Genetically Encoded Epitope Tag for Probing Lysine Acylation-Mediated Protein-Protein Interactions.

Area of Science:

• Epigenetics and Molecular Biology
• Chemical Biology

Background:

• YEATS domain proteins, like AF9, are crucial for regulating DNA-templated processes through interactions with histone lysine acetylation (Kac) and crotonylation (Kcr) marks.
• Previous work identified 2-furancarbonyllysine (Kfu) as a modification with significantly enhanced affinity for the AF9 YEATS domain compared to Kac/Kcr.
• Kfu-containing peptides served as valuable chemical tools for investigating AF9 YEATS domain-histone lysine acylation interactions.

Purpose of the Study:

• To achieve the genetic incorporation of Kfu into both bacterial (Escherichia coli) and mammalian cells.
• To develop a novel, Kfu-containing epitope tag (RAY-tag) for robust and selective detection of AF9 YEATS domain interactions.
• To demonstrate the utility of the RAY-tag for interrogating AF9 YEATS domain recruitment mediated by histone lysine acylation in diverse biological contexts.

Main Methods:

• Utilized amber codon suppression technology for the genetic incorporation of 2-furancarbonyllysine (Kfu) in Escherichia coli and mammalian cells.
• Engineered a Kfu-containing epitope tag, designated RAY-tag, designed for specific binding to the AF9 YEATS domain.
• Fused the RAY-tag to various protein modules, including fluorescent proteins and DNA-binding proteins, to assess its functionality in different cellular contexts.

Main Results:

• Successfully achieved the genetic incorporation of Kfu into both prokaryotic and eukaryotic cellular systems.
• Developed the RAY-tag, demonstrating robust and selective binding to the AF9 YEATS domain both in vitro and in cellulo.
• Validated the RAY-tag's ability to facilitate the study of AF9 YEATS domain recruitment in response to histone lysine acylation when fused to different protein effectors.

Conclusions:

• The genetic incorporation of Kfu and the development of the RAY-tag provide a powerful new tool for studying YEATS domain biology.
• The RAY-tag enables the investigation of histone lysine acylation-dependent protein recruitment in various cellular environments and biological processes.
• This technology opens avenues for deeper understanding of AF9 function and the broader roles of YEATS domain proteins in gene regulation.