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DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor ?B are not correlated.

ABSTRACT: The nuclear factor ?B (NF-?B) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-?B-binding sites (?B sites) arranged in tandem. How NF-?B activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-?B bound to an E-selectin promoter fragment with tandem ?B sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged ?B sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both ?B sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem ?B sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-?B dimers is highly complex and modulated by facilitated association-dissociation processes.

SUBMITTER: Mulero MC

PROVIDER: S-EPMC5704467 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated.

Mulero Maria Carmen MC Huang De-Bin DB Nguyen H Thien HT Wang Vivien Ya-Fan VY Li Yidan Y Biswas Tapan T Ghosh Gourisankar G

The Journal of biological chemistry 20170921 46

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with t ...[more]

PMID: 28935669

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Project description:HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti-inflammatory cytokine TGFbeta (transforming growth factor beta) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) antagonizes TGFbeta for the regulation of APOC3 gene expression in hepatocytes. TNFalpha was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFalpha-induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNFalpha involved NF-kappaB (nuclear factor kappaB). Latent membrane protein 1 of the Epstein-Barr virus, which is an established potent activator of NF-kappaB as well as wild-type forms of various NF-kappaB signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFalpha had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNFalpha and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor-gamma co-activator 1), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNFalpha, or other factors that trigger an NF-kappaB response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.

Dataset Information

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor ?B are not correlated.

Publications

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated.

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