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Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development.

Singer R, Atar S, Atias O, Oron E, Segal D, Hirsch JA, Tuller T, Orian A, Chamovitz DA - Nucleic Acids Res. (2014)

Bottom Line: While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression.These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control.While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Ecology of Plants.

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CSN7 interacts with DNA. Anisotropy binding profile for binding of full-length CSN7 to 3-nM fluorescein-labeled oligonucleotide derived from the Drosophila E2f promoter at 16°C (x). A 50-fold excess of unlabeled E2f oligonucleotide (·) at [150 nM] was added to 3 nM of fluorescein-labeled oligonucleotide as a competitor and titrated by CSN7. The KD app of CSN7 for this DNA fragment is 0.51 ± 0.03 μM with a Hill slope of 1.45, whereas the curve with unlabeled DNA had a KD app constant of 1.7 ± 0.16 μM with a Hill slope of 2.3. Due to the likelihood of multiple binding sites, caution should be exercised in overinterpreting these parameters as they represent an empirical characterization of the binding rather than providing a rigorous binding model.
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Figure 1: CSN7 interacts with DNA. Anisotropy binding profile for binding of full-length CSN7 to 3-nM fluorescein-labeled oligonucleotide derived from the Drosophila E2f promoter at 16°C (x). A 50-fold excess of unlabeled E2f oligonucleotide (·) at [150 nM] was added to 3 nM of fluorescein-labeled oligonucleotide as a competitor and titrated by CSN7. The KD app of CSN7 for this DNA fragment is 0.51 ± 0.03 μM with a Hill slope of 1.45, whereas the curve with unlabeled DNA had a KD app constant of 1.7 ± 0.16 μM with a Hill slope of 2.3. Due to the likelihood of multiple binding sites, caution should be exercised in overinterpreting these parameters as they represent an empirical characterization of the binding rather than providing a rigorous binding model.

Mentions: As earlier biophysical and cell biology studies suggested that CSN7, or the CSN in general, binds nucleic acids (25), we carried out binding assays (33) to study the potential interaction of purified full-length CSN7 protein with a fluorescently-labeled DNA fragment, 70 bp in length. Steady-state fluorescence anisotropy of fluorescein-labeled DNA, derived from E2f, was monitored upon titration with the full-length CSN7 protein. Figure 1 (x) shows a representative binding isotherm. The anisotropy increases in a saturable manner, demonstrating binding of CSN7 to this DNA fragment. The anisotropy values suggest that multiple protein copies bind this fragment.


Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development.

Singer R, Atar S, Atias O, Oron E, Segal D, Hirsch JA, Tuller T, Orian A, Chamovitz DA - Nucleic Acids Res. (2014)

CSN7 interacts with DNA. Anisotropy binding profile for binding of full-length CSN7 to 3-nM fluorescein-labeled oligonucleotide derived from the Drosophila E2f promoter at 16°C (x). A 50-fold excess of unlabeled E2f oligonucleotide (·) at [150 nM] was added to 3 nM of fluorescein-labeled oligonucleotide as a competitor and titrated by CSN7. The KD app of CSN7 for this DNA fragment is 0.51 ± 0.03 μM with a Hill slope of 1.45, whereas the curve with unlabeled DNA had a KD app constant of 1.7 ± 0.16 μM with a Hill slope of 2.3. Due to the likelihood of multiple binding sites, caution should be exercised in overinterpreting these parameters as they represent an empirical characterization of the binding rather than providing a rigorous binding model.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150811&req=5

Figure 1: CSN7 interacts with DNA. Anisotropy binding profile for binding of full-length CSN7 to 3-nM fluorescein-labeled oligonucleotide derived from the Drosophila E2f promoter at 16°C (x). A 50-fold excess of unlabeled E2f oligonucleotide (·) at [150 nM] was added to 3 nM of fluorescein-labeled oligonucleotide as a competitor and titrated by CSN7. The KD app of CSN7 for this DNA fragment is 0.51 ± 0.03 μM with a Hill slope of 1.45, whereas the curve with unlabeled DNA had a KD app constant of 1.7 ± 0.16 μM with a Hill slope of 2.3. Due to the likelihood of multiple binding sites, caution should be exercised in overinterpreting these parameters as they represent an empirical characterization of the binding rather than providing a rigorous binding model.
Mentions: As earlier biophysical and cell biology studies suggested that CSN7, or the CSN in general, binds nucleic acids (25), we carried out binding assays (33) to study the potential interaction of purified full-length CSN7 protein with a fluorescently-labeled DNA fragment, 70 bp in length. Steady-state fluorescence anisotropy of fluorescein-labeled DNA, derived from E2f, was monitored upon titration with the full-length CSN7 protein. Figure 1 (x) shows a representative binding isotherm. The anisotropy increases in a saturable manner, demonstrating binding of CSN7 to this DNA fragment. The anisotropy values suggest that multiple protein copies bind this fragment.

Bottom Line: While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression.These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control.While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Ecology of Plants.

Show MeSH