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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 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE73823 | GEO | 2016/01/01

SECONDARY ACCESSION(S): PRJNA298050

REPOSITORIES: GEO

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Genetic code expansion in stable cell lines enables encoded chromatin modification

Project description: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 NÎµ-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. mRNA was sequenced using polyA-enrichment and Truseq library preparation protocol. Two biological replicates were sequences for each cell line and condition

2016-07-03 | E-GEOD-73823 | biostudies-arrayexpress

Chimeric design of Pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion

Project description:An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a key prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems that allow unnatural amino acids with distinct scaffolds and functionalities to be incorporated into a wide range of living organisms efficiently. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. In addition, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification. To our knowledge, most of these molecules have never been shown to be incorporated with fully orthogonal pairs. Therefore, these chimera pairs offer the potential for incorporation of de novo unnatural amino acids into target proteins for a variety of applications.

2020-05-25 | PXD018660 | Pride

Defining the chromatin compaction state of all histone post-translational modifications

Project description:By performing metabolic incorporation of heavy labeled amino acids in cell culture, we estimated the turnover rates of histones decorated with different PTMs, and correlated this information with how “active” chromatin is where these PTMs reside.

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Site-specifically acetylated G6PD

Project description:Site-specifically acetylated G6PD expressed in bacteria or cultured mammalian cells by genetically encoding the incorporation of acetylated lysine. Purified G6PD (expressed in E. coli) or immunopurified G6PD (expressed in HEK293T cells) were subjected to LC-MS/MS analysis.

2023-10-24 | PXD044906 | Pride

G6PD K403 acetylation-dependent PTM

Project description:To identify acetylation-dependent posttranslational modifications (PTMs) of G6PD, site-specifically acetylated and Flag-tagged G6PD was expressed in HEK293T cells by genetically encoding the incorporation of acetylated lysine in response to an in-frame TAG stop codon. K403-acetylated G6PD (sample) and K414-acetylated G6PD (control) were co-expressed with WT Fyn kinase and a catalytically inactive mutant of Fyn (FynDN). G6PD was immunoprecipitated using anti-Flag beads before MS analysis.

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Uncover New Reactivity of Genetically Encoded Alkyl Bromide Unnatural Amino Acids

Project description:Genetically encoded unnatural amino acids (Uaas) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These Uaas, including a series of alkyl bromide-based Uaas, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based Uaa, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

2022-04-04 | PXD031159 | Pride

OTS Development and E. coli Proteome LC-MSMS

Project description:Over the past twenty years, the development of orthogonal biological systems has sparked a revolution in our ability to study cellular physiology. Orthogonal translation systems (OTSs) enable site-specific incorporation of hundreds of non-standard amino acids, offering unprecedented access to the study of cellular mechanisms modulated by post-translational modifications (e.g. protein phosphorylation). Although development of phosphoserine-OTSs (pSerOTS) has been significant, little work has focused on the biology of OTS development and utilization. To better understand the impact of OTSs on host physiology, we utilize pSerOTS as a model to systematically explore the extent to which OTS components interact with Escherichia coli. Using this information, we constructed pSerOTS variants designed to enhance OTS orthogonality by minimizing interactions with host processes and decreasing stress response activation. Our expanded understanding of OTS:host interactions enables informed OTS design practices which minimize the negative impact of OTSs while improving OTS performance across a range of experimental settings.

2023-07-27 | PXD026217 | Pride

Haloferax volcanii SILAC study of Oxidative stress

Project description:SILAC was used to monitor proteome changes to Haloferax volcanii after treatment with oxidizing agent NaOCl. SILAC amino acids used were Lysine(+8) and Arginine(+6). For treatment and control groups 4 replicates were used. In replicates 1 and 3, the control group had supplementation of both light lysine and argninie to the medium and the treatment group was supplemented with both heavy lysine(+8) and arginine(+6). In replicates 2 and 4, the labeling was switched, with the control containing heavy lysine(+8) and arginine(+6) and the treatement was supplemented with light lysine and arginine. After treatment, cells were mixed at a 1:1 ratio (control:treatment) and proteins were extracted and digested with trypsin. For the incorporation test, cells were grown in meduim containing heavy lysine(+8) and argnine(+6) and allowed to grow for 24 hours, then subcultured twice, sequentially after 24 h of growth. To test for incorporation of the heavy amino acids, cells were harvested, proteins extracted, and digested with trypsin.

2018-01-09 | PXD006877 | Pride

Identification of PD-L1 interacting partners

Project description:Unnatural amino acids were incorporated into PD-L1 to capture the covalent complexes. Purified the covalent complexes were analyzed using Mass spectrum.

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