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

Histone lysine acetylation (Kac) and crotonylation (Kcr) marks mediate the recruitment of YEATS domains to chromatin. In this way, YEATS domain-containing proteins such as AF9 participate in the regulation of DNA-templated processes. Our previous study showed that the replacement of Kac/Kcr by a 2-furancarbonyllysine (Kfu) residue led to greatly enhanced affinity toward the AF9 YEATS domain, rendering Kfu-containing peptides useful chemical tools to probe the AF9 YEATS–Kac/Kcr interactions. Here, we report the genetic incorporation of Kfu in Escherichia coli and mammalian cells through the amber codon suppression technology. We develop a Kfu-containing epitope tag, termed RAY-tag, which can robustly and selectively engage with the AF9 YEATS domain in vitro and in cellulo. We further demonstrate that the fusion of RAY-tag to different protein modules, including fluorescent proteins and DNA binding proteins, can facilitate the interrogation of the histone lysine acylation-mediated recruitment of the AF9 YEATS domain in different biological contexts.