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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

Effect of the small molecules BAY11-7082 and ethyl pyruvate on NF-κB activity in yeast.The strains containing the REs M2, RE4 and RelBCons were grown overnight (16 hours) in selective media containing low levels of galactose (0.008%) with or without the addition of different concentrations of BAY11-7082 (10μM, 20μM) and EP (1.5mM, 2.5mM, 5mM, 10mM). A-C) Average luciferase assays and standard errors of four biological repeats are presented. Results obtained with cells transformed with an empty expression vector are included to take into account the impact of the small molecules on the NF-κB-independent, basal expression of the reporter. D, E) Western blot images showing p50TAD, p50TAD + p65 and p65 protein levels in BAY and EP treated and untreated cells. PGK1 was used as a loading control.
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pone.0130170.g006: Effect of the small molecules BAY11-7082 and ethyl pyruvate on NF-κB activity in yeast.The strains containing the REs M2, RE4 and RelBCons were grown overnight (16 hours) in selective media containing low levels of galactose (0.008%) with or without the addition of different concentrations of BAY11-7082 (10μM, 20μM) and EP (1.5mM, 2.5mM, 5mM, 10mM). A-C) Average luciferase assays and standard errors of four biological repeats are presented. Results obtained with cells transformed with an empty expression vector are included to take into account the impact of the small molecules on the NF-κB-independent, basal expression of the reporter. D, E) Western blot images showing p50TAD, p50TAD + p65 and p65 protein levels in BAY and EP treated and untreated cells. PGK1 was used as a loading control.

Mentions: We also explored the use of the yeast-based assay for assessing the activity of known mammalian NF-κB inhibitors BAY [9,11,45], ethyl-pyruvate (EP) [8] and parthenolide [46]. Luciferase assays were conducted in M2, RE4 and RelBCons strains as they exhibited different relative responsiveness to either p50TAD or p65 alone or to their co-expression at a low level of galactose (0.008%) (Fig 5). BAY and EP treatments led to a dose-dependent inhibition of p65-mediated transactivation. The effect was less evident with p50TAD (Fig 6). Parthenolide had no impact on p65- or p50TAD-mediated transactivation (Fig B in S1 File). As controls, the effect of the molecules on the NF-κB-independent, basal expression of the reporter, or on the steady-state levels of p65 or p50TAD proteins was examined (Fig 6D and 6E). To test the generality of the EP effect, we examined its impact on p53-mediated transcription, considering that p53 is expressed in yeast from the same GAL1 promoter system used for NF-κB [15,30]. Unlike what was observed with p65 and p50TAD, p53 transactivation was unaffected by EP up to the 5mM dose and only slightly reduced at 10mM, while at the highest dose (20mM) it was completely inhibited. Further, no impact on p53 protein levels was seen after 2.5 or 5mM EP treatment (Fig C in S1 File). Overall, while at high doses, indirect effects could potentially bias the results, the yeast transactivation system provides a tool to study specific small molecule inhibitors of NF-κB transactivation.


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)

Effect of the small molecules BAY11-7082 and ethyl pyruvate on NF-κB activity in yeast.The strains containing the REs M2, RE4 and RelBCons were grown overnight (16 hours) in selective media containing low levels of galactose (0.008%) with or without the addition of different concentrations of BAY11-7082 (10μM, 20μM) and EP (1.5mM, 2.5mM, 5mM, 10mM). A-C) Average luciferase assays and standard errors of four biological repeats are presented. Results obtained with cells transformed with an empty expression vector are included to take into account the impact of the small molecules on the NF-κB-independent, basal expression of the reporter. D, E) Western blot images showing p50TAD, p50TAD + p65 and p65 protein levels in BAY and EP treated and untreated cells. PGK1 was used as a loading control.
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Related In: Results  -  Collection

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pone.0130170.g006: Effect of the small molecules BAY11-7082 and ethyl pyruvate on NF-κB activity in yeast.The strains containing the REs M2, RE4 and RelBCons were grown overnight (16 hours) in selective media containing low levels of galactose (0.008%) with or without the addition of different concentrations of BAY11-7082 (10μM, 20μM) and EP (1.5mM, 2.5mM, 5mM, 10mM). A-C) Average luciferase assays and standard errors of four biological repeats are presented. Results obtained with cells transformed with an empty expression vector are included to take into account the impact of the small molecules on the NF-κB-independent, basal expression of the reporter. D, E) Western blot images showing p50TAD, p50TAD + p65 and p65 protein levels in BAY and EP treated and untreated cells. PGK1 was used as a loading control.
Mentions: We also explored the use of the yeast-based assay for assessing the activity of known mammalian NF-κB inhibitors BAY [9,11,45], ethyl-pyruvate (EP) [8] and parthenolide [46]. Luciferase assays were conducted in M2, RE4 and RelBCons strains as they exhibited different relative responsiveness to either p50TAD or p65 alone or to their co-expression at a low level of galactose (0.008%) (Fig 5). BAY and EP treatments led to a dose-dependent inhibition of p65-mediated transactivation. The effect was less evident with p50TAD (Fig 6). Parthenolide had no impact on p65- or p50TAD-mediated transactivation (Fig B in S1 File). As controls, the effect of the molecules on the NF-κB-independent, basal expression of the reporter, or on the steady-state levels of p65 or p50TAD proteins was examined (Fig 6D and 6E). To test the generality of the EP effect, we examined its impact on p53-mediated transcription, considering that p53 is expressed in yeast from the same GAL1 promoter system used for NF-κB [15,30]. Unlike what was observed with p65 and p50TAD, p53 transactivation was unaffected by EP up to the 5mM dose and only slightly reduced at 10mM, while at the highest dose (20mM) it was completely inhibited. Further, no impact on p53 protein levels was seen after 2.5 or 5mM EP treatment (Fig C in S1 File). Overall, while at high doses, indirect effects could potentially bias the results, the yeast transactivation system provides a tool to study specific small molecule inhibitors of NF-κB transactivation.

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