Prediction and experimental validation of a putative non-consensus binding site for transcription factor STAT3 in serum amyloid A gene promoter.
Prediction and experimental validation of a putative non-consensus binding site for transcription factor STAT3 in serum amyloid A gene promoter.
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DOI：
10.1016/j.bbagen.2013.01.024
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发表时间：
2013-06
期刊：
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通讯作者：
Prabha Tiwari;Lokesh P. Tripathi;Teppei Nishikawa-Matsumura;Shandar Ahmad;Soken-Nakazawa J. Song;Tomoyasu Isobe;K. Mizuguchi;K. Yoshizaki
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作者：
Prabha Tiwari;Lokesh P. Tripathi;Teppei Nishikawa-Matsumura;Shandar Ahmad;Soken-Nakazawa J. Song;Tomoyasu Isobe;K. Mizuguchi;K. Yoshizaki
我们之前证明，尽管人类SAA1基因在其启动子区域没有典型的STAT3应答元件（STAT3-RE），但STAT3和核因子（NF-κB） p65首先在白细胞介素IL-1和IL-6 （IL-1+6）刺激下形成复合物，之后STAT3与SAA1启动子中NF-κB RE下游的区域相互作用。在这项研究中，我们采用了一种基于蛋白质- dna接触的间接读出的计算方法来确定一组非共识STAT3转录因子结合位点（TFBSs）的候选位点。通过双荧光素酶测定和DNA亲和层析，实验证实了STAT3与预测的非一致性TFBSs之一的结合。本研究在SAA1启动子区NF-κB p65和STAT3激活人HepG2肝细胞SAA1转录所需的NF-κB p65和STAT3下游nt - 75/−66处定义了一个新的STAT3非共识TFBS。我们的分析建立在目前对STAT3功能的理解之上，提出了STAT3在炎症反应中功能的更广泛的机制，并为研究具有潜在治疗意义的新的tf靶点关联提供了一个有用的框架。
We previously demonstrated that though the human SAA1 gene shows no typical STAT3 response element (STAT3-RE) in its promoter region, STAT3 and the nuclear factor (NF-κB) p65 first form a complex following interleukin IL-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with a region downstream of the NF-κB RE in the SAA1 promoter. In this study, we employed a computational approach based on indirect read outs of protein–DNA contacts to identify a set of candidates for non-consensus STAT3 transcription factor binding sites (TFBSs). The binding of STAT3 to one of the predicted non-consensus TFBSs was experimentally confirmed through a dual luciferase assay and DNA affinity chromatography. The present study defines a novel STAT3 non-consensus TFBS at nt −75/−66 downstream of the NF-κB RE in the SAA1 promoter region that is required for NF-κB p65 and STAT3 to activate SAA1 transcription in human HepG2 liver cells. Our analysis builds upon the current understanding of STAT3 function, suggesting a wider array of mechanisms of STAT3 function in inflammatory response, and provides a useful framework for investigating novel TF-target associations with potential therapeutic implications.