Genetic code expansion in stable cell lines enables encoded chromatin modification.
Genetic code expansion in stable cell lines enables encoded chromatin modification.
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DOI：
10.1038/nmeth.3701
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发表时间：
2016-02
期刊：
影响因子：
48
通讯作者：
Chin JW
中科院分区：
文献类型：
--
作者：
Elsässer SJ;Ernst RJ;Walker OS;Chin JW
基因编码的非天然氨基酸为调节哺乳动物细胞中蛋白质的分子功能提供了强有力的策略。然而，这种方法尚未与全基因组测量相结合，因为有效的非天然氨基酸掺入仅限于瞬时表达环境，从而导致非常不同的表达。我们证明了在哺乳动物基因组中稳定整合玛氏甲烷链霉菌的吡咯酰-tRNA合成酶(PYLS)/tRNAPylCUA对(及其衍生物)能够使不同哺乳动物细胞中的非天然氨基酸高效、均一地整合到靶蛋白中，并揭示了ES细胞和MEF对琥珀抑制的不同转录反应。通过基因编码N-ε-乙酰赖氨酸而不是组蛋白H3中的六个赖氨酸残基，可以通过与酶修饰正交的途径将预乙酰化的组蛋白沉积到细胞染色质中。在合成编码天然修饰位点的赖氨酸乙酰化后，我们确定了组蛋白中特定氨基酸的乙酰化对基因表达的影响。
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 transient expression settings that lead to very heterogeneous expression. We demonstrate that stable \ integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylS)/tRNAPylCUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous unnatural amino acid incorporation into target proteins in diverse mammalian 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 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. Upon synthetically encoding lysine-acetylation at natural modification sites we determine the consequences of acetylation at specific amino acids in histones on gene expression.