT2KIT* mediated control of c-KIT expression, by searching for proteins that can interact with this motif when folded into G4. Surprisingly, the intermediate filament protein vimentin displayed high affinity for the G4 motif comprising both KIT2 and KIT*, with no binding to the isolated G4 units. This pointed to a distinct role of the higher-order G4 arrangement adopted by KIT2KIT*. Although the biological implications of vimentin recruitment at this site are still to be addressed, this work provides an opposite perspective about the role exerted by G4 structures at the promoter of c-KIT. Indeed, not only they can prevent the binding of the transcription factor SP1, thus leading to a reduction of c-KIT expression, but also, they can trigger the onset of distinct physiological processes that rely on vimentin recruitment at this site.
B-DNA TO G-QUADRUPLEX TRANSITION AT THE C-KIT PROXIMAL PROMOTER: EVALUATION OF THE EFFECTS ON PROTEIN RECRUITMENT AND PROMOTER ACTIVITY / Ceschi, Silvia. - (2022 Mar 21).
B-DNA TO G-QUADRUPLEX TRANSITION AT THE C-KIT PROXIMAL PROMOTER: EVALUATION OF THE EFFECTS ON PROTEIN RECRUITMENT AND PROMOTER ACTIVITY
CESCHI, SILVIA
2022
Abstract
It is already well established that DNA can undergo a variety of conformational transitions, adopting structures other than the canonical B form. Among these, G-quadruplexes (G4s) are tetra-helical structures that originate at guanine-rich genomic sites, including telomeres and gene promoters. For many years, G4s were thought to constitute obstacles to replicational and transcriptional machineries and therefore they were considered as interesting targets for the selective downregulation of oncogene expression. However, recent findings suggested an active role of G4s in promoting gene expression. Therefore, a clear understanding of the physiological role of G4s is lacking. This work aims to investigate the effect B-DNA to G-quadruplex transition at the proximal promoter of the oncogene c-KIT. Here, three G4 forming motifs have been characterized, namely KIT1, KIT2 and KIT*. They are all closely located in the region spanning the first 160 bp upstream of the transcription start site (TSS). Both KIT1 and KIT* adopt a unique well-defined G-quadruplex topology, while KIT2 is more polymorphic. Indeed, KIT2 can adopt multiple G4 topologies, some being kinetically favored and others being thermodynamically stable. The close proximity between KIT2 and KIT* allows the two G4s to interact through stacking interactions between the external tetrads, arising a further level of structural complexity. In this study, point mutations were introduced at these three G4 forming motifs, to disrupt G-quadruplex folding. This resulted in an increase of the promoter activity that was proved to be mostly dependent on the increased recruitment of the transcription factor SP1 at the region harboring KIT2 and KIT* G-quadruplexes. Therefore, the B-DNA to G-quadruplex transition at KIT2 and KIT* seemed to prevent SP1 recruitment at this site and thus appeared as a feature that can be exploited to reach the downregulation of the oncogene expression. This promoted the search for ligands that could stabilize KIT2 and KIT* G4s. Given that transcription factors bind to their consensus sites on a timescale of seconds, the kinetically favored G4 topology adopted by KIT2, whose folding occurs within few seconds, was selected as preferential target. The perylene derivative K20 was proved to efficiently bind to this arrangement and promote the conformational selection of this topology, against the more thermodynamically stable one. The same ligand also displayed a good binding profile towards KIT* G4. Therefore, K20 can be used as a probe to further validate the effect of G4 folding at KIT2 and KIT* on SP1 displacement and consequent downregulation of c-KIT, with a particular focus on the kinetically favored conformation adopted by KIT2. Eventually, the latest work herein presented aimed to provide a wider perspective on the KIT2KIT* mediated control of c-KIT expression, by searching for proteins that can interact with this motif when folded into G4. Surprisingly, the intermediate filament protein vimentin displayed high affinity for the G4 motif comprising both KIT2 and KIT*, with no binding to the isolated G4 units. This pointed to a distinct role of the higher-order G4 arrangement adopted by KIT2KIT*. Although the biological implications of vimentin recruitment at this site are still to be addressed, this work provides an opposite perspective about the role exerted by G4 structures at the promoter of c-KIT. Indeed, not only they can prevent the binding of the transcription factor SP1, thus leading to a reduction of c-KIT expression, but also, they can trigger the onset of distinct physiological processes that rely on vimentin recruitment at this site.File | Dimensione | Formato | |
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