RESUMEN
The field of 3D genome organization produces large amounts of sequencing data from Hi-C and a rapidly-expanding set of other chromosome conformation protocols (3C+). Massive and heterogeneous 3C+ data require high-performance and flexible processing of sequenced reads into contact pairs. To meet these challenges, we present pairtools-a flexible suite of tools for contact extraction from sequencing data. Pairtools provides modular command-line interface (CLI) tools that can be flexibly chained into data processing pipelines. The core operations provided by pairtools are parsing of.sam alignments into Hi-C pairs, sorting and removal of PCR duplicates. In addition, pairtools provides auxiliary tools for building feature-rich 3C+ pipelines, including contact pair manipulation, filtration, and quality control. Benchmarking pairtools against popular 3C+ data pipelines shows advantages of pairtools for high-performance and flexible 3C+ analysis. Finally, pairtools provides protocol-specific tools for restriction-based protocols, haplotype-resolved contacts, and single-cell Hi-C. The combination of CLI tools and tight integration with Python data analysis libraries makes pairtools a versatile foundation for a broad range of 3C+ pipelines.
Asunto(s)
Cromosomas , Biología Computacional , Programas Informáticos , Cromosomas/genética , Cromosomas/química , Biología Computacional/métodos , Humanos , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mapeo Cromosómico/métodosRESUMEN
Chromosome conformation capture (3C) technologies reveal the incredible complexity of genome organization. Maps of increasing size, depth, and resolution are now used to probe genome architecture across cell states, types, and organisms. Larger datasets add challenges at each step of computational analysis, from storage and memory constraints to researchers' time; however, analysis tools that meet these increased resource demands have not kept pace. Furthermore, existing tools offer limited support for customizing analysis for specific use cases or new biology. Here we introduce cooltools (https://github.com/open2c/cooltools), a suite of computational tools that enables flexible, scalable, and reproducible analysis of high-resolution contact frequency data. Cooltools leverages the widely-adopted cooler format which handles storage and access for high-resolution datasets. Cooltools provides a paired command line interface (CLI) and Python application programming interface (API), which respectively facilitate workflows on high-performance computing clusters and in interactive analysis environments. In short, cooltools enables the effective use of the latest and largest genome folding datasets.
Asunto(s)
Biología Computacional , Programas Informáticos , Biología Computacional/métodos , Lenguajes de Programación , Genómica/métodos , Genoma/genética , Mapeo Cromosómico/métodos , HumanosRESUMEN
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Asunto(s)
Cromosomas , ADN-Topoisomerasas de Tipo II , ADN-Topoisomerasas de Tipo II/genética , Cromosomas/genética , Mitosis/genética , Interfase/genética , PolímerosRESUMEN
Whole chromosome and arm-level copy number alterations occur at high frequencies in tumors, but their selective advantages, if any, are poorly understood. Here, utilizing unbiased whole chromosome genetic screens combined with in vitro evolution to generate arm- and subarm-level events, we iteratively selected the fittest karyotypes from aneuploidized human renal and mammary epithelial cells. Proliferation-based karyotype selection in these epithelial lines modeled tissue-specific tumor aneuploidy patterns in patient cohorts in the absence of driver mutations. Hi-C-based translocation mapping revealed that arm-level events usually emerged in multiples of two via centromeric translocations and occurred more frequently in tetraploids than diploids, contributing to the increased diversity in evolving tetraploid populations. Isogenic clonal lineages enabled elucidation of pro-tumorigenic mechanisms associated with common copy number alterations, revealing Notch signaling potentiation as a driver of 1q gain in breast cancer. We propose that intrinsic, tissue-specific proliferative effects underlie tumor copy number patterns in cancer.
Asunto(s)
Aneuploidia , Humanos , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Variaciones en el Número de Copia de ADN , Neoplasias/genética , Neoplasias/patología , Translocación Genética , Evolución Molecular , Proliferación Celular/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Especificidad de Órganos/genética , Células Epiteliales/metabolismo , Células Epiteliales/patologíaRESUMEN
MOTIVATION: Genomic intervals are one of the most prevalent data structures in computational genome biology, and used to represent features ranging from genes, to DNA binding sites, to disease variants. Operations on genomic intervals provide a language for asking questions about relationships between features. While there are excellent interval arithmetic tools for the command line, they are not smoothly integrated into Python, one of the most popular general-purpose computational and visualization environments. RESULTS: Bioframe is a library to enable flexible and performant operations on genomic interval dataframes in Python. Bioframe extends the Python data science stack to use cases for computational genome biology by building directly on top of two of the most commonly-used Python libraries, NumPy and Pandas. The bioframe API enables flexible name and column orders, and decouples operations from data formats to avoid unnecessary conversions, a common scourge for bioinformaticians. Bioframe achieves these goals while maintaining high performance and a rich set of features. AVAILABILITY AND IMPLEMENTATION: Bioframe is open-source under MIT license, cross-platform, and can be installed from the Python Package Index. The source code is maintained by Open2C on GitHub at https://github.com/open2c/bioframe.
Asunto(s)
Biología Computacional , Genómica , Biblioteca de Genes , Sitios de Unión , Ciencia de los DatosRESUMEN
The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.
RESUMEN
The field of 3D genome organization produces large amounts of sequencing data from Hi-C and a rapidly-expanding set of other chromosome conformation protocols (3C+). Massive and heterogeneous 3C+ data require high-performance and flexible processing of sequenced reads into contact pairs. To meet these challenges, we present pairtools - a flexible suite of tools for contact extraction from sequencing data. Pairtools provides modular command-line interface (CLI) tools that can be flexibly chained into data processing pipelines. Pairtools provides both crucial core tools as well as auxiliary tools for building feature-rich 3C+ pipelines, including contact pair manipulation, filtration, and quality control. Benchmarking pairtools against popular 3C+ data pipelines shows advantages of pairtools for high-performance and flexible 3C+ analysis. Finally, pairtools provides protocol-specific tools for multi-way contacts, haplotype-resolved contacts, and single-cell Hi-C. The combination of CLI tools and tight integration with Python data analysis libraries makes pairtools a versatile foundation for a broad range of 3C+ pipelines.
RESUMEN
Cohesin-mediated loop extrusion has been shown to be blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading, possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disrupting this traffic pattern, by removing CTCF, renders cells sensitive to knockout of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Factor de Unión a CCCTC/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , CohesinasRESUMEN
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
Asunto(s)
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromosómicas no Histona , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Humanos , Origen de Réplica/genética , Fase S , CohesinasRESUMEN
Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.
Asunto(s)
Cromatina/química , Cromosomas Humanos/química , Reactivos de Enlaces Cruzados/química , Técnicas Genéticas , Línea Celular , Cromatina/metabolismo , Bases de Datos Factuales , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/fisiología , HumanosRESUMEN
Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Cromosomas Humanos/química , Compartimento Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos/metabolismo , Semivida , Humanos , Células K562 , Cinética , CohesinasRESUMEN
The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Ratones Transgénicos , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteínas Nucleares/genética , Ftalazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Células U937 , Ensayos Antitumor por Modelo de Xenoinjerto , CohesinasRESUMEN
Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.
Asunto(s)
Momento de Replicación del ADN , Replicación del ADN , Regiones Promotoras Genéticas , Actinas/genética , Sitios de Unión , Cromatina , Cromosomas , Análisis por Conglomerados , Epigenómica , Humanos , Origen de Réplica , Globinas beta/genéticaRESUMEN
Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of â¼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.
Asunto(s)
Cromosomas Humanos/ultraestructura , Animales , Factor de Unión a CCCTC/metabolismo , Células Cultivadas , Cromatina/química , Cromosomas de los Mamíferos/ultraestructura , Células Madre Embrionarias/citología , Fibroblastos/citología , Humanos , Masculino , Mamíferos/genética , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Relación Señal-RuidoRESUMEN
Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF-CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Telofase , Adenosina Trifosfatasas/metabolismo , Compartimento Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Cromatina/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Citocinesis/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Células HeLa , Humanos , Interfase , Complejos Multiproteicos/metabolismo , Fase S , CohesinasRESUMEN
Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal-to-C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y-derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.
Asunto(s)
Asparagina/metabolismo , Retículo Endoplásmico/metabolismo , Glicoproteínas/metabolismo , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicoproteínas/genética , Glicosilación , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hexosiltransferasas/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.
Asunto(s)
Glicoproteínas/metabolismo , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteómica , Glicosilación , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , HumanosRESUMEN
The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.
Asunto(s)
Aptitud Genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Mutación Silenciosa , Sustitución de Aminoácidos , Animales , Perros , Evolución Molecular , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Modelos Moleculares , Filogenia , Pliegue del ARN , Porcinos , Replicación ViralRESUMEN
Prokaryotes evolved to thrive in an extremely diverse set of habitats, and their proteomes bear signatures of environmental conditions. Although correlations between amino acid usage and environmental temperature are well-documented, understanding of the mechanisms of thermal adaptation remains incomplete. Here, we couple the energetic costs of protein folding and protein homeostasis to build a microscopic model explaining both the overall amino acid composition and its temperature trends. Low biosynthesis costs lead to low diversity of physical interactions between amino acid residues, which in turn makes proteins less stable and drives up chaperone activity to maintain appropriate levels of folded, functional proteins. Assuming that the cost of chaperone activity is proportional to the fraction of unfolded client proteins, we simulated thermal adaptation of model proteins subject to minimization of the total cost of amino acid synthesis and chaperone activity. For the first time, we predicted both the proteome-average amino acid abundances and their temperature trends simultaneously, and found strong correlations between model predictions and 402 genomes of bacteria and archaea. The energetic constraint on protein evolution is more apparent in highly expressed proteins, selected by codon adaptation index. We found that in bacteria, highly expressed proteins are similar in composition to thermophilic ones, whereas in archaea no correlation between predicted expression level and thermostability was observed. At the same time, thermal adaptations of highly expressed proteins in bacteria and archaea are nearly identical, suggesting that universal energetic constraints prevail over the phylogenetic differences between these domains of life.
Asunto(s)
Adaptación Fisiológica/fisiología , Células Procariotas/metabolismo , Proteostasis/fisiología , Aclimatación/genética , Aclimatación/fisiología , Adaptación Fisiológica/genética , Aminoácidos/genética , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Evolución Biológica , Codón/metabolismo , Simulación por Computador , Evolución Molecular , Calor , Filogenia , Células Procariotas/fisiología , Proteoma/genética , TemperaturaRESUMEN
Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long-range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence-structure-fitness relationships in other common motifs.