Acetylated lysine residues (Kac) in histones are recognized by epigenetic reader proteins, such as Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins. Human YEATS domains bind to the acetylated N-terminal tail of histone H3; however, their Kac-binding preferences at the level of the nucleosome are unknown. Through genetic code reprogramming, here, we established a nucleosome core particle (NCP) array containing histones that were acetylated at specific residues and used it to compare the Kac-binding preferences of human YEATS domains. We found that AF9-YEATS showed basal binding to the unmodified NCP and that it bound stronger to the NCP containing a single acetylation at one of K4, K9, K14, or K27 of H3, or to histone H4 multi-acetylated between K5 and K16. Crystal structures of AF9-YEATS in complex with an H4 peptide diacetylated either at K5/K8 or K8/K12 revealed that the aromatic cage of the YEATS domain recognized the acetylated K8 residue. Interestingly, E57 and D103 of AF9, both located outside of the aromatic cage, were shown to interact with acetylated K5 and K12 of H4, respectively, consistent with the increase in AF9-YEATS binding to the H4K8-acetylated NCP upon additional acetylation at K5 or K12. Finally, we show that a mutation of E57 to alanine in AF9-YEATS reduced the binding affinity for H4 multiacetylated NCPs containing H4K5ac. Our data suggest that the Kac-binding affinity of AF9-YEATS increases additively with the number of Kac in the histone tail.

组蛋白中的乙酰化赖氨酸残基 (Kac) 可被表观遗传阅读器蛋白识别，例如Y af9、E NL、 AF9 、T af14和Sas5 (YEATS) 结构域的蛋白质。人类 YEATS 结构域与组蛋白 H3 的乙酰化 N 末端尾部结合；然而，它们在核小体水平上的 Kac 结合偏好是未知的。通过遗传密码重编程，我们建立了一个核小体核心粒子 (NCP) 阵列，其中包含在特定残基处乙酰化的组蛋白，并用它来比较人类 YEATS 结构域的 Kac 结合偏好。我们发现 AF9-YEATS 显示出与未修饰的 NCP 的基础结合，并且它与包含 H3 的 K4、K9、K14 或 K27 之一的单一乙酰化的 NCP 或在 K5 和 K16 之间多乙酰化的组蛋白 H4 结合更强. AF9-YEATS 的晶体结构与在 K5/K8 或 K8/K12 双乙酰化的 H4 肽复合，表明 YEATS 结构域的芳香笼可识别乙酰化 K8 残基。有趣的是，AF9 的 E57 和 D103 均位于芳香笼外，分别与 H4 的乙酰化 K5 和 K12 相互作用，这与在 K5 处额外乙酰化后 AF9-YEATS 与 H4K8 乙酰化 NCP 结合的增加一致或 K12。最后，我们表明 AF9-YEATS 中 E57 突变为丙氨酸降低了对含有 H4K5ac 的 H4 多乙酰化 NCP 的结合亲和力。我们的数据表明，AF9-YEATS 的 Kac 结合亲和力随着组蛋白尾中 Kac 的数量而增加。我们表明，AF9-YEATS 中 E57 突变为丙氨酸降低了对含有 H4K5ac 的 H4 多乙酰化 NCP 的结合亲和力。我们的数据表明，AF9-YEATS 的 Kac 结合亲和力随着组蛋白尾中 Kac 的数量而增加。我们表明，AF9-YEATS 中 E57 突变为丙氨酸降低了对含有 H4K5ac 的 H4 多乙酰化 NCP 的结合亲和力。我们的数据表明，AF9-YEATS 的 Kac 结合亲和力随着组蛋白尾中 Kac 的数量而增加。