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通过绿色 CUT&RUN 将定量蛋白质组学与转录因子的精确基因组分析相结合。

Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN.

机构信息

Department of Urology, Medical Center-University of Freiburg, 79016 Freiburg, Germany.

German Cancer Consortium (DKTK) partner site Freiburg, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

出版信息

Nucleic Acids Res. 2021 May 21;49(9):e49. doi: 10.1093/nar/gkab038.

DOI:10.1093/nar/gkab038
PMID:33524153
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8136828/
Abstract

Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique.

摘要

通过多种技术可以检测染色质和转录调控因子的全基因组定位。在这里，我们描述了一种用于转录调控因子全基因组分析的新方法“greenCUT&RUN”，该方法具有非常高的灵敏度、分辨率、准确性和重现性，同时保证了特异性。我们的策略从用 GFP 标记感兴趣的蛋白质开始，并利用融合到 MNase 的 GFP 特异性纳米体来分析全基因组结合事件。通过使用 GFP 纳米体，greenCUT&RUN 方法消除了抗体依赖性和变异性。使用 greenCUT&RUN 获得了稳健的基因组图谱，这些图谱对开放染色质是准确且无偏的。通过将 greenCUT&RUN 与基于纳米体的亲和纯化质谱相结合，可以在基因组范围内识别“搭便车”的 DNA 结合事件。greenCUT&RUN 的独特设计赋予了靶蛋白的灵活性，并产生了高分辨率的足迹。此外，greenCUT&RUN 允许快速分析染色质和转录蛋白的突变体。总之，greenCUT&RUN 是一种广泛适用且多功能的基因组作图技术。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/4a1ad0a6d08b/gkab038fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/2f4339c4bd8c/gkab038fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/37e32f6e5bbb/gkab038fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/d91843521210/gkab038fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/82f67200e2b0/gkab038fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/2e971e5acb54/gkab038fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/9fb2953a7c7f/gkab038fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/4a1ad0a6d08b/gkab038fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/2f4339c4bd8c/gkab038fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/37e32f6e5bbb/gkab038fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/d91843521210/gkab038fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/82f67200e2b0/gkab038fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/2e971e5acb54/gkab038fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/9fb2953a7c7f/gkab038fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f1/8136828/4a1ad0a6d08b/gkab038fig7.jpg

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