Simultaneous Mapping of DNA Binding and Nucleosome Positioning with SpLiT-ChEC.
Simultaneous Mapping of DNA Binding and Nucleosome Positioning with SpLiT-ChEC.
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使用 SpLiT-ChEC 同时绘制 DNA 结合和核小体定位。
DOI：
10.1101/2023.07.03.547581
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
McKnight,LauraE
中科院分区：
文献类型：
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作者：
Banks,OrionGB;Harms,MichaelJ;McKnight,JeffreyN;McKnight,LauraE
染色质的组织——包括核小体的位置和其他蛋白质与DNA的结合——有助于确定真核生物的转录谱。虽然像ChIP-Seq和MNase-Seq这样的技术可以分别绘制蛋白质- dna和核小体的定位，但同时捕获核小体位置和蛋白质- dna相互作用的分析可以产生染色质景观的详细图像。大多数监测染色质组织和蛋白质结合的检测依赖于抗体，抗体通常表现为非特异性结合，和/或向所研究的dna结合蛋白添加大块加合物，这可能影响其表达和活性。在这里，我们描述了SpyCatcher染色质内源性切割的连锁靶向（SpLiT-ChEC），其中一个13个氨基酸的SpyTag肽附加在感兴趣的蛋白质上，作为原位酶切的高度特异性靶向片段。SpyTag/SpyCatcher系统形成共价键，将目标蛋白与共表达的MNase-SpyCatcher融合构建物连接起来。SpyTagged蛋白从内源性基因座表达，而MNase-SpyCatcher表达是在收获培养物之前立即诱导的。mase被高浓度的钙激活，它主要消化目标蛋白结合位点附近的DNA。通过对靶向mase酶切释放的DNA片段进行测序，我们发现这种方法可以恢复蛋白质结合和近端核小体定位的信息。SpLiT-ChEC提供了精确的时间控制，我们预计可用于在各种条件下和细胞周期的不同点监测染色质。
The organization of chromatin – including the positions of nucleosomes and the binding of other proteins to DNA – helps define transcriptional profiles in eukaryotic organisms. While techniques like ChIP-Seq and MNase-Seq can map protein-DNA and nucleosome localization separately, assays designed to simultaneously capture nucleosome positions and protein-DNA interactions can produce a detailed picture of the chromatin landscape. Most assays that monitor chromatin organization and protein binding rely on antibodies, which often exhibit nonspecific binding, and/or the addition of bulky adducts to the DNA-binding protein being studied, which can affect their expression and activity. Here, we describe SpyCatcher Linked Targeting of Chromatin Endogenous Cleavage (SpLiT-ChEC), where a 13-amino acid SpyTag peptide, appended to a protein of interest, serves as a highly-specific targeting moiety for in situ enzymatic digestion. The SpyTag/SpyCatcher system forms a covalent bond, linking the target protein and a co-expressed MNase-SpyCatcher fusion construct. SpyTagged proteins are expressed from endogenous loci, whereas MNase-SpyCatcher expression is induced immediately before harvesting cultures. MNase is activated with high concentrations of calcium, which primarily digests DNA near target protein binding sites. By sequencing the DNA fragments released by targeted MNase digestion, we found that this method recovers information on protein binding and proximal nucleosome positioning. SpLiT-ChEC provides precise temporal control that we anticipate can be used to monitor chromatin under various conditions and at distinct points in the cell cycle.