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

Experimental approach of greenCUT&RUN. Panels (A and B): Schematic of experimental strategy for greenCUT&RUN (A) and CUT&RUN (B). GreenCUT&RUN is rapid and easy protocol, which involves only three steps to complete. Panel (C): The GST protein fused to the GFP nanobody and MNase (nanobody-MNase) was expressed and purified from bacteria.

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.

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Citation

Sheikh Nizamuddin, Stefanie Koidl, Tanja Bhuiyan, Tamara V Werner, Martin L Biniossek, Alexandre M J J Bonvin, Silke Lassmann, HThMarc Timmers (2022):
Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN.
Nucleic Acids Research 49(9)
https://doi.org/10.1093/nar/gkab038