RESUMO
Metazoan chromosomes are sequentially partitioned into topologically associating domains (TADs) and then into smaller sub-domains. One class of sub-domains, insulated neighborhoods, are proposed to spatially sequester and insulate the enclosed genes through self-association and chromatin looping. However, it has not been determined functionally whether promoter-enhancer interactions and gene regulation are broadly restricted to within these loops. Here, we employed published datasets from murine embryonic stem cells (mESCs) to identify insulated neighborhoods that confine promoter-enhancer interactions and demarcate gene regulatory regions. To directly address the functionality of these regions, we depleted estrogen-related receptor ß (Esrrb), which binds the Mediator co-activator complex, to impair enhancers of genes within 222 insulated neighborhoods without causing mESC differentiation. Esrrb depletion reduces Mediator binding, promoter-enhancer looping, and expression of both nascent RNA and mRNA within the insulated neighborhoods without significantly affecting the flanking genes. Our data indicate that insulated neighborhoods represent functional regulons in mammalian genomes.
Assuntos
Cromossomos de Mamíferos , Elementos Facilitadores Genéticos , Elementos Isolantes , Células-Tronco Embrionárias Murinas/fisiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Bases de Dados Genéticas , Regulação para Baixo , Camundongos , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , CoesinasRESUMO
The maintenance of pluripotency in mouse embryonic stem cells (ESCs) is governed by the action of an interconnected network of transcription factors. Among them, only Oct4 and Sox2 have been shown to be strictly required for the self-renewal of ESCs and pluripotency, particularly in culture conditions in which differentiation cues are chemically inhibited. Here, we report that the conjunct activity of two orphan nuclear receptors, Esrrb and Nr5a2, parallels the importance of that of Oct4 and Sox2 in naïve mouse ESCs. By occupying a large common set of regulatory elements, these two factors control the binding of Oct4, Sox2 and Nanog to DNA. Consequently, in their absence the pluripotency network collapses and the transcriptome is substantially deregulated, leading to the differentiation of ESCs. Altogether, this work identifies orphan nuclear receptors, previously thought to be performing supportive functions, as a set of core regulators of naïve pluripotency.
Assuntos
Células-Tronco Embrionárias Murinas/citologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Diferenciação Celular , Autorrenovação Celular , Redes Reguladoras de Genes , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Receptores de Estrogênio/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice.
Assuntos
Células-Tronco Embrionárias , Endoderma , Endoderma/metabolismo , Diferenciação Celular/genéticaRESUMO
Gastric cancer (GC) is a common malignant tumor, posing a great threat to human's health and life. Previous studies have suggested aberrant expression of long non-coding RNAs (lncRNAs) in GC. This study elucidated the effects of lncRNA ACTA2-AS1 on the biological characteristics of GC. Gene expression in stomach adenocarcinoma (STAD) samples compared with normal tissues and the correlation between gene expression and prognosis of STAD patients were analyzed using bioinformatic tools. Gene expression at protein and mRNA levels in GC and normal cells was tested by western blotting and RT-qPCR. The subcellular localization of ACTA2-AS1 in AGS and HGC27 cells was identified by nuclear-cytoplasmic fractionation and FISH assay. EdU, CCK-8, flow cytometry analysis, TUNEL staining assays were conducted to evaluate the role of ACTA2-AS1 and ESRRB on GC cellular behaviors. The binding relationship among ACTA2-AS1, miR-6720-5p and ESRRB was verified by RNA pulldown, luciferase reporter assay and RIP assay. LncRNA ACTA2-AS1 was underexpressed in GC tissues and cell lines. ACTA2-AS1 elevation suppressed GC cell proliferation and induced apoptosis. Mechanistically, ACTA2-AS1 directly bound to miR-6720-5p and subsequently promoted the expression of target gene ESRRB in GC cells. Furthermore, ESRRB knockdown reversed the influence of ACTA2-AS1 overexpression on GC proliferation and apoptosis. ACTA2-AS1 plays an antioncogenic role in GC via binding with miR-6720-5p to regulate ESRRB expression.
Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias Gástricas , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Receptores de Estrogênio/genética , Actinas/genéticaRESUMO
DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.
Assuntos
Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Animais , Linhagem Celular , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Conformação de Ácido Nucleico , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Autosomal recessive nonsyndromic hearing loss (DFNB) is relatively frequent in Pakistan, which is thought to be mainly due to relatively frequent consanguinity. DFNB genes vary widely in their kinds and functions making molecular diagnosis difficult. This study determined the genetic causes in five Pakistani DFNB families with prelingual onset. The familial genetic analysis identified four pathogenic or likely pathogenic homozygous mutations by whole exome sequencing: two splicing donor site mutations of c.787+1G>A in ESRRB (DFNB35) and c.637+1G>T in CABP2 (DFNB93) and two missense mutations of c.7814A>G (p.Asn2605Ser) in CDH23 (DFNB12) and c.242G>A (p.Arg81His) in TMIE (DFNB6). The ESRRB and TMIE mutations were novel, and the TMIE mutation was observed in two families. The two missense mutations were located at well conserved sites and in silico analysis predicted their pathogenicity. This study identified four homozygous mutations as the underlying cause of DFNB including two novel mutations. This study will be helpful for the exact molecular diagnosis and treatment of deafness patients.
Assuntos
Caderinas/genética , Proteínas de Ligação ao Cálcio/genética , Surdez/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Receptores de Estrogênio/genética , Adolescente , Adulto , Proteínas Relacionadas a Caderinas , Criança , Pré-Escolar , Consanguinidade , Surdez/epidemiologia , Feminino , Perda Auditiva Neurossensorial/epidemiologia , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Paquistão/epidemiologiaRESUMO
The estrogen-related receptor b (ESRRB) is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency. In mouse ES cells, overexpression of ESRRB can maintain LIF-independent self-renewal in the absence of Nanog. However, the fundamental features of porcine ESRRB remain elusive. In this study, we revealed the expression profiles of ESRRB in both porcine pluripotent stem cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor that enhanced porcine pluripotent gene activation. Addition of ESRRB into the cocktail of core pluripotent factors Oct4, Sox2, Klf4, and c-Myc (OSKM + E) could significantly enhance the reprograming efficiency and the formation of alkaline phosphatase positive colonies. Conversely, knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes, minimized the alkaline phosphatase activity, and initiated the porcine induced pluripotent stem cell differentiation. Therefore, porcine ESRRB is a crucial transcription factor to improve the self-renewal of piPSCs.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Estrogênio/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Autorrenovação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Receptores de Estrogênio/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Sus scrofa , TransfecçãoRESUMO
Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2-dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers.
Assuntos
Técnicas de Reprogramação Celular/métodos , Proteínas de Ligação a DNA , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Complexo Repressor Polycomb 2 , Receptores de Estrogênio , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Fator 4 Semelhante a Kruppel , Camundongos , Complexo Repressor Polycomb 2/biossíntese , Complexo Repressor Polycomb 2/genética , Proteínas de Ligação a RNA , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Among transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP-flanked (floxed) alleles either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre-implantation embryos at the onset of the four-cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J x 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knockin and additional targeted alleles have been generated.
Assuntos
Alelos , Receptores de Estrogênio/genética , Animais , Linhagem Celular , Deleção de Genes , Técnicas de Transferência de Genes , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Recombinação Genética , Proteínas Virais/genética , Zigoto/metabolismoRESUMO
Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1-Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb. Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 promoter is activated by Esrrb in association with Ncoa3, and Dax1 inhibited activities of Esrrb and Ncoa3, resulting maintenance of the undifferentiated status of ES cells.
Assuntos
Receptor Nuclear Órfão DAX-1/genética , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica , Coativador 3 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/genética , Receptores de Estrogênio/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Receptor Nuclear Órfão DAX-1/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator de Transcrição GATA6/metabolismo , Células HEK293 , Humanos , Camundongos , Mutação , Coativador 3 de Receptor Nuclear/metabolismo , Motivos de Nucleotídeos/genética , Ligação Proteica , Receptores de Estrogênio/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: A familial and genetic predisposition for the development of rotator cuff tearing has been identified. The purpose of this study was to determine if a familial predisposition exists for healing after rotator cuff repair and if the reported significant association with a single-nucleotide polymorphism (SNP) in the ESRRB gene is present in patients who fail to heal. MATERIALS AND METHODS: The study recruited 72 patients undergoing arthroscopic rotator cuff repair for a full-thickness posterosuperior tear. Magnetic resonance imaging studies were performed at a minimum of 1 year postoperatively (average, 2.6 years). Healing failures were classified as lateral or medial. Self-reported family history of rotator cuff tearing data and genome-wide genotypes were available. Characteristics of cases with and without a family history of rotator cuff tearing were compared, and a comparison of the frequency of SNP 1758384 (in ESRRB) was performed between patients who healed and those who failed to heal. RESULTS: Of the rotator cuff repairs, 42% failed to heal; 42% of patients reported a family history of rotator cuff tear. Multivariate regression analysis showed a significant association between familiality and overall healing failure (medial and lateral failures) (P = .036) and lateral failures independently (P = .006). An increased risk for the presence of a rare allele for SNP rs17583842 was present in lateral failures compared with those that healed (P = .005). CONCLUSIONS: Individuals with a family history of rotator cuff tearing were more likely to have repair failures. Significant association of a SNP variant in the ESRRB gene was also observed with lateral failure.
Assuntos
Receptores de Estrogênio/genética , Lesões do Manguito Rotador/genética , Lesões do Manguito Rotador/cirurgia , Manguito Rotador/cirurgia , Cicatrização/genética , Adulto , Idoso , Alelos , Artroscopia , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Estudos Prospectivos , Fatores de Risco , Falha de Tratamento , Resultado do Tratamento , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND: The precise etiology of rotator cuff disease is unknown, but prior evidence suggests a role for genetic factors. Variants of estrogen-related receptor-ß (ESRRB) have been previously associated with rotator cuff disease. The purpose of the present study was to confirm the association between multiple candidate genes, including ESRRB, and rotator cuff disease in an independent set of patients with rotator cuff tear. MATERIALS AND METHODS: The Illumina 5M (Illumina Inc, San Diego, CA, USA) single nucleotide polymorphism (SNP) platform was used to genotype 175 patients with rotator cuff tear. Genotypes were used to select a set of 2595 genetically matched Caucasian controls available from the Illumina iControls database. Tests of association were performed with Genome-wide Efficient Mixed Model Association (GEMMA) software at 69 SNPs that fell within 20 kb of 6 candidate genes (DEFB1, DENND2C, ESRRB, FGF3, FGF10, and FGFR1). RESULTS: Tests of association revealed 1 significantly associated SNP occurring in ESRRB (rs17583842; P = 4.4E-4). Another SNP within ESRRB (rs7157192) had a nominal P value of 7.8E-3. FastPHASE software estimated 2 frequent haplotypes among 54 individuals who carried both risk alleles at these 2 SNPs. The first haplotype had a frequency of 13.9% (n = 15) in risk-allele carriers and only 2.2% in controls (odds ratio, 6.9; 95% confidence interval, 3.9-2.2). The second haplotype had a frequency of 12.9% in risk-allele carriers and only 2.7% in controls (odds ratio, 5.3; 95% confidence interval, 3.0-9.5). CONCLUSIONS: The significant association and the presence of high-risk haplotypes identified in the ESRRB gene confirm the association of variants in ESRRB and rotator cuff disease.
Assuntos
Receptores de Estrogênio/genética , Lesões do Manguito Rotador , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Haplótipos , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Ruptura/genéticaRESUMO
BACKGROUND: Hereditary hearing loss (HHL) is a common heterogeneous disorder affecting all ages, ethnicities, and genders. The most common form of HHL is autosomal recessive non-syndromic hearing loss (ARNSHL), in which there is no genotype-phenotype correlation in the majority of cases. This study aimed to identify the genetic causes of hearing loss (HL) in a family with Iranian Azeri Turkish ethnicity negative for gap junction beta-2 (GJB2), gap junction beta-6 (GJB6), and mitochondrially encoded 12S rRNA (MT-RNR1) deleterious mutations. METHODS: Targeted genome sequencing method was applied to detect genetic causes of HL in the family. Sanger sequencing was employed to verify the segregation of the variant. Finally, we used bioinformatics tools and American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines to determine whether the detected variant might affect the corresponding protein or not. RESULTS: A novel homozygous missense mutation, c.499G>A (p.G167R), was identified in exon 5 of the ESRRB (estrogen-related receptor beta) gene. Healthy and affected family members confirmed the co-segregation of the variant with ARNSHL. Eventually, the variant's pathogenicity was confirmed by the in silico analysis and the ACMG/AMP guidelines. CONCLUSION: The study suggests that the detected variant, c.499G>A, plays a crucial role in the development of ARNSHL, emphasizing the clinical significance of the ESRRB gene in ARNSHL patients. Additionally, it would be helpful for genetic counseling and clinical management of ARNSHL patients and providing preventive opportunities.
Assuntos
Surdez , Perda Auditiva Neurossensorial , Perda Auditiva , Conexinas/genética , Surdez/genética , Feminino , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Humanos , Irã (Geográfico) , Masculino , Mutação , Mutação de Sentido Incorreto , Linhagem , Receptores de EstrogênioRESUMO
Primordial germ cells (PGCs) arise from cells of the post-implantation epiblast in response to cytokine signaling. PGC development can be recapitulated in vitro by differentiating epiblast-like cells (EpiLCs) into PGC-like cells (PGCLCs) through cytokine exposure. Interestingly, the cytokine requirement for PGCLC induction can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that NANOG mediates Otx2 downregulation in the absence of cytokines and that this is essential for PGCLC induction by NANOG. Moreover, the direct NANOG target gene Esrrb, which can substitute for several NANOG functions, does not downregulate Otx2 when overexpressed in EpiLCs and cannot promote PGCLC specification. However, expression of ESRRB in Otx2+/- EpiLCs rescues emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteína Homeobox Nanog/genética , Fatores de Transcrição Otx/genética , Receptores de Estrogênio/genética , Biomarcadores , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Heterogeneidade Genética , Células Germinativas/citologia , Imunofenotipagem , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição Otx/metabolismo , Ligação Proteica , Receptores de Estrogênio/metabolismoRESUMO
Cell cycle and differentiation decisions are linked; however, the underlying principles that drive these decisions are unclear. Here, we combined cell-cycle reporter system and single-cell RNA sequencing (scRNA-seq) profiling to study the transcriptomes of embryonic stem cells (ESCs) in the context of cell-cycle states and differentiation. By applying retinoic acid, to G1 and G2/M ESCs, we show that, while both populations can differentiate toward epiblast stem cells (EpiSCs), only G2/M ESCs could differentiate into extraembryonic endoderm cells. We identified Esrrb, a pluripotency factor that is upregulated during G2/M, as a driver of extraembryonic endoderm stem cell (XEN) differentiation. Furthermore, enhancer chromatin states based on wild-type (WT) and ESRRB knockout (KO) ESCs show association of ESRRB with XEN poised enhancers. G1 cells overexpressing Esrrb allow ESCs to produce XENs, while ESRRB-KO ESCs lost their potential to differentiate into XEN. Overall, this study reveals a vital link between Esrrb and cell-cycle states during the exit from pluripotency.
Assuntos
Células-Tronco Embrionárias , Endoderma , Ciclo Celular/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Camadas GerminativasRESUMO
Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.
RESUMO
Precise gene regulation is critical during embryo development. Long terminal repeat elements (LTRs) of endogenous retroviruses (ERVs) are dynamically expressed in blastocysts of mammalian embryos. However, the expression pattern of LTRs in monkey blastocyst is still unknown. By single-cell RNA-sequencing (seq) data of cynomolgus monkeys, we found that LTRs of several ERV families, including MacERV6, MacERV3, MacERV2, MacERVK1, and MacERVK2, were highly expressed in pre-implantation embryo cells including epiblast (EPI), trophectoderm (TrB), and primitive endoderm (PrE), but were depleted in post-implantation. We knocked down MacERV6-LTR1a in cynomolgus monkeys with a short hairpin RNA (shRNA) strategy to examine the potential function of MacERV6-LTR1a in the early development of monkey embryos. The silence of MacERV6-LTR1a mainly postpones the differentiation of TrB, EPI, and PrE cells in embryos at day 7 compared to control. Moreover, we confirmed MacERV6-LTR1a could recruit Estrogen Related Receptor Beta (ESRRB), which plays an important role in the maintenance of self-renewal and pluripotency of embryonic and trophoblast stem cells through different signaling pathways including FGF and Wnt signaling pathways. In summary, these results suggest that MacERV6-LTR1a is involved in gene regulation of the pre-implantation embryo of the cynomolgus monkeys.
Assuntos
Blastocisto/metabolismo , Retrovirus Endógenos/genética , Sequências Repetidas Terminais/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ontologia Genética , Macaca fascicularis , Células-Tronco Pluripotentes/metabolismo , Fatores de Tempo , Transcriptoma/genéticaRESUMO
Trophoblast stem cells (TSCs), which can be derived from the trophoectoderm of a blastocyst, have the ability to sustain self-renewal and differentiate into various placental trophoblast cell types. Meanwhile, essential insights into the molecular mechanisms controlling the placental development can be gained by using TSCs as the cell model. Esrrb is a transcription factor that has been shown to play pivotal roles in both embryonic stem cell (ESC) and TSC, but the precise mechanism whereby Esrrb regulates TSC-specific transcriptome during differentiation and reprogramming is still largely unknown. In the present study, we elucidate the function of Esrrb in self-renewal and differentiation of TSCs, as well as during the induced TSC (iTSC) reprogramming. We demonstrate that the precise level of Esrrb is critical for stem state maintenance and further trophoblast differentiation of TSCs, as ectopically expressed Esrrb can partially block the rapid differentiation of TSCs in the absence of fibroblast growth factor 4. However, Esrrb depletion results in downregulation of certain key TSC-specific transcription factors, consequently causing a rapid differentiation of TSCs and these Esrrb-deficient TSCs lose the ability of hemorrhagic lesion formation in vivo. This function of Esrrb is exerted by directly binding and activating a core set of TSC-specific target genes including Cdx2, Eomes, Sox2, Fgfr4, and Bmp4. Furthermore, we show that Esrrb overexpression can facilitate the MEF-to-iTSC conversion. Moreover, Esrrb can substitute for Eomes to generate GEsTM-iTSCs. Thus, our findings provide a better understanding of the molecular mechanism of Esrrb in maintaining TSC self-renewal and during iTSC reprogramming.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Estrogênio/metabolismo , Trofoblastos/metabolismo , Animais , Técnicas de Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Transgênicos , Receptores de Estrogênio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/citologiaRESUMO
Following fertilization, totipotent cells undergo asymmetric cell divisions, resulting in three distinct cell types in the late pre-implantation blastocyst: epiblast (Epi), primitive endoderm (PrE), and trophectoderm (TE). Here, we aim to understand whether these three cell types can be induced from fibroblasts by one combination of transcription factors. By utilizing a sophisticated fluorescent knockin reporter system, we identified a combination of five transcription factors, Gata3, Eomes, Tfap2c, Myc, and Esrrb, that can reprogram fibroblasts into induced pluripotent stem cells (iPSCs), induced trophoblast stem cells (iTSCs), and induced extraembryonic endoderm stem cells (iXENs), concomitantly. In-depth transcriptomic, chromatin, and epigenetic analyses provide insights into the molecular mechanisms that underlie the reprogramming process toward the three cell types. Mechanistically, we show that the interplay between Esrrb and Eomes during the reprogramming process determines cell fate, where high levels of Esrrb induce a XEN-like state that drives pluripotency and high levels of Eomes drive trophectodermal fate.