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

Chromatin modifiers, transcription factors and cofactors regulate gene expression through engagement with chromatin and DNA, which can be detected by a variety of genomic techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulatory factors, which has higher sensitivity, resolution, accuracy and reproducibility than existing methods, whilst assuring specificity as assessed for the gene specific transcription factors NFY and c-FOS, and for the basal transcription factor TBP. Our strategy begins with tagging the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. Since the nanobody targets GFP, the greenCUT&RUN approach eliminates dependency on protein-specific antibodies, which makes it widely applicable. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate as defined by the presence of known transcription factors' consensus motifs and unbiased towards open chromatin regions. By integrating greenCUT&RUN with nanobody-based affinity purification massspectrometry, 'piggy-back' DNA binding events were identified on a genomic scale. The unique design of greenCUT&RUN grants target flexibility, a high chromatin accessibility to MNase and high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription regulatory proteins. In conclusion, greenCUT&RUN is a widely applicable superior genome-mapping technique with opportunities to integrate with quantitative massspectrometry.