Genetic code expansion in stable cell lines enables encoded chromatin modification

Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However this approach has not been coupled to genome-wide measurements, because efficient unnatural amino acid incorporation is limited to readily transfectable cells and leads to very heterogeneous expression. We demonstrate that rapid piggybac integration of the orthogonal pyrrolysyl-tRNA synthetase (PylS)/tRNAPyl CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous unnatural amino acid incorporation into target proteins in diverse cells, and we reveal the distinct transcriptional responses of ES cells and MEFs to amber suppression. Genetically encoding Nε-acetyl-lysine in place of six lysine residues in histone H3, that are known to be post-translationally acetylated, enables deposition of pre-acetylated histones into cellular chromatin, via a synthetic pathway that is orthogonal to enzymatic modification, allowing us to determine the consequences of acetylation at specific amino acids in histones on gene expression.

遗传编码的非天然氨基酸为调控哺乳动物细胞内蛋白质的分子功能提供了强有力的研究策略。然而该方法尚未与全基因组检测手段相结合，原因在于高效的非天然氨基酸掺入仅局限于易于转染的细胞，且会导致靶蛋白表达水平呈现高度异质性。本研究证实，将正交的吡咯赖氨酰-tRNA合成酶（pyrrolysyl-tRNA synthetase, PylS）/tRNAPyl CUA对（及其衍生物）通过piggyBac转座子系统快速整合至哺乳动物基因组，可使多种细胞中的靶蛋白实现高效且均一的非天然氨基酸掺入；同时我们揭示了胚胎干细胞（ES cells）与小鼠胚胎成纤维细胞（MEFs）对琥珀密码子抑制（amber suppression）的显著不同转录响应。将组蛋白H3（histone H3）中六个已知存在翻译后乙酰化修饰的赖氨酸残基替换为遗传编码的Nε-乙酰赖氨酸（Nε-acetyl-lysine），可通过一条与酶促修饰正交的合成途径，将预乙酰化的组蛋白沉积至细胞染色质中，从而使我们能够解析组蛋白特定氨基酸位点的乙酰化对基因表达的调控效应。