Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins
Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins
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DOI：
10.1101/gr.114850.110
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发表时间：
2011-05-01
期刊：
影响因子：
7
通讯作者：
Mitra, Robi David
中科院分区：
文献类型：
--
作者：
Wang, Haoyi;Mayhew, David;Mitra, Robi David
转录因子指导基因的表达，因此在全基因组范围内定位它们的结合位置是非常有意义的。目前的方法不允许TF结合的多重分析，这限制了它们的通量。我们描述了一种新的方法，用于同时确定多个转录因子的基因组靶基因。DNA结合蛋白被赋予将转座子插入到基因组中靠近它们结合的地方的能力。转座子变成了一张“名片”，标志着DNA结合蛋白到达该位置。转座子中的独特序列“条形码”将其与指导其插入的DNA结合蛋白相匹配。转座子侧翼的DNA序列（揭示转座子在基因组中的位置）和转座子内的条形码（识别将其置于那里的TF）通过大规模平行DNA测序来确定。为了证明该方法的可行性，我们在一个实验中确定了8个转录因子的基因组靶点。Calling Card方法有望显著降低在不同环境条件和遗传背景下确定许多转录因子的基因组靶点所需的成本和劳动力。
Transcription factors direct gene expression, so there is much interest in mapping their genome-wide binding locations. Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a "Calling Card'' marking the visit of the DNA-binding protein to that location. A unique sequence "barcode'' in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively parallel DNA sequencing. To demonstrate the method's feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds.