RESUMEN
Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state.
Asunto(s)
Células Madre Embrionarias/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Células Madre Embrionarias/citología , Chaperonas de Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Estructuras R-Loop , Proteínas de Unión al ARN/metabolismo , Línea Celular , Cromatina/metabolismo , Humanos , Estrés Fisiológico , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (ESC), observing widespread G4 formation at active promoters, active and poised enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mouse ESCs. Upon differentiation to neural progenitor cells (NPC), enhancer G4s are lost. Further, performing R-loop CUT&Tag, we demonstrate the genome-wide co-occurrence of single-stranded DNA, G4s and R loops at promoters and enhancers. We confirm that G4 structures exist independent of ongoing transcription, suggesting an intricate relationship between transcription and non-canonical DNA structures.
Asunto(s)
G-Cuádruplex , Animales , ADN/química , ADN/genética , Inestabilidad Genómica , Mamíferos , Ratones , Estructuras R-Loop , ARNRESUMEN
DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/metabolismo , Animales , Citosina/metabolismo , Desmetilación del ADN , Metilación de ADN , Proteínas de Unión al ADN/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Expresión Génica , Mamíferos/genética , Ratones , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT-seq) to define both coding and non-coding transcription units (TUs) in these three pluripotent states and combine TT-seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome-wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome-wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome-wide changes in transcription kinetics invoke ground and paused pluripotent states.
Asunto(s)
ARN Polimerasa II , Transcriptoma , Animales , Cinética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term 'EvoChromo'. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution.
Asunto(s)
Cromatina/genética , Evolución Molecular , Animales , Genoma , HumanosRESUMEN
Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models.
Asunto(s)
Aminoácidos/metabolismo , Encéfalo/metabolismo , Ingeniería Celular , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , Aminoácidos/genética , Código Genético , HumanosRESUMEN
Transposable elements comprise roughly 40% of mammalian genomes. They have an active role in genetic variation, adaptation and evolution through the duplication or deletion of genes or their regulatory elements, and transposable elements themselves can act as alternative promoters for nearby genes, resulting in non-canonical regulation of transcription. However, transposable element activity can lead to detrimental genome instability, and hosts have evolved mechanisms to silence transposable element mobility appropriately. Recent studies have demonstrated that a subset of transposable elements, endogenous retroviral elements (ERVs) containing long terminal repeats (LTRs), are silenced through trimethylation of histone H3 on lysine 9 (H3K9me3) by ESET (also known as SETDB1 or KMT1E) and a co-repressor complex containing KRAB-associated protein 1 (KAP1; also known as TRIM28) in mouse embryonic stem cells. Here we show that the replacement histone variant H3.3 is enriched at class I and class II ERVs, notably those of the early transposon (ETn)/MusD family and intracisternal A-type particles (IAPs). Deposition at a subset of these elements is dependent upon the H3.3 chaperone complex containing α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX). We demonstrate that recruitment of DAXX, H3.3 and KAP1 to ERVs is co-dependent and occurs upstream of ESET, linking H3.3 to ERV-associated H3K9me3. Importantly, H3K9me3 is reduced at ERVs upon H3.3 deletion, resulting in derepression and dysregulation of adjacent, endogenous genes, along with increased retrotransposition of IAPs. Our study identifies a unique heterochromatin state marked by the presence of both H3.3 and H3K9me3, and establishes an important role for H3.3 in control of ERV retrotransposition in embryonic stem cells.
Asunto(s)
Células Madre Embrionarias/virología , Retrovirus Endógenos/genética , Silenciador del Gen , Histonas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas Co-Represoras , ADN Helicasas/metabolismo , Inestabilidad Genómica , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metilación , Ratones , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas/química , Animales , Células HEK293 , Humanos , Microscopía Fluorescente , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
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-É-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.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Cromatina/fisiología , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Methanosarcina/enzimología , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Animales , Línea Celular , Regulación Enzimológica de la Expresión Génica/fisiología , Ingeniería Genética , Células HEK293 , Humanos , Methanosarcina/metabolismo , RatonesRESUMEN
We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co-expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver-specific genes co-expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver-specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver-specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin-like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.
Asunto(s)
Carcinoma Hepatocelular/genética , Acido Graso Sintasa Tipo I/genética , Redes Reguladoras de Genes , Neoplasias Hepáticas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Biología de Sistemas/métodos , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Células Hep G2 , Humanos , Células K562 , Hígado/química , Hígado/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Especificidad de Órganos , Mapas de Interacción de Proteínas , Análisis de Secuencia de ARNRESUMEN
Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly 90 in H3.3 and Glu 225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.
Asunto(s)
ADN/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Co-Represoras , Cristalografía por Rayos X , ADN/química , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Conformación Proteica , Multimerización de Proteína , Especificidad por Sustrato , Agua/química , Agua/metabolismoRESUMEN
Histones are among the most conserved proteins in eukaryotes: the structural constraints of the nucleosome pose a challenge to evolving novel function. Nevertheless, confined histone surfaces have diversified, allowing the modulation of basic chromatin function through specialized histone chaperones. Recent structures of three histone-chaperone complexes, DAXX, HJURP, and Scm3, exemplify a common parsimonious solution to the restricted evolutionary space of histone recognition by their cognate histone chaperones: the reutilization of existing themes in histone structural biology.
Asunto(s)
Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Co-Represoras , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Histonas/química , Histonas/genética , Humanos , Chaperonas Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/química , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation.
Asunto(s)
Epigénesis Genética/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Proteínas Mutantes/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Procesos Fotoquímicos , Arginina/metabolismo , Células HEK293 , Humanos , Isocitrato Deshidrogenasa/química , Modelos Moleculares , Estructura Molecular , Proteínas Mutantes/genética , Mutación , Neoplasias/enzimologíaRESUMEN
Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.
Asunto(s)
Chaperonas de Histonas/química , Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutación , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Desplegamiento ProteicoRESUMEN
Histone chaperones can be broadly defined as histone-binding proteins that influence chromatin dynamics in an ATP-independent manner. Their existence reflects the importance of chromatin homeostasis and the unique and unusual biochemistry of the histone proteins. Histone supply and demand at chromatin is regulated by a network of structurally and functionally diverse histone chaperones. At the core of this network is a mechanistic variability that is only beginning to be appreciated. In this review, we highlight the challenges in determining histone chaperone mechanism and discuss possible mechanisms in the context of nucleosome thermodynamics. We discuss how histone chaperones prevent promiscuous histone interactions, and consider if this activity represents the full extent of histone chaperone function in governing chromatin dynamics. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.
Asunto(s)
Chaperonas de Histonas/fisiología , Animales , Ensamble y Desensamble de Cromatina/fisiología , Chaperonas de Histonas/química , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleosomas/química , Nucleosomas/metabolismoRESUMEN
The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
Asunto(s)
Aminoácidos/metabolismo , Lisina-ARNt Ligasa/metabolismo , Pirroles/química , ARN de Transferencia/metabolismo , Lisina-ARNt Ligasa/químicaRESUMEN
We demonstrate the evolution of the PylRS/tRNA(CUA) pair for genetically encoding photocaged cysteine. By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, we establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. We demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.
Asunto(s)
Cisteína/química , Cisteína/genética , Endopeptidasas/metabolismo , Luz , Animales , Endopeptidasas/química , Endopeptidasas/genética , Activación Enzimática/efectos de la radiación , Evolución Molecular , Transferencia Resonante de Energía de Fluorescencia , Código Genético , Células HEK293 , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.