RESUMO
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.
Assuntos
Blastocisto , Diferenciação Celular , Endoderma , Animais , Endoderma/metabolismo , Endoderma/citologia , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Linhagem da Célula , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Transdução de Sinais , Desenvolvimento Embrionário , Janus Quinases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição/metabolismo , Feminino , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.
Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores de Estrogênio/metabolismo , Ativação Transcricional/fisiologia , Animais , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/genética , Domínios Proteicos , Receptores de Estrogênio/genéticaRESUMO
The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.
Assuntos
Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Evolução Molecular , Animais , Sítios de Ligação , Ilhas de CpG , Metilação de DNA , Elementos de DNA Transponíveis/genética , Desoxirribonuclease I/metabolismo , Regulação para Baixo , Desenvolvimento Embrionário , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Zigoto/metabolismoRESUMO
The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.
Assuntos
Diferenciação Celular , Reprogramação Celular , Fator 3 de Transcrição de Octâmero , Oxirredução , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Animais , Camundongos , Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , HumanosRESUMO
Under defined differentiation conditions, human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate, the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening, a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification, and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2, increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus, we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE.
Assuntos
Autofagia , Cílios/metabolismo , Células-Tronco Embrionárias/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Ciclo Celular , Proteínas de Homeodomínio/genética , Humanos , Proteína Homeobox Nanog , Placa Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismoRESUMO
Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap â¼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.
Assuntos
Cromatina , Nucleossomos , Fator 3 de Transcrição de Octâmero , Proteínas de Ligação a RNA , Humanos , Sequência de Bases , Reprogramação Celular , Cromatina/genética , DNA/metabolismo , Nucleossomos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Assuntos
DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única , Animais , Imunoprecipitação da Cromatina , Epigênese Genética , Estudo de Associação Genômica Ampla , Cinética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate how a single signalling pathway can both initiate and secure transition between cell states.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes , Animais , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genéticaRESUMO
Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.
Assuntos
Carcinogênese/genética , Metilação de DNA/genética , Neoplasias/genética , Fator 3 de Transcrição de Octâmero/genética , Animais , Reprogramação Celular/genética , Dano ao DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Camundongos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Recidiva , Transdução de Sinais/genéticaRESUMO
The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.
Assuntos
Sistemas CRISPR-Cas/genética , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição PAX6/genética , Animais , Sítios de Ligação/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Fatores de Transcrição/genética , Ativação Transcricional/genéticaRESUMO
Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.
Assuntos
Envelhecimento/genética , Reprogramação Celular/genética , Metilação de DNA , Epigênese Genética , Olho , Regeneração Nervosa/genética , Visão Ocular/genética , Visão Ocular/fisiologia , Envelhecimento/fisiologia , Animais , Axônios/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Dioxigenases , Modelos Animais de Doenças , Olho/citologia , Olho/inervação , Olho/patologia , Feminino , Vetores Genéticos/genética , Glaucoma/genética , Glaucoma/patologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/genética , Traumatismos do Nervo Óptico/genética , Proteínas Proto-Oncogênicas/genética , Células Ganglionares da Retina/citologia , Fatores de Transcrição SOXB1/genética , Transcriptoma/genéticaRESUMO
During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.
Assuntos
Genoma , Camadas Germinativas , Células-Tronco Embrionárias Murinas , Fator 3 de Transcrição de Octâmero , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Genoma/genética , Regulação da Expressão Gênica no Desenvolvimento , Cromatina/metabolismo , Cromatina/genética , Diferenciação Celular/genética , Imagem Individual de Molécula/métodos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Histonas/metabolismo , Histonas/genética , Montagem e Desmontagem da CromatinaRESUMO
Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Fatores Associados à Proteína de Ligação a TATA , Animais , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Camundongos , Desenvolvimento Embrionário/genética , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIID/genética , Feminino , Blastocisto/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Gastrulação/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Embrião de Mamíferos/metabolismoRESUMO
Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.
Assuntos
Camadas Germinativas/citologia , Camundongos/embriologia , Células-Tronco/citologia , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/análise , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
X-chromosome inactivation, or the silencing of one X chromosome that occurs initially in the female somatic four-cell-stage embryo, is reversed during embryonic development first at the time of inner cell mass formation and again during formation of germ cell precursors. Such X-chromosome reactivation in the mouse implies the silencing of the Xist gene and the transcription of its antisense partner, Tsix, from both X chromosomes. In murine embryonic stem cells, both genes are under the transcriptional control of a series of critical pluripotency factors, namely, OCT3/4, NANOG, SOX2, KLF4, C-MYC and REX1. Although the inactive/active status of the two X chromosomes present in female human embryonic stem cells remains controversial, the reactivation of X-chromosome inactivation seems to be a signature for the naive pluripotent state.
Assuntos
Células-Tronco Pluripotentes/fisiologia , Inativação do Cromossomo X , Cromossomo X/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de Sinais/fisiologia , Cromossomo X/genéticaRESUMO
The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.
Assuntos
Desdiferenciação Celular , Reprogramação Celular , Degeneração do Disco Intervertebral , Notocorda , Núcleo Pulposo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/patologia , Animais , Reprogramação Celular/genética , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Ratos , Notocorda/metabolismo , Notocorda/citologia , Humanos , Modelos Animais de Doenças , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Células CultivadasRESUMO
Autophagy is required for benign hepatic tumors to progress into malignant hepatocellular carcinoma. However, the mechanism is unclear. Here, we report that mitophagy, the selective removal of mitochondria by autophagy, positively regulates hepatic cancer stem cells (CSCs) by suppressing the tumor suppressor p53. When mitophagy is enhanced, p53 co-localizes with mitochondria and is removed by a mitophagy-dependent manner. However, when mitophagy is inhibited, p53 is phosphorylated at serine-392 by PINK1, a kinase associated with mitophagy, on mitochondria and translocated into the nucleus, where it binds to the NANOG promoter to prevent OCT4 and SOX2 transcription factors from activating the expression of NANOG, a transcription factor critical for maintaining the stemness and the self-renewal ability of CSCs, resulting in the reduction of hepatic CSC populations. These results demonstrate that mitophagy controls the activities of p53 to maintain hepatic CSCs and provide an explanation as to why autophagy is required to promote hepatocarcinogenesis.
Assuntos
Neoplasias Hepáticas/metabolismo , Mitofagia , Células-Tronco Neoplásicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteína Homeobox Nanog/biossíntese , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying mechanism remains unclear. Although wild-type SOX17 was incapable of inducing pluripotency, SOX17FNV outperformed SOX2 in mouse and human pluripotency reprogramming. In embryonic stem cells, SOX17FNV could replace SOX2 to maintain pluripotency despite considerable sequence differences and upregulated genes expressed in cleavage-stage embryos. Mechanistically, SOX17FNV co-bound OCT4 more cooperatively than SOX2 in the context of the canonical SoxOct DNA element. SOX2, SOX17, and SOX17FNV were all able to bind nucleosome core particles in vitro, which is a prerequisite for pioneer transcription factors. Experiments using purified proteins and in cellular contexts showed that SOX17 variants phase-separated more efficiently than SOX2, suggesting an enhanced ability to self-organise. Systematic deletion analyses showed that the N-terminus of SOX17FNV was dispensable for its reprogramming activity. However, the C-terminus encodes essential domains indicating multivalent interactions that drive transactivation and reprogramming. We defined a minimal SOX17FNV (miniSOX) that can support reprogramming with high activity, reducing the payload of reprogramming cassettes. This study uncovers the mechanisms behind SOX17FNV-induced pluripotency and establishes engineered SOX factors as powerful cell engineering tools.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias/metabolismo , DNA/metabolismo , Mutação Puntual , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero , Animais , Blastocisto/metabolismo , Bovinos , Endoderma/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Mamíferos/genética , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
The success of Yamanaka factor reprogramming of somatic cells into induced pluripotent stem cells suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state. How factor-dependent chromatin opening occurs remains unclear. Using FRAP and ATAC-seq, we found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal-to-epithelial transition. A mutation in the Oct4 linker, L80A, which shows impaired interaction with the BAF complex component Brg1, is inactive in heterochromatin loosening. Oct4-L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4-L80A by enhancing chromatin opening and Klf4 binding. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination.