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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

Relative transactivation potential of p50 and p65 homodimers towards a panel of κB Response Elements.A) κB target site preferences of p50TAD and p65 homodimers. Luciferase assays were performed to quantify relative transactivation capacity of p50TAD and p65 homodimers towards 17 different κB-REs. Reporter strains were grown in selective media containing 0.032% galactose for 16 hours reaching near stationary phase. For each isogenic reporter strain, the luciferase activity is calculated as fold-induction with respect to the values obtained with empty vector transformants. The average normalized activity and the standard error of four biological repeats are presented. κB-REs are ranked based on increasing transactivation potential with p50TAD. The same rank is used to plot the results obtained with p65 (lower panel). To the right are presented κB-RE sequences that are selectively responsive to either p50TAD or p65 (see text for sequence match to optimized consensus for p50 or p65). B, C) Web logo representations of the groups of κB-REs that were active or inactive with p50TAD and p65, respectively. D) Western blots presenting the relative expression of p50TAD and p65 proteins at different amounts of galactose. Yeast cells transformed with the GAL1-based expression vectors for NF-κB proteins were cultured for 16 hours at the indicated concentrations of galactose. An antibody directed against the p65 transactivation domain, which is also present in the p50TAD construct, was used for immunodetection. PGK1 endogenous protein provides a loading control.
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pone.0130170.g001: Relative transactivation potential of p50 and p65 homodimers towards a panel of κB Response Elements.A) κB target site preferences of p50TAD and p65 homodimers. Luciferase assays were performed to quantify relative transactivation capacity of p50TAD and p65 homodimers towards 17 different κB-REs. Reporter strains were grown in selective media containing 0.032% galactose for 16 hours reaching near stationary phase. For each isogenic reporter strain, the luciferase activity is calculated as fold-induction with respect to the values obtained with empty vector transformants. The average normalized activity and the standard error of four biological repeats are presented. κB-REs are ranked based on increasing transactivation potential with p50TAD. The same rank is used to plot the results obtained with p65 (lower panel). To the right are presented κB-RE sequences that are selectively responsive to either p50TAD or p65 (see text for sequence match to optimized consensus for p50 or p65). B, C) Web logo representations of the groups of κB-REs that were active or inactive with p50TAD and p65, respectively. D) Western blots presenting the relative expression of p50TAD and p65 proteins at different amounts of galactose. Yeast cells transformed with the GAL1-based expression vectors for NF-κB proteins were cultured for 16 hours at the indicated concentrations of galactose. An antibody directed against the p65 transactivation domain, which is also present in the p50TAD construct, was used for immunodetection. PGK1 endogenous protein provides a loading control.

Mentions: Presented in Fig 1A are the results of transactivation at the seventeen REs following moderate levels of induction of p65 or p50TAD (0.032% gal, as described in Fig 1D). p50TAD activated transcription of eight of the seventeen REs (Fig 1A). This result establishes that p50 can establish specific interactions with target κB-RE sequences when expressed in yeast. p65 was active towards ten κB-REs, but the relative transactivation potentials and pattern of specificities differed remarkably from that of p50TAD (Fig 1A). Five κB-REs were inactive both with p65 and p50TAD. One RE (LIF) was specifically responsive to p50TAD, while three (RANTES, RE2, M1) were selectively responsive to p65. Some of the κB-REs are named from the corresponding human NF-κB target genes. However, in our assay only the decameric κB motif is studied.


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)

Relative transactivation potential of p50 and p65 homodimers towards a panel of κB Response Elements.A) κB target site preferences of p50TAD and p65 homodimers. Luciferase assays were performed to quantify relative transactivation capacity of p50TAD and p65 homodimers towards 17 different κB-REs. Reporter strains were grown in selective media containing 0.032% galactose for 16 hours reaching near stationary phase. For each isogenic reporter strain, the luciferase activity is calculated as fold-induction with respect to the values obtained with empty vector transformants. The average normalized activity and the standard error of four biological repeats are presented. κB-REs are ranked based on increasing transactivation potential with p50TAD. The same rank is used to plot the results obtained with p65 (lower panel). To the right are presented κB-RE sequences that are selectively responsive to either p50TAD or p65 (see text for sequence match to optimized consensus for p50 or p65). B, C) Web logo representations of the groups of κB-REs that were active or inactive with p50TAD and p65, respectively. D) Western blots presenting the relative expression of p50TAD and p65 proteins at different amounts of galactose. Yeast cells transformed with the GAL1-based expression vectors for NF-κB proteins were cultured for 16 hours at the indicated concentrations of galactose. An antibody directed against the p65 transactivation domain, which is also present in the p50TAD construct, was used for immunodetection. PGK1 endogenous protein provides a loading control.
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pone.0130170.g001: Relative transactivation potential of p50 and p65 homodimers towards a panel of κB Response Elements.A) κB target site preferences of p50TAD and p65 homodimers. Luciferase assays were performed to quantify relative transactivation capacity of p50TAD and p65 homodimers towards 17 different κB-REs. Reporter strains were grown in selective media containing 0.032% galactose for 16 hours reaching near stationary phase. For each isogenic reporter strain, the luciferase activity is calculated as fold-induction with respect to the values obtained with empty vector transformants. The average normalized activity and the standard error of four biological repeats are presented. κB-REs are ranked based on increasing transactivation potential with p50TAD. The same rank is used to plot the results obtained with p65 (lower panel). To the right are presented κB-RE sequences that are selectively responsive to either p50TAD or p65 (see text for sequence match to optimized consensus for p50 or p65). B, C) Web logo representations of the groups of κB-REs that were active or inactive with p50TAD and p65, respectively. D) Western blots presenting the relative expression of p50TAD and p65 proteins at different amounts of galactose. Yeast cells transformed with the GAL1-based expression vectors for NF-κB proteins were cultured for 16 hours at the indicated concentrations of galactose. An antibody directed against the p65 transactivation domain, which is also present in the p50TAD construct, was used for immunodetection. PGK1 endogenous protein provides a loading control.
Mentions: Presented in Fig 1A are the results of transactivation at the seventeen REs following moderate levels of induction of p65 or p50TAD (0.032% gal, as described in Fig 1D). p50TAD activated transcription of eight of the seventeen REs (Fig 1A). This result establishes that p50 can establish specific interactions with target κB-RE sequences when expressed in yeast. p65 was active towards ten κB-REs, but the relative transactivation potentials and pattern of specificities differed remarkably from that of p50TAD (Fig 1A). Five κB-REs were inactive both with p65 and p50TAD. One RE (LIF) was specifically responsive to p50TAD, while three (RANTES, RE2, M1) were selectively responsive to p65. Some of the κB-REs are named from the corresponding human NF-κB target genes. However, in our assay only the decameric κB motif is studied.

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