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首页> 外文期刊>BMC Genomics >A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays
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A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

机译：使用HaloCHIP芯片和高通量报告基因检测功能分析CREB信号通路

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Background Regulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells. Results In analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1. Conclusion These data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.
机译：背景技术基因表达的调节对于正常发育和细胞生长至关重要。转录事件在空间和时间上都受到特定DNA-蛋白质相互作用的严格控制。在这项研究中，我们在所有已知的和预测的人类启动子上精确定位了CREB蛋白的全基因组靶标，并使用高通量报告基因分析法表征了这些结合事件的子集的功能后果。为了测量CREB的结合，我们使用了HaloCHIP（Chlo方法的一种无抗体替代方法，该方法利用HaloTag融合蛋白），还使用了高通量启动子-荧光素酶报告基因检测法，该方法可快速，定量地筛选启动子以进行转录激活或抑制。活细胞。结果在使用人类启动子的全面DNA微阵列分析CREB基因组范围内的结合事件时，我们首次观察到CREB对双向启动子的结合具有强烈的偏好，而与单向启动子不同，这些结合事件通常发生在转录开始的下游网站。 HaloCHIP芯片和ChIP芯片数据之间的比较表明，这两种方法都是正确的，这表明这并不是所选技术的偏向。从启动子荧光素酶报道分子阵列获得的转录数据还显示，在存在共激活蛋白TORC1的情况下，CREB结合的启动子的激活水平空前高。结论这些数据首次表明，当CREB结合在双向启动子上时，TORC1提供了方向信息，并且在初始转录激活后CREB蛋白可能暂停。而且，这种组合方法证明了更广泛地表征CREB蛋白-DNA相互作用的能力，其中不仅发现了DNA结合位点，而且还评估了启动子序列对CREB应答的潜力。

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