Interrogating transcriptional regulatory sequences in Tol2-mediated Xenopus transgenics.
Interrogating transcriptional regulatory sequences in Tol2-mediated Xenopus transgenics.
复制标题
DOI：
10.1371/journal.pone.0068548
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Harland RM
中科院分区：
文献类型：
--
作者：
Loots GG;Bergmann A;Hum NR;Oldenburg CE;Wills AE;Hu N;Ovcharenko I;Harland RM
在脊椎动物中识别基因调控元件及其靶基因仍然是一个巨大的挑战。现在已经认识到，转录调控序列在脊椎动物发育过程中和成年组织中对组织特异性基因表达的动态调控至关重要，并且这些元件可以相对于它们控制的基因的启动子定位在很远的距离。虽然通过结合染色质免疫沉淀和下一代测序来定位DNA结合区已经取得了重大进展，但功能验证仍然是提高我们将计算机预测与生物功能相关联的能力的限制步骤。我们最近开发了一种计算方法，它协同结合全基因组基因表达谱、脊椎动物基因组比较和转录因子结合位点分析来预测人类基因组中的组织特异性增强子。我们将这种方法应用于270个在骨骼肌中高表达的基因，并预测了190个假定的顺式调节模块。此外，我们优化了非洲爪哇的Tol2转基因构建物，以询问其中20个元素在胚胎发育过程中作为骨骼肌特异转录增强子的能力。我们发现，这些成分中的45%只在非洲爪哇蝌蚪的快速肌肉纤维中表达，这些纤维定位于高度组织化的人字形结构。转录因子结合位点分析在6个已验证的增强子中发现了∼200bp区域内的>；2Mef2/MyoD位点，并对这些位点进行了系统的突变，结果表明它们对增强子功能至关重要。这里描述的数据介绍了一种新的报告系统，适用于询问组织特异性的顺式调节元件，该系统允许实时监测非洲爪哇胚胎发育的整个早期阶段的增强子活性。
Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate in silico predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative cis-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in Xenopus laevis to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the Xenopus laevis tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ∼200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific cis-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in Xenopus.
登录
查看更多内容
影响因子：
4.6
作者：
Grimaldi, A;Tettamanti, G;Hughes, SM
通讯作者：
Hughes, SM
影响因子：
1.5
作者：
Hamlet, Michelle R. Johnson;Yergeau, Donald A.;Mead, Paul E.
通讯作者：
Mead, Paul E.
影响因子：
5.5
作者：
Momken, I;Lechêne, P;Ventura-Clapier, R
通讯作者：
Ventura-Clapier, R
影响因子：
7
作者：
Birnbaum RY;Clowney EJ;Agamy O;Kim MJ;Zhao J;Yamanaka T;Pappalardo Z;Clarke SL;Wenger AM;Nguyen L;Gurrieri F;Everman DB;Schwartz CE;Birk OS;Bejerano G;Lomvardas S;Ahituv N
通讯作者：
Ahituv N
影响因子：
11.8
作者：
Kawakami, K;Takeda, H;Mishina, M
通讯作者：
Mishina, M