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Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

Sharma V, Jordan JJ, Ciribilli Y, Resnick MA, Bisio A, Inga A - PLoS ONE (2015)

Bottom Line: For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα.The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65.Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.

ABSTRACT
The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

No MeSH data available.


Related in: MedlinePlus

Functional evaluation of four selected κB-REs in MCF7 cells.A-B) MCF7 cells were transiently transfected with four pGL4.26 derived vectors containing different κB-REs along with a control pGL4.26 empty vector. Twenty-four hours after transfection cells were treated for 4 or 8 hours with TNFα (10ng/ml for panel A or 50ng/ml for panel B) alone or in combination with BAY11-7082 (20μM for 8 hours, only for panel B). Presented are the average fold-induction relative to the empty pGL4.26 vector and the standard deviation of at least three independent biological replicates. C) Western blot analysis showing p50 and p65 protein levels from nuclear-cytoplasmic fractions after the indicated treatments at the following doses: TNFα (50ng/ml) and BAY11-7082 (20μM). GAPDH and Histone 3 were used as reference controls for the cytoplasmic and nuclear fraction respectively.
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pone.0130170.g004: Functional evaluation of four selected κB-REs in MCF7 cells.A-B) MCF7 cells were transiently transfected with four pGL4.26 derived vectors containing different κB-REs along with a control pGL4.26 empty vector. Twenty-four hours after transfection cells were treated for 4 or 8 hours with TNFα (10ng/ml for panel A or 50ng/ml for panel B) alone or in combination with BAY11-7082 (20μM for 8 hours, only for panel B). Presented are the average fold-induction relative to the empty pGL4.26 vector and the standard deviation of at least three independent biological replicates. C) Western blot analysis showing p50 and p65 protein levels from nuclear-cytoplasmic fractions after the indicated treatments at the following doses: TNFα (50ng/ml) and BAY11-7082 (20μM). GAPDH and Histone 3 were used as reference controls for the cytoplasmic and nuclear fraction respectively.

Mentions: To examine whether the differences in transactivation capacity observed in yeast were predictive of variable responsiveness in mammalian cells upon NF-κB activation, κB-dependent gene reporter assays were carried out in the human MCF7 cells. We selected 4 different κB-REs whose transactivation potential driven by co-expressed p65 and p50TAD ranked from high (M2, RelBCons) to medium (RE4) and to low (M1) in yeast. Those κB-REs were placed upstream of the Firefly luciferase in the pGL4.26 vector for transient transfection experiments. Twenty-four hours post-transfection cells were treated with 10ng/ml (Fig 4A) or 50ng/ml (Fig 4B) TNFα and/or with 20μM BAY11-7082 (BAY) respectively to activate or repress the NF-κB pathway. Results demonstrated that differences in relative transactivation potential measured in yeast were confirmed in human cells. In fact the M2 κB-RE was the most responsive, followed by RelBCons, with M1 being the least responsive. Time- and concentration-dependent TNFα responsiveness and repression by BAY were apparent. TNFα treatment led to a strong increase in p65 protein in nuclear extracts, while the p50 protein was already nuclear in mock condition and its abundance did not change significantly after treatment (Fig 4C). This observation suggests that the increase in M2 κB-RE responsiveness observed already in mock condition might be dependent primarily on p50 homodimers, while the enhanced responsiveness after TNFα treatment would be related to the increase of p65 in the nucleus.


Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

Sharma V, Jordan JJ, Ciribilli Y, Resnick MA, Bisio A, Inga A - PLoS ONE (2015)

Functional evaluation of four selected κB-REs in MCF7 cells.A-B) MCF7 cells were transiently transfected with four pGL4.26 derived vectors containing different κB-REs along with a control pGL4.26 empty vector. Twenty-four hours after transfection cells were treated for 4 or 8 hours with TNFα (10ng/ml for panel A or 50ng/ml for panel B) alone or in combination with BAY11-7082 (20μM for 8 hours, only for panel B). Presented are the average fold-induction relative to the empty pGL4.26 vector and the standard deviation of at least three independent biological replicates. C) Western blot analysis showing p50 and p65 protein levels from nuclear-cytoplasmic fractions after the indicated treatments at the following doses: TNFα (50ng/ml) and BAY11-7082 (20μM). GAPDH and Histone 3 were used as reference controls for the cytoplasmic and nuclear fraction respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493129&req=5

pone.0130170.g004: Functional evaluation of four selected κB-REs in MCF7 cells.A-B) MCF7 cells were transiently transfected with four pGL4.26 derived vectors containing different κB-REs along with a control pGL4.26 empty vector. Twenty-four hours after transfection cells were treated for 4 or 8 hours with TNFα (10ng/ml for panel A or 50ng/ml for panel B) alone or in combination with BAY11-7082 (20μM for 8 hours, only for panel B). Presented are the average fold-induction relative to the empty pGL4.26 vector and the standard deviation of at least three independent biological replicates. C) Western blot analysis showing p50 and p65 protein levels from nuclear-cytoplasmic fractions after the indicated treatments at the following doses: TNFα (50ng/ml) and BAY11-7082 (20μM). GAPDH and Histone 3 were used as reference controls for the cytoplasmic and nuclear fraction respectively.
Mentions: To examine whether the differences in transactivation capacity observed in yeast were predictive of variable responsiveness in mammalian cells upon NF-κB activation, κB-dependent gene reporter assays were carried out in the human MCF7 cells. We selected 4 different κB-REs whose transactivation potential driven by co-expressed p65 and p50TAD ranked from high (M2, RelBCons) to medium (RE4) and to low (M1) in yeast. Those κB-REs were placed upstream of the Firefly luciferase in the pGL4.26 vector for transient transfection experiments. Twenty-four hours post-transfection cells were treated with 10ng/ml (Fig 4A) or 50ng/ml (Fig 4B) TNFα and/or with 20μM BAY11-7082 (BAY) respectively to activate or repress the NF-κB pathway. Results demonstrated that differences in relative transactivation potential measured in yeast were confirmed in human cells. In fact the M2 κB-RE was the most responsive, followed by RelBCons, with M1 being the least responsive. Time- and concentration-dependent TNFα responsiveness and repression by BAY were apparent. TNFα treatment led to a strong increase in p65 protein in nuclear extracts, while the p50 protein was already nuclear in mock condition and its abundance did not change significantly after treatment (Fig 4C). This observation suggests that the increase in M2 κB-RE responsiveness observed already in mock condition might be dependent primarily on p50 homodimers, while the enhanced responsiveness after TNFα treatment would be related to the increase of p65 in the nucleus.

Bottom Line: For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα.The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65.Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy.

ABSTRACT
The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD), a chimeric construct containing the TAD derived from p65 was also generated (p50TAD) to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs) where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

No MeSH data available.


Related in: MedlinePlus