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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

Additive or weak cooperative transactivation between adjacent κB REs.A) Yeast-based luciferase assays were performed at moderate to high expression levels of p50TAD or p65. Results were normalized and plotted as in Fig 1. The impact of tandem duplication (2d) of the decameric (1d) κB-RE or the indicated combination of two different κB-REs was evaluated. The REs were chosen based on the results from Fig 1 to include sequences exhibiting different levels of transactivation potentials. B) RE1 and RE6 were inactive as decameric κB-REs and there was no transactivation for two decamers in tandem or high expression of NF-κB proteins (up to 1% galactose). C) Functional interactions between two different κB-REs derived from the MCP-1 promoter. The M1 and M2 decameric κB-REs exhibited different responsiveness to p50TAD and p65, when examined separately, but are both derived from the MCP-1 promoter and they are located in close distance (19 nucleotide spacer) in the natural context. The combined responsiveness of the M1 and M2 κB-REs was examined, taking into account the impact of the distance between the two decamers and orientation relative to the transcriptional start site. Tandem repeats of M1 or M2 were included as controls. Yeast reporter strains, transformed with the indicated expression vector were cultured for 16 hrs with the indicated low amount of galactose. Relative activity refers to the average light units normalized for cell number (measured by optical density at 600nm). Average and standard error of four biological repeats are presented. The NF-κB-independent reporter activity (empty vector) is also presented as reference. Interestingly, the M2+M1 strains exhibited higher basal level of reporter expression. The M1+M2 sp strain contains the M1 and M2 κB decamers separated by 18 nt as in the human gene (see Table A in S1 File).
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pone.0130170.g002: Additive or weak cooperative transactivation between adjacent κB REs.A) Yeast-based luciferase assays were performed at moderate to high expression levels of p50TAD or p65. Results were normalized and plotted as in Fig 1. The impact of tandem duplication (2d) of the decameric (1d) κB-RE or the indicated combination of two different κB-REs was evaluated. The REs were chosen based on the results from Fig 1 to include sequences exhibiting different levels of transactivation potentials. B) RE1 and RE6 were inactive as decameric κB-REs and there was no transactivation for two decamers in tandem or high expression of NF-κB proteins (up to 1% galactose). C) Functional interactions between two different κB-REs derived from the MCP-1 promoter. The M1 and M2 decameric κB-REs exhibited different responsiveness to p50TAD and p65, when examined separately, but are both derived from the MCP-1 promoter and they are located in close distance (19 nucleotide spacer) in the natural context. The combined responsiveness of the M1 and M2 κB-REs was examined, taking into account the impact of the distance between the two decamers and orientation relative to the transcriptional start site. Tandem repeats of M1 or M2 were included as controls. Yeast reporter strains, transformed with the indicated expression vector were cultured for 16 hrs with the indicated low amount of galactose. Relative activity refers to the average light units normalized for cell number (measured by optical density at 600nm). Average and standard error of four biological repeats are presented. The NF-κB-independent reporter activity (empty vector) is also presented as reference. Interestingly, the M2+M1 strains exhibited higher basal level of reporter expression. The M1+M2 sp strain contains the M1 and M2 κB decamers separated by 18 nt as in the human gene (see Table A in S1 File).

Mentions: Having established that both p65 and the chimeric p50TAD could act as sequence specific TFs in yeast, we explored whether two adjacent κB-REs could lead to synergistic induction of transactivation. Some natural NF-κB target sites contain pairs of κB-REs, whose sequence features can impact on gene transcription [20]. Functional interactions between nearby κB-RE sites have been reported [22] and the potential for cooperative interactions between NF-κB proteins at clustered binding sites has been inferred [41], but not directly addressed under a defined experimental system. The experiments were performed using two different amounts of galactose to titrate the transactivation response. The M2 and I1 REs, which were highly responsive to p65 and moderately responsive to p50TAD, were chosen. Reporter strains containing two adjacent RE copies had an additive effect when analyzed with p50TAD for both κB-REs. A higher amount of galactose (0.1%) did not lead to higher transactivation levels with p50TAD, suggesting that the maximal level of responsiveness for these strains was reached at 0.032% galactose. Instead, p65 showed a galactose-dependent transactivation ability and mainly additive effects for one vs two REs number, with the exception of the M2 RE at the lower galactose concentration (Fig 2A). We also tested RE6 and the combination of RE6 and RE1, which were inactive both with p65 and p50TAD when studied separately. Two copies of the non-responsive RE6 did not lead to any transactivation either with p50TAD or p65 even using very high expression levels (1% galactose) (Fig 2B). The same was true for the combination of RE6 and RE1 except for weak responsiveness to p65.


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)

Additive or weak cooperative transactivation between adjacent κB REs.A) Yeast-based luciferase assays were performed at moderate to high expression levels of p50TAD or p65. Results were normalized and plotted as in Fig 1. The impact of tandem duplication (2d) of the decameric (1d) κB-RE or the indicated combination of two different κB-REs was evaluated. The REs were chosen based on the results from Fig 1 to include sequences exhibiting different levels of transactivation potentials. B) RE1 and RE6 were inactive as decameric κB-REs and there was no transactivation for two decamers in tandem or high expression of NF-κB proteins (up to 1% galactose). C) Functional interactions between two different κB-REs derived from the MCP-1 promoter. The M1 and M2 decameric κB-REs exhibited different responsiveness to p50TAD and p65, when examined separately, but are both derived from the MCP-1 promoter and they are located in close distance (19 nucleotide spacer) in the natural context. The combined responsiveness of the M1 and M2 κB-REs was examined, taking into account the impact of the distance between the two decamers and orientation relative to the transcriptional start site. Tandem repeats of M1 or M2 were included as controls. Yeast reporter strains, transformed with the indicated expression vector were cultured for 16 hrs with the indicated low amount of galactose. Relative activity refers to the average light units normalized for cell number (measured by optical density at 600nm). Average and standard error of four biological repeats are presented. The NF-κB-independent reporter activity (empty vector) is also presented as reference. Interestingly, the M2+M1 strains exhibited higher basal level of reporter expression. The M1+M2 sp strain contains the M1 and M2 κB decamers separated by 18 nt as in the human gene (see Table A in S1 File).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493129&req=5

pone.0130170.g002: Additive or weak cooperative transactivation between adjacent κB REs.A) Yeast-based luciferase assays were performed at moderate to high expression levels of p50TAD or p65. Results were normalized and plotted as in Fig 1. The impact of tandem duplication (2d) of the decameric (1d) κB-RE or the indicated combination of two different κB-REs was evaluated. The REs were chosen based on the results from Fig 1 to include sequences exhibiting different levels of transactivation potentials. B) RE1 and RE6 were inactive as decameric κB-REs and there was no transactivation for two decamers in tandem or high expression of NF-κB proteins (up to 1% galactose). C) Functional interactions between two different κB-REs derived from the MCP-1 promoter. The M1 and M2 decameric κB-REs exhibited different responsiveness to p50TAD and p65, when examined separately, but are both derived from the MCP-1 promoter and they are located in close distance (19 nucleotide spacer) in the natural context. The combined responsiveness of the M1 and M2 κB-REs was examined, taking into account the impact of the distance between the two decamers and orientation relative to the transcriptional start site. Tandem repeats of M1 or M2 were included as controls. Yeast reporter strains, transformed with the indicated expression vector were cultured for 16 hrs with the indicated low amount of galactose. Relative activity refers to the average light units normalized for cell number (measured by optical density at 600nm). Average and standard error of four biological repeats are presented. The NF-κB-independent reporter activity (empty vector) is also presented as reference. Interestingly, the M2+M1 strains exhibited higher basal level of reporter expression. The M1+M2 sp strain contains the M1 and M2 κB decamers separated by 18 nt as in the human gene (see Table A in S1 File).
Mentions: Having established that both p65 and the chimeric p50TAD could act as sequence specific TFs in yeast, we explored whether two adjacent κB-REs could lead to synergistic induction of transactivation. Some natural NF-κB target sites contain pairs of κB-REs, whose sequence features can impact on gene transcription [20]. Functional interactions between nearby κB-RE sites have been reported [22] and the potential for cooperative interactions between NF-κB proteins at clustered binding sites has been inferred [41], but not directly addressed under a defined experimental system. The experiments were performed using two different amounts of galactose to titrate the transactivation response. The M2 and I1 REs, which were highly responsive to p65 and moderately responsive to p50TAD, were chosen. Reporter strains containing two adjacent RE copies had an additive effect when analyzed with p50TAD for both κB-REs. A higher amount of galactose (0.1%) did not lead to higher transactivation levels with p50TAD, suggesting that the maximal level of responsiveness for these strains was reached at 0.032% galactose. Instead, p65 showed a galactose-dependent transactivation ability and mainly additive effects for one vs two REs number, with the exception of the M2 RE at the lower galactose concentration (Fig 2A). We also tested RE6 and the combination of RE6 and RE1, which were inactive both with p65 and p50TAD when studied separately. Two copies of the non-responsive RE6 did not lead to any transactivation either with p50TAD or p65 even using very high expression levels (1% galactose) (Fig 2B). The same was true for the combination of RE6 and RE1 except for weak responsiveness to p65.

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