Limits...
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.

Show MeSH
(A) qRT-PCR analyses of CSN7-DamID target genes in RNA isolated from Kc167 cells following transfection with an RNAi-CSN7 construct. The levels of CycE, E2f and Scalloped were downregulated relative to cells transfected with a control RNA. The columns show the geometric mean of the expression, with the error bars representing the minimal and maximal deviations. (B) ChIP. Chromatin was isolated from cross-linked Kc167 cells and subjected to immuno-precipitation (IP) with αCSN7, αH3 or NGS followed by qRT-PCR. PCR primers were designed around the predicted binding regions for CSN7 in the Sd, E2F and CycE genes (Supplementary Table S2), and around a region ∼2.5 kb downstream from the predicted CSN7 target in CycE. The predicted binding regions of all three genes were precipitated with the positive control αH3, and with αCSN7, significantly beyond the signal for the negative control NGS. (P-values for significance above background: Sd, 9.72e-04; E2F, 0.01; CycE, 0.01). Moreover, while αH3 also precipitated the ORF of Cyclin E, αCSN7 did not, showing specificity for interaction with the promoter region only, as identified by the DamID experiment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150811&req=5

Figure 4: (A) qRT-PCR analyses of CSN7-DamID target genes in RNA isolated from Kc167 cells following transfection with an RNAi-CSN7 construct. The levels of CycE, E2f and Scalloped were downregulated relative to cells transfected with a control RNA. The columns show the geometric mean of the expression, with the error bars representing the minimal and maximal deviations. (B) ChIP. Chromatin was isolated from cross-linked Kc167 cells and subjected to immuno-precipitation (IP) with αCSN7, αH3 or NGS followed by qRT-PCR. PCR primers were designed around the predicted binding regions for CSN7 in the Sd, E2F and CycE genes (Supplementary Table S2), and around a region ∼2.5 kb downstream from the predicted CSN7 target in CycE. The predicted binding regions of all three genes were precipitated with the positive control αH3, and with αCSN7, significantly beyond the signal for the negative control NGS. (P-values for significance above background: Sd, 9.72e-04; E2F, 0.01; CycE, 0.01). Moreover, while αH3 also precipitated the ORF of Cyclin E, αCSN7 did not, showing specificity for interaction with the promoter region only, as identified by the DamID experiment.

Mentions: The bioinformatic analysis above clearly illustrated that CSN-dependent genes are enriched in the CSN7-DamID dataset. To further connect between the genes bound by CSN7 and the phenotypes described above, we determined if the expression of the genes within the CSN7-DamID loci is also misregulated upon RNAi-mediated reduction of CSN7. We examined by qRT-PCR the mRNA levels of three key developmental genes involved in cell-cycle regulation and wing disc development, CycE, E2f and Scalloped (Sd). mRNA levels were checked in Kc167 and S2 cells where we reduced CSN7 levels via RNAi. As shown in Figure 4A and Supplementary Figure S6, the levels of CycE and E2f were reduced when Csn7 was silenced in both cell types, while Sd was reduced only in Kc167 cells.


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)

(A) qRT-PCR analyses of CSN7-DamID target genes in RNA isolated from Kc167 cells following transfection with an RNAi-CSN7 construct. The levels of CycE, E2f and Scalloped were downregulated relative to cells transfected with a control RNA. The columns show the geometric mean of the expression, with the error bars representing the minimal and maximal deviations. (B) ChIP. Chromatin was isolated from cross-linked Kc167 cells and subjected to immuno-precipitation (IP) with αCSN7, αH3 or NGS followed by qRT-PCR. PCR primers were designed around the predicted binding regions for CSN7 in the Sd, E2F and CycE genes (Supplementary Table S2), and around a region ∼2.5 kb downstream from the predicted CSN7 target in CycE. The predicted binding regions of all three genes were precipitated with the positive control αH3, and with αCSN7, significantly beyond the signal for the negative control NGS. (P-values for significance above background: Sd, 9.72e-04; E2F, 0.01; CycE, 0.01). Moreover, while αH3 also precipitated the ORF of Cyclin E, αCSN7 did not, showing specificity for interaction with the promoter region only, as identified by the DamID experiment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: (A) qRT-PCR analyses of CSN7-DamID target genes in RNA isolated from Kc167 cells following transfection with an RNAi-CSN7 construct. The levels of CycE, E2f and Scalloped were downregulated relative to cells transfected with a control RNA. The columns show the geometric mean of the expression, with the error bars representing the minimal and maximal deviations. (B) ChIP. Chromatin was isolated from cross-linked Kc167 cells and subjected to immuno-precipitation (IP) with αCSN7, αH3 or NGS followed by qRT-PCR. PCR primers were designed around the predicted binding regions for CSN7 in the Sd, E2F and CycE genes (Supplementary Table S2), and around a region ∼2.5 kb downstream from the predicted CSN7 target in CycE. The predicted binding regions of all three genes were precipitated with the positive control αH3, and with αCSN7, significantly beyond the signal for the negative control NGS. (P-values for significance above background: Sd, 9.72e-04; E2F, 0.01; CycE, 0.01). Moreover, while αH3 also precipitated the ORF of Cyclin E, αCSN7 did not, showing specificity for interaction with the promoter region only, as identified by the DamID experiment.
Mentions: The bioinformatic analysis above clearly illustrated that CSN-dependent genes are enriched in the CSN7-DamID dataset. To further connect between the genes bound by CSN7 and the phenotypes described above, we determined if the expression of the genes within the CSN7-DamID loci is also misregulated upon RNAi-mediated reduction of CSN7. We examined by qRT-PCR the mRNA levels of three key developmental genes involved in cell-cycle regulation and wing disc development, CycE, E2f and Scalloped (Sd). mRNA levels were checked in Kc167 and S2 cells where we reduced CSN7 levels via RNAi. As shown in Figure 4A and Supplementary Figure S6, the levels of CycE and E2f were reduced when Csn7 was silenced in both cell types, while Sd was reduced only in Kc167 cells.

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