Limits...
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

IκBα inhibits p65-dependent transactivation in yeast.The highly responsive M2 strain was used to test the impact of co-expressing IκBα with the NF-κB proteins p50TAD or p65. A) Luciferase assays results were obtained and plotted as described in Fig 1. Control transformants lacking the IκBα expression construct were obtained using the pRS315 empty vector. Cells were cultured in 0.032% galactose for 16 hours to achieve moderate expression of p50TAD or p65. IκBα is expressed under the constitutive ADH1 promoter. For all conditions the light units were normalize for the optical density of the cultures. The relative luciferase activity, obtained with cells transformed with empty vectors was set to 1 and used to obtained the fold of reporter induction due to the expression of NF-κB proteins. Bars plot the average and standard errors of four biological replicates. B) A western blot image revealing the impact of IκBα on p50TAD or p65 protein levels. Transformants with two p50TAD expression vectors that differ for the selection marker gene (LEU2 for p50TAD #1 and TRP1 for p50TAD #2) were included. PGK1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493129&req=5

pone.0130170.g005: IκBα inhibits p65-dependent transactivation in yeast.The highly responsive M2 strain was used to test the impact of co-expressing IκBα with the NF-κB proteins p50TAD or p65. A) Luciferase assays results were obtained and plotted as described in Fig 1. Control transformants lacking the IκBα expression construct were obtained using the pRS315 empty vector. Cells were cultured in 0.032% galactose for 16 hours to achieve moderate expression of p50TAD or p65. IκBα is expressed under the constitutive ADH1 promoter. For all conditions the light units were normalize for the optical density of the cultures. The relative luciferase activity, obtained with cells transformed with empty vectors was set to 1 and used to obtained the fold of reporter induction due to the expression of NF-κB proteins. Bars plot the average and standard errors of four biological replicates. B) A western blot image revealing the impact of IκBα on p50TAD or p65 protein levels. Transformants with two p50TAD expression vectors that differ for the selection marker gene (LEU2 for p50TAD #1 and TRP1 for p50TAD #2) were included. PGK1 was used as loading control.

Mentions: We cloned the human IκBα cDNA into a constitutive expression vector exploiting the ADH1 constitutive moderate promoter and the LEU2 selection marker. This enabled us to select double transformants. The M2 κB-RE, which was the most responsive to p65 and moderately responsive to p50TAD, was chosen for these experiments. There was a significant reduction in luciferase activity with p65-IκBα double transformant cells, compared to transformants with p65 alone (Fig 5A). Interestingly, there was no effect of IκBα on the basal, constitutive level of reporter expression or on the activity of the reporter dependent on p50TAD. Immunoblots indicated that in the presence of the IκBα expression plasmid, protein levels of both p50TAD and p65 from whole cell extracts were comparable or even higher (Fig 5B), suggesting that the impact of IκBα might actually be underestimated, and that IκBα might stabilize p50TAD and especially p65 proteins by forming stable complexes as reported for mammalian cells [43,44].


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)

IκBα inhibits p65-dependent transactivation in yeast.The highly responsive M2 strain was used to test the impact of co-expressing IκBα with the NF-κB proteins p50TAD or p65. A) Luciferase assays results were obtained and plotted as described in Fig 1. Control transformants lacking the IκBα expression construct were obtained using the pRS315 empty vector. Cells were cultured in 0.032% galactose for 16 hours to achieve moderate expression of p50TAD or p65. IκBα is expressed under the constitutive ADH1 promoter. For all conditions the light units were normalize for the optical density of the cultures. The relative luciferase activity, obtained with cells transformed with empty vectors was set to 1 and used to obtained the fold of reporter induction due to the expression of NF-κB proteins. Bars plot the average and standard errors of four biological replicates. B) A western blot image revealing the impact of IκBα on p50TAD or p65 protein levels. Transformants with two p50TAD expression vectors that differ for the selection marker gene (LEU2 for p50TAD #1 and TRP1 for p50TAD #2) were included. PGK1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130170.g005: IκBα inhibits p65-dependent transactivation in yeast.The highly responsive M2 strain was used to test the impact of co-expressing IκBα with the NF-κB proteins p50TAD or p65. A) Luciferase assays results were obtained and plotted as described in Fig 1. Control transformants lacking the IκBα expression construct were obtained using the pRS315 empty vector. Cells were cultured in 0.032% galactose for 16 hours to achieve moderate expression of p50TAD or p65. IκBα is expressed under the constitutive ADH1 promoter. For all conditions the light units were normalize for the optical density of the cultures. The relative luciferase activity, obtained with cells transformed with empty vectors was set to 1 and used to obtained the fold of reporter induction due to the expression of NF-κB proteins. Bars plot the average and standard errors of four biological replicates. B) A western blot image revealing the impact of IκBα on p50TAD or p65 protein levels. Transformants with two p50TAD expression vectors that differ for the selection marker gene (LEU2 for p50TAD #1 and TRP1 for p50TAD #2) were included. PGK1 was used as loading control.
Mentions: We cloned the human IκBα cDNA into a constitutive expression vector exploiting the ADH1 constitutive moderate promoter and the LEU2 selection marker. This enabled us to select double transformants. The M2 κB-RE, which was the most responsive to p65 and moderately responsive to p50TAD, was chosen for these experiments. There was a significant reduction in luciferase activity with p65-IκBα double transformant cells, compared to transformants with p65 alone (Fig 5A). Interestingly, there was no effect of IκBα on the basal, constitutive level of reporter expression or on the activity of the reporter dependent on p50TAD. Immunoblots indicated that in the presence of the IκBα expression plasmid, protein levels of both p50TAD and p65 from whole cell extracts were comparable or even higher (Fig 5B), suggesting that the impact of IκBα might actually be underestimated, and that IκBα might stabilize p50TAD and especially p65 proteins by forming stable complexes as reported for mammalian cells [43,44].

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