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The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.
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Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.
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Genetic and genomic techniques have proven incredibly powerful for identifying and studying molecular players implicated in the epigenetic regulation of DNA-templated processes such as transcription. However, achieving a mechanistic understanding of how these molecules interact with chromatin to elicit a functional output is non-trivial, owing to the tremendous complexity of the biochemical networks involved. Advances in protein engineering have enabled the reconstitution of 'designer' chromatin containing customized post-translational modification patterns, which, when used in conjunction with sophisticated biochemical and biophysical methods, allow many mechanistic questions to be addressed. In this Review, we discuss how such tools complement established 'omics' techniques to answer fundamental questions on chromatin regulation, focusing on chromatin mark establishment and protein-chromatin interactions.
Asunto(s)
Cromatina , Epigénesis Genética , Cromatina/genética , Histonas/genética , Procesamiento Proteico-Postraduccional , Replicación del ADNRESUMEN
The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.
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The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions BCR/ABL and DNAJB1/PRKACA where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as novel therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.
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Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.
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Núcleo Celular , Cromatina , Reactivos de Enlaces Cruzados , Humanos , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Reactivos de Enlaces Cruzados/análisis , Reactivos de Enlaces Cruzados/química , Transferencia de Energía , Epigenómica , Inteínas , Iridio , Mutación , Neoplasias/genética , Fármacos Fotosensibilizantes , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates "snapshots" of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.
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Luz , Proteínas , HumanosRESUMEN
Cell differentiation and tissue specialization lead to unique cellular surface landscapes and exacerbated or loss of expression patterns can result in further heterogenicity distinctive of pathological phenotypes1-3. Immunotherapies and emerging protein therapeutics seek to exploit such differences by engaging cell populations selectively based on their surface markers. Since a single surface antigen rarely defines a specific cell type4,5, the development of programmable molecular systems that integrate multiple cell surface features to convert on-target inputs to user-defined outputs is highly desirable. Here, we describe an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive fragments. We show that this protein actuator platform can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. Due to its intrinsic modularity and tunability, this technology is expected to be compatible with different types of inputs, targeting modalities and functional outputs, and as such will have broad application in the synthetic biology and biotechnology areas.
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Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.
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Histonas , Nucleosomas , Animales , Histonas/genética , Histonas/química , Glutamina , Heterocromatina , Proteína Glutamina Gamma Glutamiltransferasa 2 , Cromatina/genética , ADN/química , MamíferosRESUMEN
Split intein-mediated protein trans-splicing (PTS) is widely applied in chemical biology and biotechnology to carry out traceless and specific protein ligation. However, the external residues immediately flanking the intein (exteins) can reduce the splicing rate, thereby limiting certain applications of PTS. Splicing by a recently developed intein with atypical split architecture ("Cat") exhibits a stark dependence on the sequence of its N-terminal extein residues. Here, we further developed Cat using error-prone polymerase chain reaction (PCR) and a cell-based selection assay to produce Cat*, which exhibits greatly enhanced PTS activity in the presence of unfavorable N-extein residues. We then applied solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to explore how the dynamics of a conserved B-block histidine residue (His78) contribute to this extein dependence. The enhanced extein tolerance of Cat* reported here should expand the applicability of atypically split inteins, and the mechanism highlights common principles that contribute to extein dependence.
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Exteínas , Inteínas , Histidina/metabolismo , Empalme de Proteína , Proteínas/metabolismoRESUMEN
Work over the last decade has uncovered a new layer of epigenetic dysregulation. It is now appreciated that somatic missense mutations in histones, the packaging agents of genomic DNA, are often associated with human pathologies, especially cancer. Although some of these "oncohistone" mutations are thought to be key drivers of cancer, the impacts of the majority of them on disease onset and progression remain to be elucidated. Here, we survey this rapidly expanding research field with particular emphasis on how histone mutants, even at low dosage, can corrupt chromatin states. This work is unveiling the remarkable intricacies of epigenetic control mechanisms. Throughout, we highlight how studies of oncohistones have leveraged, and in some cases fueled, the advances in our ability to manipulate and interrogate chromatin at the molecular level.
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Epigénesis Genética , Neoplasias , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Mutación , Neoplasias/genética , Neoplasias/patologíaRESUMEN
In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.
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Proteínas Bacterianas , Proteínas de la Membrana , Péptido Hidrolasas , Feromonas , Percepción de Quorum , Staphylococcus aureus , Transactivadores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/fisiología , Péptido Hidrolasas/genética , Péptido Hidrolasas/fisiología , Feromonas/biosíntesis , Percepción de Quorum/genética , Staphylococcus aureus/patogenicidad , Transactivadores/genética , Transactivadores/metabolismo , VirulenciaRESUMEN
Nucleosomes frequently exist as asymmetric species in native chromatin contexts. Current methods for the traceless generation of these heterotypic chromatin substrates are inefficient and/or difficult to implement. Here, we report an application of the SpyCatcher/SpyTag system as a convenient route to assemble desymmetrized nucleoprotein complexes. This genetically encoded covalent tethering system serves as an internal chaperone, maintained through the assembly process, affording traceless asymmetric nucleosomes following proteolytic removal of the tethers. The strategy allows for generation of nucleosomes containing asymmetric modifications on single or multiple histones, thereby providing facile access to a range of substrates. Herein, we use such constructs to interrogate how nucleosome desymmetrization caused by the incorporation of cancer-associated histone mutations alters chromatin remodeling processes. We also establish that our system provides access to asymmetric dinucleosomes, which allowed us to query the geometric/symmetry constraints of the unmodified histone H3 tail in stimulating the activity of the histone lysine demethylase, KDM5B. By providing a streamlined approach to generate these sophisticated substrates, our method expands the chemical biology toolbox available for interrogating the consequences of asymmetry on chromatin structure and function.
RESUMEN
Nucleosomes, the structural building blocks of chromatin, possess 2-fold pseudo symmetry which can be broken through differential modification or removal of one copy of a pair of sister histones. The resultant asymmetric nucleosomes and hexasomes have been implicated in gene regulation, yet the use of these noncanonical substrates in chromatin biochemistry is limited, owing to the lack of efficient methods for their preparation. Here, we report a strategy that allows the orientation of these asymmetric species to be tightly controlled relative to the underlying DNA sequence. Our approach is based on the use of truncated DNA templates to assemble oriented hexasomes followed by DNA ligation and, in the case of asymmetric nucleosomes, addition of the missing heterotypic histones. We show that this approach is compatible with multiple nucleosome positioning sequences, allowing the generation of desymmetrized mononucleosomes and oligonucleosomes with varied DNA overhangs and heterotypic histone H2A/H2B dimer compositions. Using this technology, we examine the functional consequences of asymmetry on BRG1/BRM associated factor (BAF) complex-mediated chromatin remodeling. Our results indicate that cancer-associated histone mutations can reprogram the inherent activity of BAF chromatin remodeling to induce aberrant chromatin structure.
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Cromatina/química , ADN/química , Nucleosomas/química , Histonas/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Protein-protein interactions (PPIs) in the nucleus play key roles in transcriptional regulation and ensure genomic stability. Critical to this are histone-mediated PPI networks, which are further fine-tuned through dynamic post-translational modification. Perturbation to these networks leads to genomic instability and disease, presenting epigenetic proteins as key therapeutic targets. This mini-review will describe progress in mapping the combinatorial histone PTM landscape, and recent chemical biology approaches to map histone interactors. Recent advances in mapping direct interactors of histone PTMs as well as local chromatin interactomes will be highlighted, with a focus on mass-spectrometry based workflows that continue to illuminate histone-mediated PPIs in unprecedented detail.
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Histonas/metabolismo , Cristalografía por Rayos X/métodos , Espectrometría de Masas/métodos , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.
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Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Cromosómicas no Histona/química , Código de Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Mutación , Nucleosomas/química , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factores de Transcripción/químicaRESUMEN
The fundamental repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around two copies each of four canonical histone proteins. Nucleosomes possess 2-fold pseudo-symmetry that is subject to disruption in cellular contexts. For example, the post-translational modification (PTM) of histones plays an essential role in epigenetic regulation, and the introduction of a PTM on only one of the two "sister" histone copies in a given nucleosome eliminates the inherent symmetry of the complex. Similarly, the removal or swapping of histones for variants or the introduction of a histone mutant may render the two faces of the nucleosome asymmetric, creating, if you will, a type of "Janus" bioparticle. Over the past decade, many groups have detailed the discovery of asymmetric species in chromatin isolated from numerous cell types. However, in vitro biochemical and biophysical investigation of asymmetric nucleosomes has proven synthetically challenging. Whereas symmetric nucleosomes are readily formed via a stochastic combination of their histone and DNA components, asymmetric nucleosome assembly demands the selective incorporation of a single modified/mutant histone copy alongside its wild-type counterpart.Herein we describe the chemical biology tools that we and others have developed in recent years for investigating nucleosome asymmetry. Such approaches, each with its own benefits and shortcomings, fall into five broad categories. First, we discuss affinity tag-based purification methods. These enable the assembly of theoretically any asymmetric nucleosome of interest but are frequently labor-intensive and suffer from low yields. Second, we detail transient cross-linking strategies that are amenable to the preparation of histone H3- or H4-modified/mutant asymmetric species. These yield asymmetric nucleosomes in a traceless fashion, albeit through the use of more complicated synthesis techniques. Third, we describe a synthetic biology technique based on the generation of bump-hole mutant H3 histones that selectively heterodimerize. Although currently developed only for H3 and a related isoform, this method uniquely allows for the interrogation of nucleosome asymmetry in yeast. Fourth, we outline a method for generating H2A- or H2B-modified/mutant asymmetric nucleosomes that relies on the differential DNA-histone contact strength inherent in the Widom 601 DNA sequence. This technique involves the initial formation of hexasomes which are then complemented with distinct H2A/H2B dimers. Finally, we review an approach that utilizes split intein technology to isolate asymmetric H2A- or H2B-modified/mutant nucleosomes. This method shares steps in common with the former but exploits tagged, intein-fused dimers for the facile purification of asymmetric products.Throughout the Account, we highlight various biological questions that drove the development of these methods and ultimately were answered by them. Though each technique has its own shortcomings, collectively these chemical biology tools provide a means to biochemically interrogate a plethora of asymmetric nucleosome species. We conclude with a discussion of remaining challenges, particularly that of endogenous asymmetric nucleosome detection.
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Nucleosomas/metabolismo , Marcadores de Afinidad , ADN/metabolismo , Histonas/metabolismo , Nucleosomas/químicaRESUMEN
ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.
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Cromatina/metabolismo , Histonas/biosíntesis , ADP-Ribosilación , Cromatina/química , Histonas/química , Conformación MolecularRESUMEN
The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.