Genetic code expansion in stable cell lines enables encoded chromatin modification

Abstract

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 incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl)CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-ɛ-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. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

“…First, we inserted the genetic‐code‐expansion machinery into the CHO genome by using a piggyback transposase 15. Second, we integrated the heavy and light chain genes and selected for high expression of the antibody.…”

Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

“…Decoding such sites with the aid of introduced o-pair may have deleterious consequences as illustrated by the Sep-OTS in E. coli [59] or Pyl-OTS in certain mammalian cell lines [60] . In E. coli , natural stop codons are not equally susceptible to o-tRNA mediated decoding and this susceptibility depends upon the presence of the appropriate release factor and/or introduction of ribosomal proteins with lower affinity for the same release factor [61] .…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…The utility of this system was shown by genetically incorporating six 1-N-acetyllysines (AcK; Fig. 1B59) into histone H3 (Elsasser et al 2016). Methodological advances have also allowed the genetic incorporation of ncAAs into proteins in various multicellular organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Streptomyces venezuelae (Greiss and Chin 2011;Bianco et al 2012;Chin 2014;He et al 2015).…”