RESUMO
Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.
Assuntos
Linfócitos B , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fator de Transcrição Ikaros , Linfopoese , Fator de Transcrição PAX5 , Transativadores , Animais , Fator de Transcrição Ikaros/metabolismo , Fator de Transcrição Ikaros/genética , Fator de Transcrição PAX5/metabolismo , Fator de Transcrição PAX5/genética , Camundongos , Transativadores/metabolismo , Transativadores/genética , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfopoese/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteólise , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Diferenciação Celular , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Fator 3 de Transcrição/metabolismo , Fator 3 de Transcrição/genética , Recombinação V(D)J , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Cadeias Leves Substitutas da Imunoglobulina/genética , Transcrição GênicaRESUMO
Dysregulated Wnt/ß-catenin signaling is a common feature of colorectal cancer (CRC). The T-cell factor/lymphoid enhancer factor (TCF/LEF; hereafter, TCF) family of transcription factors are critical regulators of Wnt/ß-catenin target gene expression. Of the four TCF family members, TCF7L1 predominantly functions as a transcriptional repressor. Although TCF7L1 has been ascribed an oncogenic role in CRC, only a few target genes whose expression it regulates have been characterized in this cancer. Through transcriptome analyses of TCF7L1 regulated genes, we noted enrichment for those associated with cellular migration. By silencing and overexpressing TCF7L1 in CRC cell lines, we demonstrated that TCF7L1 promoted migration, invasion, and adhesion. We localized TCF7L1 binding across the CRC genome and overlapped enriched regions with transcriptome data to identify candidate target genes. The growth arrest-specific 1 (GAS1) gene was among these and we demonstrated that GAS1 is a critical mediator of TCF7L1-dependent CRC cell migratory phenotypes. Together, these findings uncover a novel role for TCF7L1 in repressing GAS1 expression to enhance migration and invasion of CRC cells.
Assuntos
Movimento Celular , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Proteína 1 Semelhante ao Fator 7 de Transcrição , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Movimento Celular/genética , Linhagem Celular Tumoral , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Invasividade Neoplásica , Adesão Celular/genética , Via de Sinalização WntRESUMO
Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.
Assuntos
Células-Tronco Embrionárias Murinas , Proteína 1 Semelhante ao Fator 7 de Transcrição , Via de Sinalização Wnt , beta Catenina , Animais , Humanos , Camundongos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Proteólise/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , UbiquitinaçãoRESUMO
An important factor in the emergence and progression of osteosarcoma (OS) is the dysregulated expression of microRNAs (miRNAs). Transcription factor 7-like 1 (TCF7L1), a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family, interacts with the Wnt signaling pathway regulator ß-catenin and acts as a DNA-specific binding protein. This study sought to elucidate the impact of the interaction between miR-329-3p and TCF7L1 on the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches. MiR329-3p was significantly downregulated, while TCF7L1 was considerably up-regulated in all examined OS cell lines. Additionally, a clinical comparison study was performed using the TCGA database. Subsequently, the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments. When miR-329-3p was transfected into the OS cell line, the expression of TCF7L1 decreased, the proliferation of OS cells was inhibited, the cytoskeleton disintegrated, and the nucleus condensed to form apoptotic bodies. The expression of proteins that indicate apoptosis increased simultaneously. The cell cycle was arrested in the G0/G1 phase, and the G1/S transition was blocked. The introduction of miR-329-3p also inhibited downstream Cyclin D1 of the Wnt pathway. Xenograft experiments indicated that the overexpression of miR-329-3p significantly inhibited the growth of OS xenografts in nude mice, and the expression of TCF7L1 and c-Myc in tumor tissues decreased. MiR-329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo. Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7L1 by miR-329-3p. Summarizing these results, it can be inferred that miR-329-3p exerts anticancer effects in osteosarcoma by inhibiting TCF7L1.
Assuntos
Neoplasias Ósseas , MicroRNAs , Osteossarcoma , Proteína 1 Semelhante ao Fator 7 de Transcrição , Via de Sinalização Wnt , Animais , Humanos , Camundongos , beta Catenina/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/patologia , Via de Sinalização Wnt/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
Objective To investigate the expression of T cell factor 3 (TCF3) in hepatocellular carcinoma (HCC), its correlation with the prognosis of HCC patients, and its effect on the invasion, migration, and metastasis of HCC cells. Methods The expression of TCF3 mRNA in HCC tissues was detected with tumor public databases and the expression of TCF3 protein in HCC specimens was detected by immunohistochemical staining. Correlation between TCF3 expression and HCC patients' prognosis was analyzed. Western blot analysis was used to detect the expression of TCF3 in different human HCC cell lines, and lentivirus infection was conducted to construct TCF3-upregulated and TCF3-downregulated HCC cell lines. The effect of TCF3 on the invasion and migration of HCC cells was assessed by in vitro TranswellTM assay, and in vivo intrahepatic tumor implantation models were established to evaluate the effect of TCF3 on the metastatic capacity of HCC cells. Results The expression of TCF3 mRNA was significantly higher in HCC tissues than that in normal liver tissues, and high expression of TCF3 mRNA was closely correlated with decreased overall survival rates of HCC patients. In 120 cases of HCC tissues, the protein level of TCF3 was significantly higher than that in adjacent nontumor tissues, and patients with positive TCF3 expression had a markedly decreased overall survival rate and a higher recurrence rate compared with patients with negative TCF3 expression. In vitro TranswellTM assay indicated that TCF3 upregulation promoted the invasion and migration of PLC/PRF/5 cells, whereas knockdown of TCF3 inhibited the invasion and migration abilities of HCCLM3 cells. Intrahepatic tumor implantation models showed that TCF3 upregulation promoted the metastasis of PLC/PRF/5 cells, while TCF3 knockdown weakened the metastatic capacity of HCCLM3 cells. Conclusion TCF3 expression is significantly upregulated in human HCC tissues, and high TCF3 expression predicts a poor prognosis of HCC patients. TCF3 markedly promotes the invasion, migration, and metastasis of HCC cells.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.
Assuntos
Autorrenovação Celular , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1 de Ligação ao Facilitador Linfoide/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Animais , Benzamidas/farmacologia , Autorrenovação Celular/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Edição de Genes , Fator 1-alfa Nuclear de Hepatócito/deficiência , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Ligante Coestimulador de Linfócitos T Induzíveis/antagonistas & inibidores , Ligante Coestimulador de Linfócitos T Induzíveis/genética , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/deficiência , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Catenina/deficiência , beta Catenina/genética , Proteína AIRERESUMO
BACKGROUND: Cervical cancer (CC) is the second most common tumor in women worldwide. Studies have been accepted that genetic variations play an important role in the development of CC. The aim of this study was to evaluate the impact of TCF7L1 variants on CC risk. METHODS: 508 patients of cervical cancer and 497 healthy subjects were recruited to determine the impact of TCF7L1 polymorphisms on CC susceptibility. The associations were investigated by computing odds ratios (ORs) and 95% confidence intervals. The effect of SNP-SNP interactions on CC risk was explored by multifactor dimensionality reduction analysis. RESULTS: Our study showed that rs11904127 (OR 0.79, p = 0.010) and rs62162674 (OR 0.82, p = 0.044) of TCF7L1 significantly decreased cervical cancer risk. Stratified analysis indicated that rs11904127 and rs62162674 present decreased susceptibility to CC in age > 51 years (OR 0.74, p = 0.019; OR 0.72, p = 0.014, respectively). Haplotype analyses revealed that Grs2366264Trs11689667Crs62162674 has a lower risk to cervical cancer (OR = 0.43, p = 0.018). Besides, there is strong interaction of rs11904127 and rs2366264. CONCLUSION: Rs11904127 and rs62162674 in TCF7L1 are related to cervical cancer. We suggest that these variants can be used as prognostic markers for judging the susceptibility to cervical cancer.
Assuntos
Povo Asiático/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Neoplasias do Colo do Útero/genética , Adulto , China , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The canonical Wnt pathway transcriptional co-activator ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.
Assuntos
Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Néfrons/metabolismo , Células-Tronco/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Néfrons/citologia , Néfrons/embriologia , Regiões Promotoras Genéticas , Ligação Proteica , Células-Tronco/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Fatores de Transcrição/genéticaRESUMO
A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they still have the ability to bind to CpG islands in promoters and to interact with their protein partners via their other functional domains. We have observed that KDM2A-SF and KDM2B-SF bind to the promoters of axin 2 and cyclin D1, two canonical Wnt signaling target genes, and repress their activity. Moreover, KDM2A-SF and KDM2B-SF are both able to strongly repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to negatively affect the transcription of canonical Wnt signaling target genes by binding to their promoters and by interacting with TCF7L1 and other co-repressors.
Assuntos
Ciclina D1/metabolismo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Regiões Promotoras Genéticas , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt , Ilhas de CpG , Ciclina D1/genética , Proteínas F-Box/genética , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/genética , Lisina/metabolismo , Isoformas de Proteínas , Proteína 1 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
The Wnt/ß-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the ß-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/ß-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/ß-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/ß-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/ß-catenin signaling pathway activity.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Transcrição TCF/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Proliferação de Células , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Embrião não Mamífero/metabolismo , Humanos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição TCF/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Ativação Transcricional , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Aberrantly activated Wnt signaling causes cellular transformation that can lead to human colorectal cancer. Wnt signaling is mediated by Lymphoid Enhancer Factor/T-Cell Factor (LEF/TCF) DNA-binding factors. Here we investigate whether altered LEF/TCF expression is conserved in human colorectal tumor sample and may potentially be correlated with indicators of cancer progression. We carried out a meta-analysis of carefully selected publicly available gene expression data sets with paired tumor biopsy and adjacent matched normal tissues from colorectal cancer patients. Our meta-analysis confirms that among the four human LEF/TCF genes, LEF1 and TCF7 are preferentially expressed in tumor biopsies, while TCF7L2 and TCF7L1 in normal control tissue. We also confirm positive correlation of LEF1 and TCF7 expression with hallmarks of active Wnt signaling (i.e., AXIN2 and LGR5). We are able to correlate differential LEF/TCF gene expression with distinct transcriptomes associated with cell adhesion, extracellular matrix organization, and Wnt receptor feedback regulation. We demonstrate here in human colorectal tumor sample correlation of altered LEF/TCF gene expression with quantitatively and qualitatively different transcriptomes, suggesting LEF/TCF-specific transcriptional regulation of Wnt target genes relevant for cancer progression and survival. This bioinformatics analysis provides a foundation for future more detailed, functional, and molecular analyses aimed at dissecting such functional differences.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Fator 1 de Ligação ao Facilitador Linfoide/biossíntese , Proteínas de Neoplasias/biossíntese , Proteína 1 Semelhante ao Fator 7 de Transcrição/biossíntese , Proteína 2 Semelhante ao Fator 7 de Transcrição/biossíntese , Transcriptoma , Via de Sinalização Wnt , Adenocarcinoma/patologia , Proteína Axina/biossíntese , Proteína Axina/genética , Biópsia , Neoplasias Colorretais/patologia , Mineração de Dados , Conjuntos de Dados como Assunto , Progressão da Doença , Retroalimentação Fisiológica , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Proteínas de Neoplasias/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
Tcf7l1, which is a key effector molecule of the Wnt/ß-catenin signaling pathway, is highly expressed in various cancers, and it promotes tumor growth. In this study, we demonstrated that unlike its tumor-promoting effects in several other types of cancers, Tcf7l1 expression is downregulated in hepatocarcinoma compared with their adjacent nontumor counterparts. Underexpression of Tcf7l1 is correlated with poorer survival. In liver cancer stem cell (CSC) populations, Tcf7l1 expression is downregulated. Ectopic expression of Tcf7l1 attenuates the self-renewal abilities of liver CSCs. Mechanistically, Tcf7l1 regulates the self-renewal abilities of liver CSCs through transcriptional repression of the Nanog gene, and the effect is independent of ß-catenin. Moreover, we found that Tcf7l1 expression is controlled by extracellular insulin-like growth factor (IGF) signaling, and we demonstrated for the first time that IGF signaling stimulates Tcf7l1 phosphorylation and degradation through the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Overall, our results provide some new insights into how extracellular signals modulate the self-renewal of liver CSCs and highlight the inhibitory roles of Tcf7l1 in cancer. Stem Cells 2019;37:1389-1400.
Assuntos
Sobrevivência Celular/fisiologia , Fígado/citologia , Fígado/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Somatomedinas/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Citometria de Fluxo , Humanos , Imunoensaio , Imuno-Histoquímica , Imunoprecipitação , Técnicas In Vitro , Lentivirus , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fosforilação , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Somatomedinas/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , beta Catenina/genéticaRESUMO
Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.
Assuntos
Sinalização do Cálcio/fisiologia , Espaço Intracelular/metabolismo , Células-Tronco Embrionárias Murinas/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células-Tronco Pluripotentes/fisiologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Diferenciação Celular , Linhagem da Célula , Autorrenovação Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quinase 3 da Glicogênio Sintase/metabolismo , Homeostase , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.
Assuntos
Olho/crescimento & desenvolvimento , Morfogênese , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Proliferação de Células , Embrião não Mamífero/metabolismo , Olho/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Cinética , Masculino , Mutação/genética , Placa Neural/embriologia , Neurogênese , Penetrância , Fenótipo , Prosencéfalo/embriologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Regulação para Cima/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Zigoto/metabolismoRESUMO
Gastric cancer is one of the most common cancers worldwide and is the third leading cause of cancer-related deaths globally. Although significant progress has been made in the diagnosis and treatment for the cancer, less improvement has been made in overall survival rate. Thus, there is an urgent need for a better understanding of the biological aspects of the cancer. The transcription factor transcription factor 7-like 1 (TCF7L1) is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in gastric cancer has seldom been discussed. In the present study, by using the Cancer Genome Atlas dataset analysis, we demonstrated that patients with higher expression of TCF7L1 could be used to reflect prognosis. An examination of the mechanisms demonstrated that TCF7L1 could positively regulate antioxidant response in gastric cancer cells by positively regulating Keap1/NRF2 [Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2] pathway. Collectively, our data demonstrated that TCF7L1 is a novel marker for predicting overall survival of gastric cancer and provided the possible underlying molecular mechanism.
Assuntos
Proliferação de Células/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias Gástricas/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Humanos , Estimativa de Kaplan-Meier , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Prognóstico , Transdução de Sinais/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismoRESUMO
The T-cell factor/lymphoid enhancer factor (TCF/LEF) family protein Tcf7l1 is highly abundant in embryonic stem cells (ESCs), regulating pluripotency and preparing epiblasts for further differentiation. Defects in the cardiovascular system in Tcf7l1-null mouse were considered secondary to mesoderm malformation. Here, we used temporally controlled Tcf7l1 expression in Tcf7l1-null ESCs to address whether Tcf7l1 directly contributes to cardiac forward programming. Tcf7l1 knockout during differentiation impaired cardiomyocyte formation but did not affect mesoderm formation. Tcf7l1-null ESCs showed delay in mesoderm formation, but once completed, ectopic Tcf7l1 augmented cardiomyocyte differentiation. Further, Tcf7l1-VP16 and Tcf7l1dN showed procardiac activity whereas Tcf7l1-En was ineffective. Our results support that Tcf7l1 contributes to cardiac lineage development as a ß-catenin-independent transactivator of cardiac genes.
Assuntos
Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/metabolismo , Miócitos Cardíacos/metabolismo , Organogênese/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camadas Germinativas/citologia , Lentivirus/genética , Lentivirus/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Fatores de Tempo , Proteína 1 Semelhante ao Fator 7 de Transcrição/deficiênciaRESUMO
Adenomatous polyposis coli (APC) and Axin are core components of the ß-catenin destruction complex. How APC's function is regulated and whether Wnt signaling influences the direct APC-Axin interaction to inhibit the ß-catenin destruction complex is not clear. Through a CRISPR screen of ß-catenin stability, we have identified ICAT, a polypeptide previously known to block ß-catenin-TCF interaction, as a natural inhibitor of APC. ICAT blocks ß-catenin-APC interaction and prevents ß-catenin-mediated APC-Axin interaction, enhancing stabilization of ß-catenin in cells harboring truncated APC or stimulated with Wnt, but not in cells deprived of a Wnt signal. Using ICAT as a tool to disengage ß-catenin-mediated APC-Axin interaction, we demonstrate that Wnt quickly inhibits the direct interaction between APC and Axin. Our study highlights an important scaffolding function of ß-catenin in the assembly of the destruction complex and suggests Wnt-inhibited APC-Axin interaction as a mechanism of Wnt-dependent inhibition of the destruction complex.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Domínios e Motivos de Interação entre Proteínas/genética , beta Catenina/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína Axina/genética , Humanos , Estabilidade Proteica , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt/genéticaRESUMO
Although long noncoding RNAs (lncRNAs) are emerging as new modulators in the fate decision of pluripotent stem cells, the functions of specific lncRNAs remain unclear. Here, we found that telomeric RNA (TERRA or TelRNA), one type of lncRNAs, is highly expressed in mouse embryonic stem cells (mESCs) but declines significantly upon differentiation. TERRA is induced by the Wnt/ß-catenin signaling pathway and can reproduce its self-renewal-promoting effect when overexpressed. Further studies revealed that T cell factor 3 ( TCF3) is a potential downstream target of TERRA and mediates the effect of TERRA in mESC maintenance. TERRA inhibits TCF3 transcription, while enforced TCF3 expression abrogates the undifferentiated state of mESCs supported by TERRA. Accordingly, the transcripts of the pluripotency genes Esrrb, Tfcp2l1, and Klf2, repressed by TCF3 in mESCs, are increased in TERRA-overexpressing cells. Our study therefore highlights the important role of TERRA in mESC maintenance and also uncovers a mechanism by which TERRA promotes self-renewal. These data will expand our understanding of the pluripotent regulatory network of ESCs.
Assuntos
Autorrenovação Celular , Células-Tronco Embrionárias Murinas/metabolismo , RNA não Traduzido/metabolismo , Telômero/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Autorrenovação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos da Linhagem 129 , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA não Traduzido/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Telômero/genética , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Transcrição Gênica , Via de Sinalização WntRESUMO
BACKGROUND: The TCF7L2 (transcription factor 7 like 2) gene is strongly associated with type 2 diabetes risk. However, many people without the TCF7L2 at-risk allele develop T2D. The aim of this study was to investigate altered Tcf7l2 DNA methylation and gene expression caused by high-fat diets (HFDs). METHODS: C57BL/6 mice were fed either an HFD or normal diet for 8 weeks, and intraperitoneal glucose tolerance tests were performed. Pancreatic islets were sorted for bisulfite sequencing polymerase chain reaction to determine DNA methylation status. We cloned the Tcf7l2 promoter, methylated it with methyltransferase, and transfected this construct into MIN-6 cells to confirm the effects of promoter methylation on Tcf7l2 expression. RESULTS: Aberrant methylation at position -165 bp relative to the transcriptional start site of Tcf7l2 was present in mice fed an HFD. Accordingly, expression of Tcf7l2 mRNA and its corresponding protein was lower in the HFD group (P < .05). Methylation of the Tcf7l2 promoter suppressed gene expression in MIN-6 cells. CONCLUSION: An HFD was shown to induce aberrant methylation of the Tcf7l2 promoter in mouse islets, which resulted in diminished gene expression. This study provides an evidence of the association between nutrient consumption and gene expression.
Assuntos
Metilação de DNA , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/patologia , Regiões Promotoras Genéticas , Proteína 1 Semelhante ao Fator 7 de Transcrição/genética , Animais , Células Cultivadas , Epigênese Genética , Teste de Tolerância a Glucose , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Co-expression and cross-regulation of the four TCF/LEFs render their redundant and unique functions ambiguous. Here, we describe quadruple-knockout (QKO) mouse ESCs lacking all full-length TCF/LEFs and cell lines rescued with TCF7 or TCF7L1. QKO cells self-renew, despite gene expression patterns that differ significantly from WT, and display delayed, neurectoderm-biased, embryoid body (EB) differentiation. QKO EBs have no contracting cardiomyocytes and differentiate poorly into mesendoderm but readily generate neuronal cells. QKO cells and TCF7L1-rescued cells cannot efficiently activate TCF reporters, whereas TCF7-rescued cells exhibit significant reporter responsiveness. Surprisingly, despite dramatically different transactivation capacities, re-expression of TCF7L1 or TCF7 in QKO cells restores their tri-lineage differentiation ability, with similar lineage marker expression patterns and beating cardiomyocyte frequencies observed in EBs. Both factors also similarly affect the transcriptome of QKO cells. Our data reveal that a single TCF, regardless of its activation capacity, is sufficient for effective trilineage differentiation of ESCs.