He Qiye, Johnston Jeff, Zeitlinger Julia
Stowers Institute for Medical Research, Kansas City, Missouri, USA.
1] Stowers Institute for Medical Research, Kansas City, Missouri, USA. [2] Department of Pathology, Kansas University Medical Center, Kansas City, Kansas, USA.
Nat Biotechnol. 2015 Apr;33(4):395-401. doi: 10.1038/nbt.3121. Epub 2015 Mar 9.
Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins--human TBP and Drosophila NFkB, Twist and Max--shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.
了解真核生物增强子如何被转录因子的特定组合所结合和调控仍然是一个重大挑战。为了在体内以核苷酸分辨率更好地全基因组定位转录因子结合情况，我们开发了一种强大的ChIP-exo方案，称为ChIP-nexus（通过核酸外切酶、独特条形码和单连接实现核苷酸分辨率的染色质免疫沉淀实验），该方案在文库制备过程中利用了高效的DNA自环化步骤。将ChIP-nexus应用于四种蛋白质——人类TBP和果蝇NFkB、Twist和Max——表明它在分辨率和特异性方面优于现有的ChIP方案，能够精确确定含有多个结合基序的增强子内的相关结合位点，并允许对体内结合特异性进行分析。值得注意的是，我们发现Max经常与其基序旁边的DNA序列相互作用，并且这种结合模式与局部DNA序列特征（如DNA形状）相关。ChIP-nexus将广泛应用于研究体内转录因子结合特异性及其与人类和模式生物中顺式调控变化的关系。
