ChEC-seq2: an improved Chromatin Endogenous Cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.
ChEC-seq2: an improved Chromatin Endogenous Cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.
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ChEC-seq2：一种改进的染色质内源切割测序方法和生物信息学分析流程，用于绘制体内蛋白质-DNA 相互作用图谱。
DOI：
10.1101/2023.10.15.562421
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Brickner,JasonH
中科院分区：
文献类型：
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作者：
VanBelzen,Jake;Duan,Chengzhe;Brickner,DonnaGarvey;Brickner,JasonH
转录因子(TF)活体DNA结合特异性的确定几乎完全依赖于染色质免疫沉淀(CHIP)。虽然CHIP显示了TF结合模式，但其分辨率较低。使用核酸酶的高分辨率方法，如ChIP-exO、染色质内源切割(ChEC-SEQ)和CUT&RUN，既能解决TF占位问题，又能保护结合位点。CHEC-SEQ将内源性转铁蛋白与微球菌核酸酶融合，既不需要固定，也不需要抗体。然而，Check过程中DNA裂解的特异性低于ChIP或ChIP-exo鉴定的峰的特异性，可能反映了转录因子与DNA的非特异性结合。我们已经简化了CHEC-SEQ方案，以最大限度地减少核酸酶消化，同时增加切割DNA的产量。Check-Seq2裂解模式在复制之间以及与已公布的Check-Seq数据之间具有高度重复性。结合一种新的去除非特定切割位点的生物信息学管道DoubleChEC，check-seq2确定了三种不同酵母TF的高置信度切割位点，这些切割位点因其已知结合位点而高度丰富，并且毗邻已知的目标基因。
Defining thein vivoDNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.