RESUMEN
Humans can flexibly use metacognition to monitor their own knowledge and strategically acquire new information when needed. While humans can deploy these skills across a variety of contexts, most evidence for metacognition in animals has focused on simple situations, such as seeking out information about the location of food. Here, we examine the flexibility, breadth, and limits of this skill in chimpanzees. We tested semi-free-ranging chimpanzees on a novel task where they could seek information by standing up to peer into different containers. In Study 1, we tested n = 47 chimpanzees to assess if chimpanzees would spontaneously engage in information-seeking without prior experience, as well as to characterize individual variation in this propensity. We found that many chimpanzees engaged in information-seeking with minimal experience, and that younger chimpanzees and females were more likely to do so. In two subsequent studies, we then further tested chimpanzees who initially showed robust information-seeking on new variations of this task, to disentangle the cognitive processing shaping their behaviors. In Study 2, we examined how a subset of n = 12 chimpanzees applied these skills to seek information about the location versus the identity of rewards, and found that chimpanzees were equally adept at seeking out location and identity information. In Study 3, we examined whether a subset of n = 6 chimpanzees could apply these skills to make more efficacious decisions when faced with uncertainty about reward payoffs. Chimpanzees were able to use information-seeking to resolve risk and choose more optimally when faced with uncertain payoffs, although they often also engaged in information-seeking when it was not strictly necessary. These results identify core features of flexible metacognition that chimpanzees share with humans, as well as constraints that may represent key evolutionary shifts in human cognition.
Asunto(s)
Conducta en la Búsqueda de Información , Pan troglodytes , Animales , Femenino , Masculino , Conducta en la Búsqueda de Información/fisiología , Metacognición/fisiología , Conducta Animal/fisiología , RecompensaRESUMEN
Though common among humans, social play by adults is an uncommon occurrence in most animals, even between parents and offspring.1,2,3 The most common explanation for why adult play is so rare is that its function and benefits are largely limited to development, so that social play has little value later in life.3,4,5,6 Here, we draw from 10 years of behavioral data collected by the Kibale Chimpanzee Project to consider an alternative hypothesis: that despite its benefits, adult play in non-humans is ecologically constrained by energy shortage or time limitations. We further hypothesized that, since they may be the only available partners for their young offspring, mother chimpanzees pay greater costs of play than other adults. Our analysis of nearly 4,000 adult play bouts revealed that adult chimpanzees played both among themselves and with immature partners. Social play was infrequent when diet quality was low but increased with the proportion of high-quality fruits in the diet. This suggests that adults engage in play facultatively when they have more energy and/or time to do so. However, when diet quality was low and most adult play fell to near zero, play persisted between mothers and offspring. Increased use of play by adult chimpanzees during periods of resource abundance suggests that play retains value as a social currency beyond development but that its costs constrain its use. At the same time, when ecological conditions constrain opportunities for young to play, play by mothers fills a critical role to promote healthy offspring development.
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Hominidae , Pan troglodytes , Animales , Femenino , Humanos , Dieta , Conducta Animal , Madres , Conducta SocialRESUMEN
Undergraduate research and laboratory experiences provide a wide range of benefits to student learning in science and are integral to imbed authentic research experiences in biology labs. While the benefit of courses with research experience is widely accepted, it can be challenging to measure conceptual research skills in a quick and easily scalable manner. We developed a card-sorting task to differentiate between novice and expert conceptualization of research principles. There were significant differences in the way faculty/postdocs, graduate students, and undergraduate students organized their information, with faculty/postdocs more likely to use deep feature sorting patterns related to research approach. When provided scaffolding of group names reflecting expert-like organization, participant groups were better able to sort by that organization, but undergraduate students did not reach expert levels. Undergraduates with Advanced Placement experience were more likely to display expert-like thinking than undergraduates without Advanced Placement Biology experience and non-PEER (persons excluded because of their Ethnicity or Race) students displayed more expert-like thinking than PEER students. We found evidence of undergraduates in various stages of development toward expert-like thinking in written responses. This card-sorting task can provide a framework for analyzing student's conceptualizations of research and identify areas to provide added scaffolding to help shift from novice-like to expert-like thinking.
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Formación de Concepto , Estudiantes , Humanos , Etnicidad , Docentes , LaboratoriosRESUMEN
Pathogen surveillance for great ape health monitoring has typically been performed on non-invasive samples, primarily feces, in wild apes and blood in sanctuary-housed apes. However, many important primate pathogens, including known zoonoses, are shed in saliva and transmitted via oral fluids. Using metagenomic methods, we identified viruses in saliva samples from 46 wild-born, sanctuary-housed chimpanzees at two African sanctuaries in Republic of Congo and Uganda. In total, we identified 20 viruses. All but one, an unclassified CRESS DNA virus, are classified in five families: Circoviridae, Herpesviridae, Papillomaviridae, Picobirnaviridae, and Retroviridae. Overall, viral prevalence ranged from 4.2% to 87.5%. Many of these viruses are ubiquitous in primates and known to replicate in the oral cavity (simian foamy viruses, Retroviridae; a cytomegalovirus and lymphocryptovirus; Herpesviridae; and alpha and gamma papillomaviruses, Papillomaviridae). None of the viruses identified have been shown to cause disease in chimpanzees or, to our knowledge, in humans. These data suggest that the risk of zoonotic viral disease from chimpanzee oral fluids in sanctuaries may be lower than commonly assumed.
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Pan troglodytes , Saliva , Animales , Humanos , Congo , Uganda , Zoonosis/epidemiología , RetroviridaeRESUMEN
Course-based undergraduate research experiences (CUREs) rapidly have become more common in biology laboratory courses. The effort to implement CUREs has stimulated attempts to differentiate CUREs from other types of laboratory teaching. The Laboratory Course Assessment Survey (LCAS) was developed to measure students' perceptions of how frequently they participate in activities related to iteration, discovery, broader relevance, and collaboration in their laboratory courses. The LCAS has been proposed as an instrument that can be used to define whether a laboratory course fits the criteria for a CURE or not. However, the threshold LCAS scores needed to define a course as a CURE are unclear. As a result, we examined variation in published LCAS scores among different laboratory course types. In addition, we examined the distribution of LCAS scores for students enrolled in our research-for-credit course. Overall, we found substantial variation in scores among CUREs and broad overlap among course types in scores related to all three scales measured by the LCAS. Furthermore, the mean LCAS scores for all course types fell within the main part of the distribution of scores for our mentored research students. These results suggest that the LCAS cannot be used to easily quantify whether a course is a CURE or not. We propose that the biology education community needs to move beyond trying to quantitatively identify whether a course is a CURE. Instead, we should use tools like the LCAS to investigate what students are actually doing in their laboratory courses and how those activities impact student outcomes.
RESUMEN
Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild-born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long-term conservation strategies. We used metagenomic next-generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other "atypical" or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas-Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector-borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood-borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.
Asunto(s)
Pan troglodytes , Virosis , Animales , África/epidemiología , Pan troglodytes/virología , Virosis/epidemiología , Virosis/veterinaria , Virosis/virología , Animales de Zoológico/virologíaRESUMEN
Student self-perception is related to persistence in science. Yet how self-perception develops over time is less clear. We examined student self-perception trajectories and their relationship with gender, persons excluded due to ethnicity or race (PEER) status, and first-generation college student (FGCS) status across a yearlong introductory biology sequence. While we found similar rates of change in self-efficacy and science identity for all groups, females and PEER students had lower initial scores that failed to "catch up" to male and non-PEER scores by the end of the year. Students grouped into either high and stable or lower and decreasing trajectories for scientific community values, with first-generation college students overrepresented in the latter group. Additionally, we found no evidence for intersectionality of subgroups. We did find evidence that the relationship between gender and PEER status and science identity is likely mediated via self-efficacy. Taken together, our results suggest that introductory biology students develop self-efficacy and science identity at similar rates regardless of gender, PEER status, or FGCS status and that interventions targeting scientific community values for all students and self-efficacy of female and PEER students may be fruitful areas to pursue to increase persistence of students in the sciences and to reduce score differences between groups.
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Autoimagen , Estudiantes , Biología/educación , Etnicidad , Femenino , Humanos , Masculino , UniversidadesRESUMEN
Calls for early exposure of all undergraduates to research have led to the increased use and study of course-based research experiences (CREs). CREs have been shown to increase measures of persistence in the sciences, such as science identity, scientific self-efficacy, project ownership, scientific community values, and networking. However, implementing CREs can be challenging and resource-intensive. These barriers may be partly mitigated by the use of short-term CRE modules rather than semester- or year-long projects. One study has shown that a CRE module captures some of the known benefits of CREs as measured by the Persistence in the Sciences (PITS) survey. Here, we used this same survey to assess outcomes for introductory biology students who completed a semester of modular CREs based on faculty research at an R1 university. The results indicated levels of self-efficacy, science community values, and science identity similar to those previously reported for students in the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) full-semester CRE. Scores for project ownership (content) were between previously reported traditional lab and CRE scores, while project ownership (emotion) and networking were similar to those of traditional labs. Our results suggest that modular CREs can lead to significant gains in student affect measures that have been linked to persistence in the sciences in other studies. Although gains were not as great in all measures as with a semester-long CRE, implementation of modular CREs may be more feasible and offers the added benefits of exposing students to diverse research fields and lab techniques.
RESUMEN
Chimpanzees (Pan troglodytes) are a crucial model for understanding the evolution of human health and longevity. Cardiovascular disease is a major source of mortality during ageing in humans and therefore a key issue for comparative research. Current data indicate that compared to humans, chimpanzees have proatherogenic blood lipid profiles, an important risk factor for cardiovascular disease in humans. However, most work to date on chimpanzee lipids come from laboratory-living populations where lifestyles diverge from a wild context. Here, we examined cardiovascular profiles in chimpanzees living in African sanctuaries, who range semi-free in large forested enclosures, consume a naturalistic diet, and generally experience conditions more similar to a wild chimpanzee lifestyle. We measured blood lipids, body weight and body fat in 75 sanctuary chimpanzees and compared them to publicly available data from laboratory-living chimpanzees from the Primate Aging Database. We found that semi-free-ranging chimpanzees exhibited lower body weight and lower levels of lipids that are risk factors for human cardiovascular disease, and that some of these disparities increased with age. Our findings support the hypothesis that lifestyle can shape health indices in chimpanzees, similar to effects observed across human populations, and contribute to an emerging understanding of human cardiovascular health in an evolutionary context. This article is part of the theme issue 'Evolution of the primate ageing process'.
Asunto(s)
Tejido Adiposo/metabolismo , Biomarcadores , Peso Corporal , Lípidos/sangre , Longevidad , Pan troglodytes/fisiología , Animales , Animales Salvajes/fisiología , Animales de Zoológico/fisiología , Enfermedades Cardiovasculares , Sistema Cardiovascular/química , Congo , Femenino , Estado de Salud , Humanos , Masculino , Modelos Animales , Factores de RiesgoRESUMEN
Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu's substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.
Asunto(s)
Aminoácidos/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Ingeniería de ProteínasRESUMEN
Rationally engineering thermostability in proteins would create enzymes and receptors that function under harsh industrial applications. Several sequence-based approaches can generate thermostable variants of mesophilic proteins. To gain insight into the mechanisms by which proteins become more stable, we use structural and dynamic analyses to compare two popular approaches, ancestral sequence reconstruction (ASR) and the consensus method, used to generate thermostable variants of Elongation Factor Thermo-unstable (EF-Tu). We present crystal structures of ancestral and consensus EF-Tus, accompanied by molecular dynamics simulations aimed at probing the strategies employed to enhance thermostability. All proteins adopt crystal structures similar to extant EF-Tus, revealing no difference in average structure between the methods. Molecular dynamics reveals that ASR-generated sequences retain dynamic properties similar to extant, thermostable EF-Tu from Thermus aquaticus, while consensus EF-Tu dynamics differ from evolution-based sequences. This work highlights the advantage of ASR for engineering thermostability while preserving natural motions in multidomain proteins.
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Proteínas Bacterianas/química , Escherichia coli/metabolismo , Guanosina Difosfato/química , Factor Tu de Elongación Peptídica/química , Ingeniería de Proteínas , Thermus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , Cristalografía por Rayos X , Escherichia coli/clasificación , Escherichia coli/genética , Evolución Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Guanosina Difosfato/metabolismo , Simulación de Dinámica Molecular , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Thermus/clasificación , Thermus/genéticaRESUMEN
Reconstructing Evolutionary Adaptive Paths (REAP) is one of several methods to improve enzyme -functionality. This approach incorporates computational and theoretical aspects of protein engineering to create a focused library of protein variety with a high degree of functionality. In contrast to other -techniques like DNA shuffling, REAP allows a library to have diverse functionality among relatively few variants. REAP is a low-throughput method which takes advantage of natural selection and uses ancestral protein sequences to direct gene mutations, thereby creating a library with a high density of viable proteins. These proteins must then be assayed to characterize their functionality to identify which variants have the desired traits such as acid stability or thermostability.
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Ingeniería de Proteínas/métodos , Evolución Biológica , Enzimas/química , Enzimas/genética , Enzimas/metabolismoRESUMEN
Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system.
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Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Unión Proteica/genética , Conformación Proteica , Replegamiento Proteico , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Protein evolution relies on designing a library of sequences that capture meaningful functional diversity in a limited number of protein variants. Several approaches take advantage of the sequence space already explored through natural selection by incorporating sequence diversity available from modern genomes (and their ancestors) when designing these libraries. The success of these approaches is, partly, owing to the fact that modern sequence diversity has already been subjected to evolutionary selective forces and thus the diversity has already been deemed 'fit to survive'. Five of these approaches will be discussed in this review to highlight how protein engineers can use evolutionary sequence history/diversity of homologous proteins in unique ways to design protein libraries.
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Evolución Molecular , Ingeniería de Proteínas , Estabilidad Proteica , Proteínas/química , Proteínas/genética , Bacterias/química , Bacterias/genética , Biblioteca de Péptidos , Selección Genética , Homología de Secuencia de AminoácidoRESUMEN
Directed evolution and protein engineering approaches used to generate novel or enhanced biomolecular function often use the evolutionary sequence diversity of protein homologs to rationally guide library design. To fully capture this sequence diversity, however, libraries containing millions of variants are often necessary. Screening libraries of this size is often undesirable due to inaccuracies of high-throughput assays, costs, and time constraints. The ability to effectively cull sequence diversity while still generating the functional diversity within a library thus holds considerable value. This is particularly relevant when high-throughput assays are not amenable to select/screen for certain biomolecular properties. Here, we summarize our recent attempts to develop an evolution-guided approach, Reconstructing Evolutionary Adaptive Paths (REAP), for directed evolution and protein engineering that exploits phylogenetic and sequence analyses to identify amino acid substitutions that are likely to alter or enhance function of a protein. To demonstrate the utility of this technique, we highlight our previous work with DNA polymerases in which a REAP-designed small library was used to identify a DNA polymerase capable of accepting non-standard nucleosides. We anticipate that the REAP approach will be used in the future to facilitate the engineering of biopolymers with expanded functions and will thus have a significant impact on the developing field of 'evolutionary synthetic biology'.
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Evolución Molecular , Modelos Genéticos , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Barajamiento de ADN/métodos , ADN Polimerasa Dirigida por ADN/genética , Evolución Molecular Dirigida/métodos , Biblioteca de Genes , Variación Genética , Modelos Moleculares , Mutagénesis , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein.
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Ciclina D1/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteómica , Transcripción Genética , Alelos , Animales , Proteína de Unión a CREB/metabolismo , Inmunoprecipitación de Cromatina , Ciclina D1/deficiencia , Ciclina D1/genética , Genoma/genética , Ensayos Analíticos de Alto Rendimiento , Histona Acetiltransferasas/metabolismo , Espectrometría de Masas , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteómica/métodos , Ratas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Retina/citología , Retina/embriología , Retina/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.
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Células Madre Embrionarias/metabolismo , MicroARNs/genética , Transcripción Genética , Animales , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.
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Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción TCF/fisiología , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma , Técnicas para Inmunoenzimas , Lentivirus , Ratones , Proteína Homeótica Nanog , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1 , Transducción de Señal , Proteína 1 Similar al Factor de Transcripción 7 , Proteínas Wnt/metabolismoRESUMEN
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
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Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Complejos Multiproteicos , Proteínas de Neoplasias , Proteínas Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transducción de Señal/fisiología , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.