A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays.
A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays.
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使用Halochip-Chip和高吞吐量报告基因测定法对CREB信号通路进行功能分析。
DOI：
10.1186/1471-2164-10-497
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发表时间：
2009-10-27
期刊：
影响因子：
4.4
通讯作者：
Urh M
中科院分区：
文献类型：
--
作者：
Hartzell DD;Trinklein ND;Mendez J;Murphy N;Aldred SF;Wood K;Urh M
基因表达的调节对于正常发育和细胞生长是必不可少的。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，我们精细地绘制了CREB蛋白在所有已知和预测的人类启动子中的全基因组靶点，并使用高通量报告分析表征了这些结合事件的子集的功能后果。为了测量CREB结合，我们使用了HaloCHIP，一种利用HaloTag融合蛋白的ChIP方法的无抗体替代方法，以及高通量启动子-荧光素酶报告基因测定，其提供了活细胞中转录激活或抑制的启动子的快速和定量筛选。在分析CREB全基因组的结合事件，使用一个全面的DNA微阵列的人类启动子，我们第一次观察到，CREB有一个强烈的偏好结合在双向启动子和单向启动子不同，这些结合事件往往发生在转录起始位点的下游。HaloCHIP-芯片和ChIP-芯片数据之间的比较表明，这对于两种方法都是正确的，表明这不是所选技术的偏差。从启动子-荧光素酶报告基因阵列获得的转录数据也显示了在共激活蛋白TORC 1存在下CREB结合启动子的前所未有的高水平激活。这些数据首次表明，当CREB结合在双向启动子上时，TORC 1提供了方向信息，并可能在初始转录激活后暂停CREB蛋白。此外，这种组合的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响应CREB的潜力。
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. 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. 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.
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DOI：
10.1006/meth.1999.0879
发表时间：
1999-11-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Kuo, MH;Allis, CD
通讯作者：
Allis, CD
影响因子：
64.5
作者：
Kim, Jonghwan;Chu, Jianlin;Orkin, Stuart H.
通讯作者：
Orkin, Stuart H.
影响因子：
7
作者：
Johnson, David S.;Li, Wei;Liu, X. Shirley
通讯作者：
Liu, X. Shirley
影响因子：
64.5
作者：
Impey, S;McCorkle, SR;Goodman, RH
通讯作者：
Goodman, RH
影响因子：
7
作者：
Euskirchen, Ghia M.;Rozowsky, Joel S.;Snyder, Michael
通讯作者：
Snyder, Michael