RESUMEN
The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Genoma/genética , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Cigoto , Animales , Cromatina/metabolismo , Metilación de ADN/genética , Desarrollo Embrionario/genética , Células Madre Embrionarias , Epigénesis Genética/genética , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Ratones , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genéticaRESUMEN
Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways, including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.
Asunto(s)
Genes Homeobox , Células Madre Pluripotentes , Animales , Humanos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Gástrula/citología , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilación , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Epigenoma/genética , Eritropoyesis , Análisis Factorial , Gástrula/embriología , Gastrulación/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/análisis , Factores de Tiempo , Dedos de ZincRESUMEN
DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.
Asunto(s)
Metilación de ADN , ADN Metiltransferasa 3A , Embarazo , Femenino , Animales , Ratones , Placentación , Placenta/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis GenéticaRESUMEN
Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.
Asunto(s)
Cromatina/ultraestructura , Regulación de la Expresión Génica/genética , Sitios Genéticos , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/genética , Ciclo Celular , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Cromatina/química , Proteínas Cromosómicas no Histona/fisiología , ADN/química , ADN/metabolismo , Humanos , Conformación de Ácido Nucleico , ARN Largo no Codificante , CohesinasRESUMEN
BACKGROUND: Robust analysis of DNA sequencing data needs to include a set of quality control steps to ensure that technical bias is kept to a minimum. A metric easily obtained is the frequency of each of the nucleobases for each position across all sequencing reads. Here, we explore the differences in nucleobase compositions of various library types produced by standard experimental methodologies. Methods: We obtained the compositions of nearly 3000 publicly available datasets and subjected them to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction for a two-dimensional representation of their composition characteristics. Results: We find that most library types result in a specific composition profile. We use this to give an estimate of how strongly the composition of a test library resembles the profiles of previously published libraries, and how likely the test sample is to be of a particular type. We introduce Librarian, a user-friendly web application and command line tool which enables checking base compositions of test libraries against known library types. Conclusions: Library preparation methods strongly influence the per position nucleobase content. By comparing test libraries to a database of previously published library types we can make predictions regarding the library preparation method. Librarian is a user-friendly tool to access this information for quality assurance purposes as discrepancies can flag potential irregularities very early on.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Bibliotecólogos , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biblioteca de Genes , Análisis de Secuencia de ADN/métodos , Control de CalidadRESUMEN
Monoallelic gene expression exposes an organism to the risks associated with the unmasking of recessive mutations. A recent study by Gimelbrant and colleagues, supported by results from two methodologically different studies, demonstrated that random monoallelic expression is surprisingly widespread among autosomal genes. This raises important questions about why, when and how cells choose and tolerate monoallelism and whether functional hemizygosity might provide an unappreciated advantage.
Asunto(s)
Desequilibrio Alélico/fisiología , Impresión Genómica/fisiología , Patrón de Herencia , Alelos , Animales , Evolución Molecular , Humanos , Modelos Biológicos , Factores de TiempoRESUMEN
The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.
Asunto(s)
Regulación de la Expresión Génica , Células Madre Pluripotentes/metabolismo , Cromatina/metabolismo , Metilación de ADN , Elementos de Facilitación Genéticos , Humanos , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFß/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFß signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFß signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFß signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFß pathway function in human pluripotency spanning a developmental window from naive to primed states.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Pluripotentes/fisiología , Transducción de Señal/fisiología , Proteína Smad2/genética , Proteína smad3/genética , Factor de Crecimiento Transformador beta/genética , Línea Celular , Reprogramación Celular , Humanos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
How the epigenetic landscape is established in development is still being elucidated. Here, we uncover developmental pluripotency associated 2 and 4 (DPPA2/4) as epigenetic priming factors that establish a permissive epigenetic landscape at a subset of developmentally important bivalent promoters characterized by low expression and poised RNA-polymerase. Differentiation assays reveal that Dppa2/4 double knockout mouse embryonic stem cells fail to exit pluripotency and differentiate efficiently. DPPA2/4 bind both H3K4me3-marked and bivalent gene promoters and associate with COMPASS- and Polycomb-bound chromatin. Comparing knockout and inducible knockdown systems, we find that acute depletion of DPPA2/4 results in rapid loss of H3K4me3 from key bivalent genes, while H3K27me3 is initially more stable but lost following extended culture. Consequently, upon DPPA2/4 depletion, these promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel epigenetic priming mechanism at developmental promoters, poising them for future lineage-specific activation.
Asunto(s)
Dipeptidil Peptidasa 4/genética , Epigénesis Genética , Células Madre Embrionarias de Ratones/citología , Factores de Transcripción/genética , Animales , Diferenciación Celular , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Dipeptidil Peptidasa 4/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Histonas/genética , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In C. elegans, expression of the UPRER transcription factor xbp-1s in neurons cell non-autonomously activates the UPRER in the intestine, leading to enhanced proteostasis and lifespan. To better understand this signaling pathway, we isolated neurons from animals expressing neuronal xbp-1s for transcriptomic analysis, revealing a striking remodeling of transcripts involved in neuronal signaling. We then identified signaling molecules required for cell non-autonomous intestinal UPRER activation, including the biogenic amine tyramine. Expression of xbp-1s in just two pairs of neurons that synthesize tyramine, the RIM and RIC interneurons, induced intestinal UPRER activation and extended longevity, and exposure to stress led to splicing and activation of xbp-1 in these neurons. In addition, we found that neuronal xbp-1s modulates feeding behavior and reproduction, dependent upon tyramine synthesis. XBP-1s therefore remodels neuronal signaling to coordinately modulate intestinal physiology and stress-responsive behavior, functioning as a global regulator of organismal responses to stress.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Mucosa Intestinal/metabolismo , Neuronas/metabolismo , Tiramina/metabolismo , Respuesta de Proteína Desplegada , Animales , Caenorhabditis elegans , Conducta Alimentaria , Longevidad , Empalme del ARN , Estrés Fisiológico , TranscriptomaRESUMEN
Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the GC and Tfh cell response declines, resulting in impaired humoral immunity. We sought to discover what underpins the poor Tfh cell response in ageing and whether it is possible to correct it. Here, we demonstrate that older people and aged mice have impaired Tfh cell differentiation upon vaccination. This deficit is preceded by poor activation of conventional dendritic cells type 2 (cDC2) due to reduced type 1 interferon signalling. Importantly, the Tfh and cDC2 cell response can be boosted in aged mice by treatment with a TLR7 agonist. This demonstrates that age-associated defects in the cDC2 and Tfh cell response are not irreversible and can be enhanced to improve vaccine responses in older individuals.
Asunto(s)
Centro Germinal/fisiología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Células T Auxiliares Foliculares/fisiología , Linfocitos T Colaboradores-Inductores/fisiología , Adolescente , Traslado Adoptivo , Adulto , Anciano , Envejecimiento , Animales , Linfocitos B , Células de la Médula Ósea , Antígenos CD11/genética , Antígenos CD11/metabolismo , Quimera , Femenino , Humanos , Inmunidad Humoral , Memoria Inmunológica , Vacunas contra la Influenza/administración & dosificación , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/veterinaria , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Vacunación , Adulto JovenRESUMEN
The chromosome conformation capture technique is used to monitor intra- and intermolecular chromosomal associations. By introducing an adaptation of this technique, Ling and colleagues have identified an unexpected coassociation between two loci on separate chromosomes in mouse nuclei, the imprinted Igf2-H19 locus of chromosome 7 and the Wsb1-Nf1 locus of chromosome 11. Strikingly, this interaction is CCCTC-binding factor (CTCF)-dependent and strictly allele specific. These findings extend our appreciation for genome organization and its influence on gene expression and imprinting.
Asunto(s)
Cromosomas de los Mamíferos/genética , Regulación de la Expresión Génica , Impresión Genómica , Alelos , Animales , Factor de Unión a CCCTC , Núcleo Celular/metabolismo , Cromosomas/ultraestructura , Islas de CpG , Cartilla de ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Genoma , Ratones , Modelos Biológicos , Modelos Genéticos , Proteínas Represoras/genética , Proteínas Represoras/fisiologíaRESUMEN
The unfolded protein response of the endoplasmic reticulum (UPRER) is a crucial mediator of secretory pathway homeostasis. Expression of the spliced and active form of the UPRER transcription factor XBP-1, XBP-1s, in the nervous system triggers activation of the UPRER in the intestine of Caenorhabditis elegans (C. elegans) through release of a secreted signal, leading to increased longevity. We find that expression of XBP-1s in the neurons or intestine of the worm strikingly improves proteostasis in multiple tissues, through increased clearance of toxic proteins. To identify the mechanisms behind this enhanced proteostasis, we conducted intestine-specific RNA-seq analysis to identify genes upregulated in the intestine when XBP-1s is expressed in neurons. This revealed that neuronal XBP-1s increases the expression of genes involved in lysosome function. Lysosomes in the intestine of animals expressing neuronal XBP-1s are more acidic, and lysosomal protease activity is higher. Moreover, intestinal lysosome function is necessary for enhanced lifespan and proteostasis. These findings suggest that activation of the UPRER in the intestine through neuronal signaling can increase the activity of lysosomes, leading to extended longevity and improved proteostasis across tissues.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Lisosomas/metabolismo , Proteostasis , Respuesta de Proteína Desplegada , Animales , Retículo Endoplásmico/metabolismo , Intestinos/fisiologíaRESUMEN
Ten-eleven-translocation (TET) proteins catalyze DNA hydroxylation, playing an important role in demethylation of DNA in mammals. Remarkably, although hydroxymethylation levels are high in the mouse brain, the potential role of TET proteins in adult neurogenesis is unknown. We show here that a non-catalytic action of TET3 is essentially required for the maintenance of the neural stem cell (NSC) pool in the adult subventricular zone (SVZ) niche by preventing premature differentiation of NSCs into non-neurogenic astrocytes. This occurs through direct binding of TET3 to the paternal transcribed allele of the imprinted gene Small nuclear ribonucleoprotein-associated polypeptide N (Snrpn), contributing to transcriptional repression of the gene. The study also identifies BMP2 as an effector of the astrocytic terminal differentiation mediated by SNRPN. Our work describes a novel mechanism of control of an imprinted gene in the regulation of adult neurogenesis through an unconventional role of TET3.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células-Madre Neurales/citología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Nucleares snRNP/metabolismo , Animales , Astrocitos/citología , Encéfalo/metabolismo , Catálisis , Dioxigenasas , Ventrículos Laterales/metabolismo , Ratones , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Transcription of protein coding genes is accompanied by recruitment of COMPASS to promoter-proximal chromatin, which methylates histone H3 lysine 4 (H3K4) to form H3K4me1, H3K4me2 and H3K4me3. Here, we determine the importance of COMPASS in maintaining gene expression across lifespan in budding yeast. We find that COMPASS mutations reduce replicative lifespan and cause expression defects in almost 500 genes. Although H3K4 methylation is reported to act primarily in gene repression, particularly in yeast, repressive functions are progressively lost with age while hundreds of genes become dependent on H3K4me3 for full expression. Basal and inducible expression of these genes is also impaired in young cells lacking COMPASS components Swd1 or Spp1. Gene induction during ageing is associated with increasing promoter H3K4me3, but H3K4me3 also accumulates in non-promoter regions and the ribosomal DNA. Our results provide clear evidence that H3K4me3 is required to maintain normal expression of many genes across organismal lifespan.
Asunto(s)
Envejecimiento/genética , N-Metiltransferasa de Histona-Lisina/genética , Procesamiento Proteico-Postraduccional/genética , Transcripción Genética , Cromatina/genética , Regulación Fúngica de la Expresión Génica/genética , Histonas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.
Asunto(s)
Expresión Génica/fisiología , Regiones Promotoras Genéticas/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Activación Transcripcional/genética , Regulación de la Expresión Génica/genética , Regulación hacia ArribaRESUMEN
We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse 'tet-on' phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.
Asunto(s)
Silenciador del Gen , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Línea Celular , Dimerización , Escherichia coli/metabolismo , Células HeLa , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Transactivadores/antagonistas & inhibidoresRESUMEN
Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome.
Asunto(s)
Retrovirus Endógenos/genética , Epigénesis Genética , Genoma , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/genética , Transcriptoma , Animales , Ciclo Celular/genética , Reprogramación Celular , Cromatina/química , Cromatina/metabolismo , Metilación de ADN , Metilasas de Modificación del ADN/deficiencia , Metilasas de Modificación del ADN/genética , Retrovirus Endógenos/metabolismo , Impresión Genómica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Familia de Multigenes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
A set of Tet repressor (TetR) based eukaryotic transactivators that respond to 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) but no longer to tetracycline (tc) is presented. The novel transactivators exhibit high activation in absence of an effector and a 200-fold reduction of reporter gene activity in the presence of cmt3. The most cmt3-sensitive mutant was coexpressed with a tc-responsive Tet transregulator harbouring an altered DNA recognition specificity. Use of cmt3 and tc yields independent control of expression of two genes in the same cell without crosstalk.