DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor B are not correlated
DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor B are not correlated
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DOI：
10.1074/jbc.m117.813980
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发表时间：
2017-11-17
影响因子：
4.8
通讯作者：
Ghosh, Gourisankar
中科院分区：
文献类型：
--
作者：
Mulero, Maria Carmen;Huang, De-Bin;Ghosh, Gourisankar
核因子B （NF-B）转录因子家族调节参与细胞增殖和炎症的基因。这些基因的启动子通常含有串联排列的nf -B结合位点（B位点）。NF-B如何通过这些多个位点激活转录尚不完全清楚。我们在这里报道了一个同型二聚体的x射线晶体结构，它包括NF-B中含有Rel同源区域（RHR）的RelA dna结合域，该区域与具有串联B位点的e -选择素启动子片段结合。这种结构揭示了两个二聚体不对称地结合到对称排列的B位点上，在B位点上，一个二聚体与相应DNA之间的多个同源接触被破坏。由于RelA-RHR二聚体与溶液中串联位点的同时结合是反合作的，我们推断，接触较少的不对称RelA-RHR结合可能表明一种解离结合模式。我们发现这两个B位点对于全长RelA同型二聚体的报告基因激活至关重要，这表明二聚体促进DNA相互结合，即使它们的稳定共占没有得到促进。串联B位点的间距和方向发生改变的启动子变异显示出意想不到的报告活性，这无法用RelA-RHR的溶液结合模式来解释。值得注意的是，全长RelA结合所有dna的亲和力和特异性较弱。此外，反活化结构域在DNA结合中起负作用。这些观察结果表明，其他核因子通过中和反活化结构域的负面效应来影响全长RelA与DNA的结合。我们认为NF-B二聚体的DNA结合是高度复杂的，并通过促进的结合-解离过程进行调节。
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.