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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders I

ABSTRACT: Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE283704 | GEO | 2024/12/15

REPOSITORIES: GEO

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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders III

Project description:Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.

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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders II

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