RESUMEN
Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.
Asunto(s)
Blastocisto , Diferenciación Celular , Endodermo , Animales , Endodermo/metabolismo , Endodermo/citología , Ratones , Blastocisto/metabolismo , Blastocisto/citología , Linaje de la Célula , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Transducción de Señal , Desarrollo Embrionario , Quinasas Janus/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción STAT/metabolismo , Factores de Transcripción/metabolismo , Femenino , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citologíaRESUMEN
Although adult pluripotent stem cells (aPSCs) are found in many animal lineages, mechanisms for their formation during embryogenesis are unknown. Here, we leveraged Hofstenia miamia, a regenerative worm that possesses collectively pluripotent aPSCs called neoblasts and produces manipulable embryos. Lineage tracing and functional experiments revealed that one pair of blastomeres gives rise to cells that resemble neoblasts in distribution, behavior, and gene expression. In Hofstenia, aPSCs include transcriptionally distinct subpopulations that express markers associated with differentiated tissues; our data suggest that despite their heterogeneity, aPSCs are derived from one lineage, not from multiple tissue-specific lineages during development. Next, we combined single-cell transcriptome profiling across development with neoblast cell-lineage tracing and identified a molecular trajectory for neoblast formation that includes transcription factors Hes, FoxO, and Tbx. This identification of a cellular mechanism and molecular trajectory for aPSC formation opens the door for in vivo studies of aPSC regulation and evolution.
Asunto(s)
Células Madre Adultas , Eucariontes , Células Madre Pluripotentes , Animales , Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes/fisiología , Eucariontes/clasificación , Eucariontes/citologíaRESUMEN
In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.
Asunto(s)
Células Madre Embrionarias , Gastrulación , Animales , Diferenciación Celular/fisiología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario , Endodermo , Mamíferos , RatonesRESUMEN
Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.
Asunto(s)
Blastocisto/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Animales , Femenino , RatonesRESUMEN
The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Análisis de la Célula Individual , Vía de Señalización WntRESUMEN
Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.
Asunto(s)
Desarrollo Embrionario , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Modelos Biológicos , Filogenia , Cigoto/metabolismoRESUMEN
Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.
Asunto(s)
Reprogramación Celular , Ensamble y Desensamble de Cromatina , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Poliadenilación , Transducción de Señal , Animales , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HEK293 , Humanos , RatonesRESUMEN
Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.
Asunto(s)
Reprogramación Celular , Factores de Transcripción/metabolismo , Animales , Cromatina/metabolismo , Fibroblastos/metabolismo , Código de Histonas , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Elementos Reguladores de la Transcripción , Factores de Transcripción SOXB1/metabolismo , Elementos Silenciadores TranscripcionalesRESUMEN
During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.
Asunto(s)
Implantación del Embrión , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Activación Transcripcional , Animales , Implantación del Embrión/genética , Ratones , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Fosforilación , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Femenino , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de SeñalRESUMEN
The nucleus is composed of functionally distinct membraneless compartments that undergo phase separation (PS). However, whether different subnuclear compartments are connected remains elusive. We identified a type of nuclear body with PS features composed of BAZ2A that associates with active chromatin. BAZ2A bodies depend on RNA transcription and BAZ2A non-disordered RNA-binding TAM domain. Although BAZ2A and H3K27me3 occupancies anticorrelate in the linear genome, in the nuclear space, BAZ2A bodies contact H3K27me3 bodies. BAZ2A-body disruption promotes BAZ2A invasion into H3K27me3 domains, causing H3K27me3-body loss and gene upregulation. Weak BAZ2A-RNA interactions, such as with nascent transcripts, promote BAZ2A bodies, whereas the strong binder long non-coding RNA (lncRNA) Malat1 impairs them while mediating BAZ2A association to chromatin at nuclear speckles. In addition to unraveling a direct connection between nuclear active and repressive compartments through PS mechanisms, the results also showed that the strength of RNA-protein interactions regulates this process, contributing to nuclear organization and the regulation of chromatin and gene expression.
Asunto(s)
Cromatina , Histonas , ARN Largo no Codificante , Cromatina/metabolismo , Cromatina/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Histonas/metabolismo , Histonas/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células HeLa , Transcripción Genética , ARN/metabolismo , ARN/genética , Animales , Regulación de la Expresión GénicaRESUMEN
Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.
Asunto(s)
Células Madre Embrionarias de Ratones , Secuencias Reguladoras de Ácidos Nucleicos , Ratones , Animales , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos de Facilitación GenéticosRESUMEN
Nucleosome organization influences gene activity by controlling DNA accessibility to transcription machinery. Here, we develop a chemical biology approach to determine mammalian nucleosome positions genome-wide. We uncovered surprising features of nucleosome organization in mouse embryonic stem cells. In contrast to the prevailing model, we observe that for nearly all mouse genes, a class of fragile nucleosomes occupies previously designated nucleosome-depleted regions around transcription start sites and transcription termination sites. We show that nucleosomes occupy DNA targets for a subset of DNA-binding proteins, including CCCTC-binding factor (CTCF) and pluripotency factors. Furthermore, we provide evidence that promoter-proximal nucleosomes, with the +1 nucleosome in particular, contribute to the pausing of RNA polymerase II. Lastly, we find a characteristic preference for nucleosomes at exon-intron junctions. Taken together, we establish an accurate method for defining the nucleosome landscape and provide a valuable resource for studying nucleosome-mediated gene regulation in mammalian cells.
Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/genética , Animales , Factor de Unión a CCCTC , Estudio de Asociación del Genoma Completo , Ratones , ARN Polimerasa II/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
As chimeras transform from beasts of Greek mythology into tools of contemporary bioscience, secrets of developmental biology and evolutionary divergence are being revealed. Recent advances in stem cell biology and interspecies chimerism have generated new models with extensive basic and translational applications, including generation of transplantable, patient-specific organs.
Asunto(s)
Quimera/metabolismo , Mamíferos/metabolismo , Animales , Humanos , Organogénesis , Especificidad de la EspecieRESUMEN
Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.
Asunto(s)
Timina ADN Glicosilasa , Proteína p53 Supresora de Tumor , Animales , Ratones , Ciclo Celular/genética , Línea Celular , Regulación de la Expresión Génica , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Transcription factors are defined by their sequence-specific binding to DNA and by their selective impacts on gene expression, depending on specific binding sites. The factor binding motifs in the DNA should thus represent a blueprint of regulatory logic, suggesting that transcription factor binding patterns on the genome (e.g., measured by ChIP-seq) should indicate which target genes the factors are directly controlling. However, although genetic data confirm high impacts of transcription factor perturbation in embryology, transcription factors bind to far more sites than the number of genes they dynamically regulate, when measured by direct perturbation in a given cell type. Also, deletion of carefully chosen transcription factor binding sites often gives disappointingly weak results. In a new study in the previous issue of Genes & Development, Lo and colleagues (pp. 1079-1095) reconcile these contradictions by using an elegant experimental system to directly compare the roles of transcription factor-binding site interaction in gene regulation maintenance with roles of the same factor-site interactions in gene regulation through developmental change. They examine Oct4:Sox2 shared target genes under maintained versus reinduced pluripotency conditions within the same cell clone. The results show that the same factor-site interaction impacts can appear modest in assays in developmental steady-state but are far more important as regulatory catalysts of developmental change.
Asunto(s)
Células Madre Embrionarias , Factores de Transcripción , Factores de Transcripción/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Sitios de Unión , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ADN/metabolismo , Factores de Transcripción SOXB1/genética , Diferenciación Celular/genéticaRESUMEN
Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.
Asunto(s)
Aprendizaje , Mamíferos , Animales , Activación Transcripcional , Sitios de Unión , MutagénesisRESUMEN
Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.
Asunto(s)
Proteínas Portadoras , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas Portadoras/genética , Expresión Génica , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been predominantly studied in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss of Pol II pausing stalls pluripotent state transitions within the epiblast of the early mouse embryo. Using Nelfb -/- mice and a NELFB degron mouse pluripotent stem cell model, we show that embryonic stem cells (ESCs) representing the naïve state of pluripotency successfully initiate a transition program but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. We found an increase in chromatin-associated NELF during transition from the naïve to later pluripotent states. Overall, our work defines the acute and long-term molecular consequences of NELF loss and reveals a role for Pol II pausing in the pluripotency continuum as a modulator of cell state transitions.
RESUMEN
Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.
Asunto(s)
Elementos de Facilitación Genéticos , Factor 5 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exones , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/genética , Histonas/metabolismo , Intrones , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Transcripción Genética , Proteína Fluorescente RojaRESUMEN
Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.