RESUMO
Transcriptional regulation controls cellular functions through interactions between transcription factors (TFs) and their chromosomal targets. However, understanding the fate conversion potential of multiple TFs in an inducible manner remains limited. Here, we introduce iTF-seq as a method for identifying individual TFs that can alter cell fate toward specific lineages at a single-cell level. iTF-seq enables time course monitoring of transcriptome changes, and with biotinylated individual TFs, it provides a multi-omics approach to understanding the mechanisms behind TF-mediated cell fate changes. Our iTF-seq study in mouse embryonic stem cells identified multiple TFs that trigger rapid transcriptome changes indicative of differentiation within a day of induction. Moreover, cells expressing these potent TFs often show a slower cell cycle and increased cell death. Further analysis using bioChIP-seq revealed that GCM1 and OTX2 act as pioneer factors and activators by increasing gene accessibility and activating the expression of lineage specification genes during cell fate conversion. iTF-seq has utility in both mapping cell fate conversion and understanding cell fate conversion mechanisms.
Assuntos
Diferenciação Celular , Fatores de Transcrição , Animais , Camundongos , Diferenciação Celular/genética , Linhagem da Célula/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Multiômica , RNA Citoplasmático Pequeno/genética , RNA Citoplasmático Pequeno/metabolismo , RNA-Seq/métodos , Análise de Sequência de RNA/métodos , Análise da Expressão Gênica de Célula Única , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , TranscriptomaRESUMO
Although cell density is known to affect numerous biological processes including gene expression and cell fate specification, mechanistic understanding of what factors link cell density to global gene regulation is lacking. Here, we reveal that the expression of thousands of genes in mouse embryonic stem cells (mESCs) is affected by cell seeding density and that low cell density enhances the efficiency of differentiation. Mechanistically, ß-catenin is localized primarily to adherens junctions during both self-renewal and differentiation at high density. However, when mESCs differentiate at low density, ß-catenin translocates to the nucleus and associates with Tcf7l1, inducing co-occupied lineage markers. Meanwhile, Esrrb sustains the expression of pluripotency-associated genes while repressing lineage markers at high density, and its association with DNA decreases at low density. Our results provide new insights into the previously neglected but pervasive phenomenon of density-dependent gene regulation.
RESUMO
Recent genomic data analyses have revealed important underlying logics in eukaryotic gene regulation, such as CpG islands (CGIs)-dependent dual-mode gene regulation. In mammals, genes lacking CGIs at their promoters are generally regulated by interconversion between euchromatin and heterochromatin, while genes associated with CGIs constitutively remain as euchromatin. Whether a similar mode of gene regulation exists in non-mammalian species has been unknown. Here, through comparative epigenomic analyses, we demonstrate that the dual-mode gene regulation program is common in various eukaryotes, even in the species lacking CGIs. In cases of vertebrates or plants, we find that genes associated with high methylation level promoters are inactivated by forming heterochromatin and expressed in a context-dependent manner. In contrast, the genes with low methylation level promoters are broadly expressed and remain as euchromatin even when repressed by Polycomb proteins. Furthermore, we show that invertebrate animals lacking DNA methylation, such as fruit flies and nematodes, also have divergence in gene types: some genes are regulated by Polycomb proteins, while others are regulated by heterochromatin formation. Altogether, our study establishes gene type divergence and the resulting dual-mode gene regulation as fundamental features shared in a broad range of higher eukaryotic species.
Assuntos
Regulação da Expressão Gênica , Animais , Caenorhabditis elegans/genética , Ilhas de CpG , Metilação de DNA , Drosophila melanogaster/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Transcrição Gênica , Vertebrados/genéticaRESUMO
Hippo effectors YAP and TAZ control cell fate and survival through various mechanisms, including transcriptional regulation of key genes. However, much of this research has been marked by conflicting results, as well as controversy over whether YAP and TAZ are redundant. A substantial portion of the discordance stems from their contradictory roles in stem cell self-renewal vs. differentiation and cancer cell survival vs. apoptosis. In this review, we present an overview of the multiple context-dependent functions of YAP and TAZ in regulating cell fate decisions in stem cells and organoids, as well as their mechanisms of controlling programmed cell death pathways in cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias/patologia , Células-Tronco/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular , Desenvolvimento Embrionário/genética , Humanos , Neoplasias/metabolismo , RNA não Traduzido/metabolismo , Células-Tronco/citologia , Proteínas de Sinalização YAPRESUMO
Science communication and outreach are essential for training the next generation of scientists and raising public awareness for science. Providing effective science, technology, engineering, and mathematics (STEM) educational outreach to students in classrooms is challenging because of the need to form partnerships with teachers, the time commitment required for the presenting scientist, and the limited class time allotted for presentations. In our Present Your Ph.D. Thesis to a 12-Year Old outreach project, our novel solution to this problem is hosting a youth science workshop (YSW) on our university campus. The YSW is an interpersonal science communication and outreach experience in which graduate students from diverse scientific disciplines introduce middle and high school students to their cutting-edge research and mentor them to develop a white-board presentation to communicate the research to the workshop audience. Our assessment of the YSW indicated that participating young students expressed significantly more positive attitudes toward science and increased motivation to work in a STEM career after attending the workshop. Qualitative follow-up interviews with participating graduate students' show that even with minimal time commitment, an impactful science communication training experience can be achieved. The YSW is a low-cost, high-reward educational outreach event amenable to all disciplines of science. It enhances interest and support of basic science research while providing opportunities for graduate students to engage with the public, improve their science communication skills, and enhance public understanding of science. This YSW model can be easily implemented at other higher education institutions to globally enhance science outreach initiatives.
Assuntos
Relações Comunidade-Instituição , Tutoria/métodos , Ciência/educação , Estudantes , Comunicação , Humanos , Tutoria/estatística & dados numéricos , Modelos Educacionais , Motivação , Avaliação de Programas e Projetos de Saúde , Estudantes/psicologia , Estudantes/estatística & dados numéricos , Inquéritos e QuestionáriosRESUMO
Trophectoderm (TE) lineage development is pivotal for proper implantation, placentation, and healthy pregnancy. However, only a few TE-specific transcription factors (TFs) have been systematically characterized, hindering our understanding of the process. To elucidate regulatory mechanisms underlying TE development, here we map super-enhancers (SEs) in trophoblast stem cells (TSCs) as a model. We find both prominent TE-specific master TFs (Cdx2, Gata3, and Tead4), and >150 TFs that had not been previously implicated in TE lineage, that are SE-associated. Mapping targets of 27 SE-predicted TFs reveals a highly intertwined transcriptional regulatory circuitry. Intriguingly, SE-predicted TFs show 4 distinct expression patterns with dynamic alterations of their targets during TSC differentiation. Furthermore, depletion of a subset of TFs results in dysregulation of the markers for specialized cell types in placenta, suggesting a role during TE differentiation. Collectively, we characterize an expanded TE-specific regulatory network, providing a framework for understanding TE lineage development and placentation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Trofoblastos/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Camundongos , Placentação/genética , Gravidez , Fatores de Transcrição/genética , Trofoblastos/citologiaRESUMO
Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.
Assuntos
Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Animais , Sistemas CRISPR-Cas , Ciclo Celular/genética , Proliferação de Células , Reprogramação Celular , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Approximately, 30% of embryonic stem cells (ESCs) die after exiting self-renewal, but regulators of this process are not well known. Yap1 is a Hippo pathway transcriptional effector that plays numerous roles in development and cancer. However, its functions in ESC differentiation remain poorly characterized. We first reveal that ESCs lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently show that Yap1 contextually protects differentiating, but not self-renewing, ESC from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via cis-regulatory elements while mildly suppressing pro-apoptotic genes, which moderates the level of mitochondrial priming that occurs during differentiation. Individually modulating the expression of single apoptosis-related genes targeted by Yap1 is sufficient to augment or hinder survival during differentiation. Our demonstration of the context-dependent pro-survival functions of Yap1 during ESC differentiation contributes to our understanding of the balance between survival and death during cell fate changes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Autorrenovação Celular , Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Mutação , Fosfoproteínas/metabolismo , Proteínas de Sinalização YAPRESUMO
We present the complete genome sequences of four phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from beehives and beeswax products from Las Vegas, Nevada. The genomes are 50 to 55 kbp long and use the "direct terminal repeats" DNA-packaging strategy.
RESUMO
CpG islands (CGIs) have long been implicated in the regulation of vertebrate gene expression. However, the involvement of CGIs in chromosomal architectures and associated gene expression regulations has not yet been thoroughly explored. By combining large-scale integrative data analyses and experimental validations, we show that CGIs clearly reconcile two competing models explaining nuclear gene localizations. We first identify CGI-containing (CGI+) and CGI-less (CGI-) genes are non-randomly clustered within the genome, which reflects CGI-dependent spatial gene segregation in the nucleus and corresponding gene regulatory modes. Regardless of their transcriptional activities, CGI+ genes are mainly located at the nuclear center and encounter frequent long-range chromosomal interactions. Meanwhile, nuclear peripheral CGI- genes forming heterochromatin are activated and internalized into the nuclear center by local enhancer-promoter interactions. Our findings demonstrate the crucial implications of CGIs on chromosomal architectures and gene positioning, linking the critical importance of CGIs in determining distinct mechanisms of global gene regulation in three-dimensional space in the nucleus.
Assuntos
Cromossomos de Mamíferos/química , Ilhas de CpG , Regulação da Expressão Gênica , Animais , Linhagem Celular , Núcleo Celular/genética , Cromatina/química , Camundongos , Células NIH 3T3 , Transcrição GênicaRESUMO
During early development in placental mammals, proper trophoblast lineage development is essential for implantation and placentation. Defects in this lineage can cause early pregnancy failures and other pregnancy disorders. However, transcription factors controlling trophoblast development remain poorly understood. Here, we utilize Fosl1, previously implicated in trophoblast giant cell development as a member of the AP-1 complex, to trans-differentiate embryonic stem (ES) cells to trophoblast lineage-like cells. We first show that the ectopic expression of Fosl1 is sufficient to induce trophoblast-specific gene expression programs in ES cells. Surprisingly, we find that this transcriptional reprogramming occurs independently of changes in levels of ES cell core factors during the cell fate change. This suggests that Fosl1 acts in a novel way to orchestrate the ES to trophoblast cell fate conversion compared to previously known reprogramming factors. Mapping of Fosl1 targets reveals that Fosl1 directly activates TE lineage-specific genes as a pioneer factor. Our work suggests Fosl1 may be used to reprogram ES cells into differentiated cell types in trophoblast lineage, which not only enhances our knowledge of global trophoblast gene regulation but also may provide a future therapeutic tool for generating induced trophoblast cells from patient-derived pluripotent stem cells.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Trofoblastos/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Feminino , Camundongos , Células-Tronco Pluripotentes/citologia , Gravidez , Proteínas Proto-Oncogênicas c-fos/genética , Trofoblastos/citologiaRESUMO
Direct reprogramming can be achieved by forced expression of master transcription factors. Yet how such factors mediate repression of initial cell-type-specific genes while activating target cell-type-specific genes is unclear. Through embryonic stem (ES) to trophoblast stem (TS)-like cell reprogramming by introducing individual TS cell-specific 'CAG' factors (Cdx2, Arid3a and Gata3), we interrogate their chromosomal target occupancies, modulation of global transcription and chromatin accessibility at the initial stage of reprogramming. From the studies, we uncover a sequential, two-step mechanism of cellular reprogramming in which repression of pre-existing ES cell-associated gene expression program is followed by activation of TS cell-specific genes by CAG factors. Therefore, we reveal that CAG factors function as both decommission and pioneer factors during ES to TS-like cell fate conversion.
Assuntos
Fator de Transcrição CDX2/fisiologia , Técnicas de Reprogramação Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/citologia , Fator de Transcrição GATA3/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/fisiologia , Trofoblastos/citologia , Animais , Fator de Transcrição CDX2/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Meios de Cultivo Condicionados , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Fibroblastos , Fator de Transcrição GATA3/genética , Ontologia Genética , Código das Histonas , Camundongos , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38-45 kb in size and contain 68-86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the "cohesive ends with 3' overhang" DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area.
RESUMO
We present here the complete genome sequences of nine phages that infect Paenibacillus larvae, the causative agent of American foulbrood disease in honeybees. The phages were isolated from soil, propolis, and infected bees from three U.S. states. This is the largest number of P. larvae phage genomes sequenced in a single publication to date.
RESUMO
American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed.
Assuntos
Bacteriófagos/fisiologia , Abelhas/microbiologia , Paenibacillus/fisiologia , Animais , Abelhas/crescimento & desenvolvimento , Abelhas/virologia , Larva/microbiologia , Larva/virologia , Paenibacillus/virologiaRESUMO
Paenibacillus larvae is the causative agent of American foulbrood (AFB) disease which affects early larval stages during honeybee development. Due to its virulence, transmissibility, capacity to develop antibiotic resistance, and the inherent resilience of its endospores, Paenibacillus larvae is extremely difficult to eradicate from infected hives which often must be burned. AFB contributes to the worldwide decline of honeybee populations, which are crucial for pollination and the food supply. We have isolated a novel bacteriophage lysin, PlyPalA, from the genome of a novel Paenibacillus larvae bacteriophage originally extracted from an environmental sample. PlyPalA has an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and possesses lytic activity against infectious strains of Paenibacillus larvae without harming commensal bacteria known to compose the honeybee larval microbiota. A single dose of PlyPalA rescued 75% of larvae infected with endospores, showing that it represents a powerful tool for future treatment of AFB. This represents the first time that lysins have been tested for therapeutic use in invertebrates.