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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 interactions between p50 and p65 towards a panel of κB-REs.A) and B) Eight different κB-REs, each comprising two adjacent copies of the decameric κB sequences were examined for the transactivation potentials of p50TAD, p65 as well as upon co-expression of both proteins. Cells were grown in lower (0.008%, panel A) and high (0.064%, panel B) levels of galactose. C) A non-chimeric p50 construct lacking the TAD domain was also studied at the higher galactose level for all REs, except RelBCons. In all panels, luciferase assays were conducted and results plotted as described in Fig 1.
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pone.0130170.g003: Functional interactions between p50 and p65 towards a panel of κB-REs.A) and B) Eight different κB-REs, each comprising two adjacent copies of the decameric κB sequences were examined for the transactivation potentials of p50TAD, p65 as well as upon co-expression of both proteins. Cells were grown in lower (0.008%, panel A) and high (0.064%, panel B) levels of galactose. C) A non-chimeric p50 construct lacking the TAD domain was also studied at the higher galactose level for all REs, except RelBCons. In all panels, luciferase assays were conducted and results plotted as described in Fig 1.

Mentions: Since p50 and p65 are normally present at the same time in mammalian cells and the p50/p65 heterodimer is considered a prominent functional complex in vivo, we examined p50 and p65 co-expression and transactivation towards the eight κB-REs described in Fig 3A and 3B. Both genes were expressed under the same GAL1 promoter on different single copy centromere plasmids. Experiments were performed at two levels of NF-κB protein expression where co-expression of p50TAD and p65 resulted in transactivation levels that were approximately an average of those seen with the expression of either protein, with the exception of the RelBCons κB-RE. The relative proportion of homodimers and heterodimers was not determined. In fact, p50TAD or p65 alone led to similar levels of RelBCons κB-RE transactivation but the reporter was much more responsive in the co-expression experiment. The strain with the I1 and I2 κB-REs exhibited similar responsiveness to both p50TAD and p65, but co-expression of these two proteins did not have an impact on the level of transactivation of this promoter. At higher levels of galactose (Fig 3B), the differences between expression of p65 alone or co-expression were lower, with the RelBCons strain maintaining high responsiveness when the two TFs were co-expressed. The experiment was performed also at different time points of NF-κB proteins induction with comparable results (Fig A in S1 File).


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 interactions between p50 and p65 towards a panel of κB-REs.A) and B) Eight different κB-REs, each comprising two adjacent copies of the decameric κB sequences were examined for the transactivation potentials of p50TAD, p65 as well as upon co-expression of both proteins. Cells were grown in lower (0.008%, panel A) and high (0.064%, panel B) levels of galactose. C) A non-chimeric p50 construct lacking the TAD domain was also studied at the higher galactose level for all REs, except RelBCons. In all panels, luciferase assays were conducted and results plotted as described in Fig 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130170.g003: Functional interactions between p50 and p65 towards a panel of κB-REs.A) and B) Eight different κB-REs, each comprising two adjacent copies of the decameric κB sequences were examined for the transactivation potentials of p50TAD, p65 as well as upon co-expression of both proteins. Cells were grown in lower (0.008%, panel A) and high (0.064%, panel B) levels of galactose. C) A non-chimeric p50 construct lacking the TAD domain was also studied at the higher galactose level for all REs, except RelBCons. In all panels, luciferase assays were conducted and results plotted as described in Fig 1.
Mentions: Since p50 and p65 are normally present at the same time in mammalian cells and the p50/p65 heterodimer is considered a prominent functional complex in vivo, we examined p50 and p65 co-expression and transactivation towards the eight κB-REs described in Fig 3A and 3B. Both genes were expressed under the same GAL1 promoter on different single copy centromere plasmids. Experiments were performed at two levels of NF-κB protein expression where co-expression of p50TAD and p65 resulted in transactivation levels that were approximately an average of those seen with the expression of either protein, with the exception of the RelBCons κB-RE. The relative proportion of homodimers and heterodimers was not determined. In fact, p50TAD or p65 alone led to similar levels of RelBCons κB-RE transactivation but the reporter was much more responsive in the co-expression experiment. The strain with the I1 and I2 κB-REs exhibited similar responsiveness to both p50TAD and p65, but co-expression of these two proteins did not have an impact on the level of transactivation of this promoter. At higher levels of galactose (Fig 3B), the differences between expression of p65 alone or co-expression were lower, with the RelBCons strain maintaining high responsiveness when the two TFs were co-expressed. The experiment was performed also at different time points of NF-κB proteins induction with comparable results (Fig A in S1 File).

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