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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

Comparison between predicted DNA binding affinity and yeast-based transactivation.A), B) The relative binding affinities of p50 or p65 towards 17 κB-REs were obtained from (http://thebrain.bwh.harvard.edu/nfkb) and compared with the relative transactivation potential measured in yeast at moderate levels of galactose induction. REs are ordered from left to right based on increasing Z-score for DNA binding affinity. The highest affinity RE (RE5 for p50 and I1 for p65) is set to 100. REs with Z-score lower than 4, considered equivalent to background, are labeled by *. C) Similarly, Z-scores of the p50-p65 heterodimers and transactivation potentials are compared for the 6 κB-REs that were tested in the co-expression experiments in yeast.
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pone.0130170.g007: Comparison between predicted DNA binding affinity and yeast-based transactivation.A), B) The relative binding affinities of p50 or p65 towards 17 κB-REs were obtained from (http://thebrain.bwh.harvard.edu/nfkb) and compared with the relative transactivation potential measured in yeast at moderate levels of galactose induction. REs are ordered from left to right based on increasing Z-score for DNA binding affinity. The highest affinity RE (RE5 for p50 and I1 for p65) is set to 100. REs with Z-score lower than 4, considered equivalent to background, are labeled by *. C) Similarly, Z-scores of the p50-p65 heterodimers and transactivation potentials are compared for the 6 κB-REs that were tested in the co-expression experiments in yeast.

Mentions: We compared transactivation potentials with DNA binding affinities measured by in vitro gel shift assays [21] or by custom NF-κB protein binding microarray experiments and surface plasmon resonance analysis [48], as described in Fig 7. We also wanted to address various functional aspects of NF-κB target sequences, such as interactions between adjacent κB-REs and the impact of short spacers. Seventeen κB-REs were chosen for this analysis and the relative transactivation capacities were measured with p65 and p50TAD. These REs were chosen to represent a wide range of DNA binding affinities (~100-fold for p50), based on EMSA assays [21]. There were striking exceptions to an overall correlation trend between relative DNA binding affinity and relative transactivation potential both for p50 and p65+p50. For example, JunB and RE6 were extremely weak in transactivation assays but were reported to be bound with high affinity in vitro (Fig D in S1 File). The overall correlation was higher with DNA binding predictions based on protein-binding microarray experiments [48] that were obtained from an online tool (http://thebrain.bwh.harvard.edu/nfkb), particularly for p50/p65 heterodimers. There were however differences between the two parameters (e.g. LIF and RANTES for p50, RE2 and RE1 for p65) (Fig 7). These findings further validate our yeast-based approach. A recent study based on a largely unbiased ChIP-sequencing approach in mammalian cells confirmed that the central sequence motif of a κB-RE plays a critical role in deciding the transcriptional specificity by NF-κB proteins [22], emphasizing the significance of RE sequence even in an environment where additional cis-elements and trans-factors interplay to regulate transcription. Our data confirmed the impact that single nucleotide changes can have on transactivation potential (e.g. RE1 vs RE2, M1 vs M2) (Table A and Fig A in S1 File), consistent with previous reports in mammalian cells [20,21]. For example, a lentiviral-based approach demonstrated that differences in the nucleotide sequence of κB-REs embedded in about 5 kb of the MCP-1 promoter sequence could dictate the level of responsiveness to NF-κB activation by TNFα or LPS and the relative activity of different NF-κB family members [20]. In agreement with our observations in yeast, this study also established p65 as a major factor in MCP-1 promoter transactivation with a limited contribution from p50 and p52. Also, consistent with our results, the replacement of the κB-REs within MCP-1 with those present in the IP-10 promoter (I1 and I2) led to preferential responsiveness to p50 homo or hetero-dimers. However, the I1 κB-RE when tested separately exhibited a lower relative responsiveness to p50TAD in yeast.


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)

Comparison between predicted DNA binding affinity and yeast-based transactivation.A), B) The relative binding affinities of p50 or p65 towards 17 κB-REs were obtained from (http://thebrain.bwh.harvard.edu/nfkb) and compared with the relative transactivation potential measured in yeast at moderate levels of galactose induction. REs are ordered from left to right based on increasing Z-score for DNA binding affinity. The highest affinity RE (RE5 for p50 and I1 for p65) is set to 100. REs with Z-score lower than 4, considered equivalent to background, are labeled by *. C) Similarly, Z-scores of the p50-p65 heterodimers and transactivation potentials are compared for the 6 κB-REs that were tested in the co-expression experiments in yeast.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493129&req=5

pone.0130170.g007: Comparison between predicted DNA binding affinity and yeast-based transactivation.A), B) The relative binding affinities of p50 or p65 towards 17 κB-REs were obtained from (http://thebrain.bwh.harvard.edu/nfkb) and compared with the relative transactivation potential measured in yeast at moderate levels of galactose induction. REs are ordered from left to right based on increasing Z-score for DNA binding affinity. The highest affinity RE (RE5 for p50 and I1 for p65) is set to 100. REs with Z-score lower than 4, considered equivalent to background, are labeled by *. C) Similarly, Z-scores of the p50-p65 heterodimers and transactivation potentials are compared for the 6 κB-REs that were tested in the co-expression experiments in yeast.
Mentions: We compared transactivation potentials with DNA binding affinities measured by in vitro gel shift assays [21] or by custom NF-κB protein binding microarray experiments and surface plasmon resonance analysis [48], as described in Fig 7. We also wanted to address various functional aspects of NF-κB target sequences, such as interactions between adjacent κB-REs and the impact of short spacers. Seventeen κB-REs were chosen for this analysis and the relative transactivation capacities were measured with p65 and p50TAD. These REs were chosen to represent a wide range of DNA binding affinities (~100-fold for p50), based on EMSA assays [21]. There were striking exceptions to an overall correlation trend between relative DNA binding affinity and relative transactivation potential both for p50 and p65+p50. For example, JunB and RE6 were extremely weak in transactivation assays but were reported to be bound with high affinity in vitro (Fig D in S1 File). The overall correlation was higher with DNA binding predictions based on protein-binding microarray experiments [48] that were obtained from an online tool (http://thebrain.bwh.harvard.edu/nfkb), particularly for p50/p65 heterodimers. There were however differences between the two parameters (e.g. LIF and RANTES for p50, RE2 and RE1 for p65) (Fig 7). These findings further validate our yeast-based approach. A recent study based on a largely unbiased ChIP-sequencing approach in mammalian cells confirmed that the central sequence motif of a κB-RE plays a critical role in deciding the transcriptional specificity by NF-κB proteins [22], emphasizing the significance of RE sequence even in an environment where additional cis-elements and trans-factors interplay to regulate transcription. Our data confirmed the impact that single nucleotide changes can have on transactivation potential (e.g. RE1 vs RE2, M1 vs M2) (Table A and Fig A in S1 File), consistent with previous reports in mammalian cells [20,21]. For example, a lentiviral-based approach demonstrated that differences in the nucleotide sequence of κB-REs embedded in about 5 kb of the MCP-1 promoter sequence could dictate the level of responsiveness to NF-κB activation by TNFα or LPS and the relative activity of different NF-κB family members [20]. In agreement with our observations in yeast, this study also established p65 as a major factor in MCP-1 promoter transactivation with a limited contribution from p50 and p52. Also, consistent with our results, the replacement of the κB-REs within MCP-1 with those present in the IP-10 promoter (I1 and I2) led to preferential responsiveness to p50 homo or hetero-dimers. However, the I1 κB-RE when tested separately exhibited a lower relative responsiveness to p50TAD in yeast.

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