RESUMO
SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry.
Assuntos
Bass , Doenças dos Peixes , Proteínas de Peixes , Animais , Doenças dos Peixes/virologia , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Bass/genética , Bass/virologia , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição Sp3/genética , Filogenia , Nodaviridae/genética , Clonagem Molecular , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/genética , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Infecções por Vírus de DNA/genética , Sequência de AminoácidosRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic heterogeneous disease. This study explored the mechanism of specificity protein 1/3 (Sp1/3) in T-ALL cells through ß-catenin by acting as targets of miR-495-3p. Expression levels of miR-495-3p, Sp1, Sp3, and ß-catenin in the serum from T-ALL children patients, healthy controls, and the T-ALL cell lines were measured. The cell proliferation ability and apoptosis rate were detected. Levels of proliferation-related proteins proliferating cell nuclear antigen (PCNA)/cyclinD1 and apoptosis-related proteins B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) were determined. The binding of Sp1/3 and ß-catenin promoter and the targeted relationship between miR-495-3p with Sp1/3 were analyzed. Sp1/3 were upregulated in CD4+ T-cells in T-ALL and were linked with leukocyte count and risk classification. Sp1/3 interference prevented proliferation and promoted apoptosis in T-ALL cells. Sp1/3 transcription factors activated ß-catenin expression. Sp1/3 enhanced T-ALL cell proliferation by facilitating ß-catenin expression. miR-495-3p targeted and repressed Sp1/3 expressions. miR-495-3p overexpression inhibited T-ALL cell proliferation and promoted apoptosis. Conjointly, Sp1/3, as targets of miR-495-3p limit apoptosis and promote proliferation in T-ALL cells by promoting ß-catenin expression.
Assuntos
Apoptose , Proliferação de Células , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Fator de Transcrição Sp1 , Fator de Transcrição Sp3 , beta Catenina , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/metabolismoRESUMO
In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Nucleossomos , Regiões Promotoras Genéticas , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Diferenciação Celular/genética , Camundongos , Músculo Esquelético/metabolismo , Desenvolvimento Muscular/genética , Linhagem Celular , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição Sp3/genética , Fibras Musculares Esqueléticas/metabolismoRESUMO
The specificity protein (Sp) transcription factors (TFs) Sp1, Sp2, Sp3 and Sp4 exhibit structural and functional similarities in cancer cells and extensive studies of Sp1 show that it is a negative prognostic factor for patients with multiple tumor types. In this review, the role of Sp1, Sp3 and Sp4 in the development of cancer and their regulation of pro-oncogenic factors and pathways is reviewed. In addition, interactions with non-coding RNAs and the development of agents that target Sp transcription factors are also discussed. Studies on normal cell transformation into cancer cell lines show that this transformation process is accompanied by increased levels of Sp1 in most cell models, and in the transformation of muscle cells into rhabdomyosarcoma, both Sp1 and Sp3, but not Sp4, are increased. The pro-oncogenic functions of Sp1, Sp3 and Sp4 in cancer cell lines were studied in knockdown studies where silencing of each individual Sp TF decreased cancer growth, invasion and induced apoptosis. Silencing of an individual Sp TF was not compensated for by the other two and it was concluded that Sp1, Sp3 and Sp4 are examples of non-oncogene addicted genes. This conclusion was strengthened by the results of Sp TF interactions with non-coding microRNAs and long non-coding RNAs where Sp1 contributed to pro-oncogenic functions of Sp/non-coding RNAs. There are now many examples of anticancer agents and pharmaceuticals that induce downregulation/degradation of Sp1, Sp3 and Sp4, yet clinical applications of drugs specifically targeting Sp TFs are not being used. The application of agents targeting Sp TFs in combination therapies should be considered for their potential to enhance treatment efficacy and decrease toxic side effects.
Assuntos
Antineoplásicos , MicroRNAs , Rabdomiossarcoma , Humanos , Fatores de Transcrição Sp/metabolismo , Antineoplásicos/farmacologia , MicroRNAs/genética , Rabdomiossarcoma/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.
Assuntos
Reprogramação Celular , Homólogo 5 da Proteína Cromobox , Proteínas de Ligação a DNA , Inativação Gênica , Fator de Transcrição Sp3 , Animais , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismoRESUMO
G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Androgênios , Animais , Sítios de Ligação/genética , Humanos , Masculino , Camundongos , Neoplasias da Próstata/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Receptores Androgênicos/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismoRESUMO
The open reading frame 50 (ORF50) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the master regulator essential for initiating the viral lytic cycle. Previously, we have demonstrated that the ORF50 protein can cooperate with Sp3 to synergistically activate a set of viral and cellular gene promoters through highly conserved ORF50-responsive elements that harbor a Sp3-binding motif. Herein, we show that Sp3 undergoes proteolytic cleavage during the viral lytic cycle, and the cleavage of Sp3 is dependent on caspase activation. Since similar cleavage patterns of Sp3 could be detected in both KSHV-positive and KSHV-negative lymphoma cells undergoing apoptosis, the proteolytic cleavage of Sp3 could be a common event during apoptosis. Mutational analysis identifies 12 caspase cleavage sites in Sp3, which are situated at the aspartate (D) positions D17, D19, D180, D273, D275, D293, D304 (or D307), D326, D344, D530, D543, and D565. Importantly, we noticed that three stable Sp3 C-terminal fragments generated through cleavage at D530, D543, or D565 encompass an intact DNA-binding domain. Like the full-length Sp3, the C-terminal fragments of Sp3 could still retain the ability to cooperate with ORF50 protein to activate specific viral and cellular gene promoters synergistically. Collectively, our findings suggest that despite the proteolytic cleavage of Sp3 under apoptotic conditions, the resultant Sp3 fragments may retain biological activities important for the viral lytic cycle or for cellular apoptosis. IMPORTANCE The ORF50 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3. In this report, we show that Sp3 is proteolytically cleaved during the viral lytic cycle, and up to 12 caspase cleavage sites are identified in Sp3. Despite the proteolytic cleavage of Sp3, several resulting C-terminal fragments that have intact zinc-finger DNA-binding domains still retain substantial influence in the synergy with ORF50 to activate specific gene promoters. Overall, our studies elucidate the caspase-mediated cleavage of Sp3 and uncover how ORF50 utilizes the cleavage fragments of Sp3 to transactivate specific viral and cellular gene promoters.
Assuntos
Caspases/metabolismo , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiologia , Fator de Transcrição Sp3/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoptose , Caspases/genética , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/fisiopatologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma/genética , Linfoma/metabolismo , Linfoma/fisiopatologia , Linfoma/virologia , Alinhamento de Sequência , Fator de Transcrição Sp3/química , Fator de Transcrição Sp3/genética , Transativadores/genética , Transativadores/metabolismo , Latência ViralRESUMO
In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.
Assuntos
Clusterina/genética , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Expressão Gênica , Transdução de Sinais/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Engenharia Tecidual/métodos , Fator de Transcrição AP-1/metabolismo , Cicatrização/genética , Adulto , Idoso , Células Cultivadas , Criança , Clusterina/metabolismo , Epitélio Corneano/metabolismo , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Doadores de Tecidos , TransfecçãoRESUMO
Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.
Assuntos
Biotina/toxicidade , Proliferação de Células/efeitos dos fármacos , Suplementos Nutricionais/toxicidade , Hormônio Foliculoestimulante/sangue , Espermatogônias/efeitos dos fármacos , Fator de Células-Tronco/metabolismo , Testículo/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Espermatogônias/metabolismo , Espermatogônias/patologia , Testículo/metabolismo , Testículo/patologiaRESUMO
Programmable DNA methylation is required for understanding of transcriptional regulation and elucidating gene functions. We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells. ssDNA-mediated knock-in has recently been reported to completely reduce random integrations. We speculated that by changing MMEJ-to ssDNA-based knock-in, targeted DNA methylation may be achieved through a hemimethylation-symmetric methylation pathway. We herein successfully developed a new system that enables the replacement of an unmethylated promoter with a methylated ssDNA promoter through ssDNA-based knock-in. A DNA methylation ratio of approximately 100% was achieved at the cancer-associated gene SP3 in HEK293 cells. The present results provide a promising framework for artificial epigenetic modifications.
Assuntos
Metilação de DNA , DNA de Cadeia Simples/genética , Epigênese Genética , Edição de Genes/métodos , Proteínas de Neoplasias/genética , Fator de Transcrição Sp3/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Células Clonais , Citosina/metabolismo , DNA de Cadeia Simples/metabolismo , Técnicas de Introdução de Genes , Genoma , Células HEK293 , Humanos , Proteínas de Neoplasias/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Fator de Transcrição Sp3/metabolismo , Transcrição GênicaRESUMO
AIMS: Long noncoding RNA (LncRNA) urothelial cancer associated 1 (UCA1) was dysregulated in colorectal cancers (CRC) and promoted tumor progression of CRC. The aims of this study are to further investigate the underlying mechanism. MAIN METHODS: Short hairpin RNAs (shRNAs) were applied for gene knockdown. microRNA mimic and pcDNA-UCA1 plasmids were transfected for miR-495 and UCA1 overexpression, respectively. MTT was applied to determine cell viability and sensitivity of 5-fluorouracil (FU). Transwell assays were performed to evaluate cell migration/invasion. Angiogenesis was evaluated by tube formation. Western blotting and quantitative PCR were utilized for protein and mRNA detection, respectively. The interaction of UCA1, miR-495 and SP1/SP3 were explored by dual-luciferase assay. RNA pulldown was adopted to determine the UCA1/miR-495 interaction. KEY FINDINGS: UCA1 was significantly upregulated in CRC tissues. UCA1 enhanced cell proliferation, migration/invasion, angiogenesis, epithelial-mesenchymal transition, and resistance to 5-FU in CRC cell lines. MiR-495 was inversely correlated to the expression of UCA1. The results indicated that UCA1 sponged miR-495, leading to the disinhibition of SP1/SP3 expression. SP1/SP3 induced the expression of DNA methyltransferases and, in turn, contributed to UCA1 mediated tumor-promoting actions. Reduction of SP1/SP3 exerted anti-cancer effects, which can be reversed by forced expression of UCA1. SIGNIFICANCE: UCA1-miR-495-SP1/SP3 axis is dysregulated in CRC and contributed to malignant phenotypes of CRC. UCA1-SP1/SP3 may form a positive feedback loop in CRC.
Assuntos
Neoplasias Colorretais/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , RNA Longo não Codificante/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismoRESUMO
Tumor initiation, development, and relapse may be closely associated with cancer stem cells (CSCs). The complicated mechanisms underlying the maintenance of CSCs are keeping in illustration. Long noncoding RNAs (lncRNAs), due to their multifunction in various biological processes, have been indicated to play a crucial role in CSC renewal and stemness maintenance. Using lncRNA array, we identified a novel lncRNA (named lnc408) in epithelial-mesenchymal transition-related breast CSCs (BCSCs). The lnc408 is high expressed in BCSCs in vitro and in vivo. The enhanced lnc408 is critical to BCSC characteristics and tumorigenesis. Lnc408 can recruit transcript factor SP3 to CBY1 promoter to serve as an inhibitor in CBY1 transcription in BCSCs. The high expressed CBY1 in non-BCSC interacts with 14-3-3 and ß-catenin to form a ternary complex, which leads a translocation of the ternary complex into cytoplasm from nucleus and degradation of ß-catenin in phosphorylation-dependent pattern. The lnc408-mediated decrease of CBY1 in BCSCs impairs the formation of 14-3-3/ß-catenin/CBY1 complex, and keeps ß-catenin in nucleus to promote CSC-associated CD44, SOX2, Nanog, Klf4, and c-Myc expressions and contributes to mammosphere formation; however, restoration of CBY1 expression in tumor cells reduces BCSC and its enrichment, thus lnc408 plays an essential role in maintenance of BCSC stemness. In shortly, these findings highlight that the novel lnc408 functions as an oncogenic factor by recruiting SP3 to inhibit CBY1 expression and ß-catenin accumulation in nucleus to maintain stemness properties of BCSCs. Lnc408-CBY1-ß-catenin signaling axis might serve as a new diagnostic and therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp3/metabolismo , beta Catenina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Xenoenxertos , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologiaRESUMO
Elevated expression of ß4-galactosyltransferase (ß4GalT) 3 is correlated with poor clinical outcome of neuroblastoma patients. Our recent study has revealed that the transcription of the ß4GalT3 gene is activated by Specificity protein (Sp) 3 in SH-SY5Y human neuroblastoma cell line. Here we report the biological significance of the Sp3 phosphorylation in the transcriptional activation of the ß4GalT3 gene. The treatment of SH-SY5Y cells with 10% fetal bovine serum (FBS) increased the mitogen-activated protein kinase (MAPK) signaling and the promoter activity of the ß4GalT3 gene. Meanwhile, the treatment with U0126, an inhibitor for MAPK kinase, decreased the MAPK signaling and the promoter activity. These findings indicate that the transcriptional activation of the ß4GalT3 gene is mediated by the MAPK signaling. In SH-SY5Y cells cultured in the medium containing 10% FBS, the serine (Ser) residues in Sp3 were phosphorylated. Human Sp3 contains four Ser residues, Ser73, Ser563, Ser566, and Ser646, as the putative phosphorylation sites. Sp3 mutant with the mutation of Ser73 did not decrease the promoter activation of the ß4GalT3 gene, indicating that Ser73 is uninvolved in the promoter activation of the ß4GalT3 gene by Sp3. In contrast, Sp3 mutants with the mutations of Ser563, Ser566, and Ser646 significantly reduced the promoter activation by Sp3. The results suggest that the phosphorylation of these Ser residues is implicated in the promoter activation by Sp3. This study demonstrates that the phosphorylation of Sp3 plays important roles in the transcriptional activation of the ß4GalT3 gene in human neuroblastoma.
Assuntos
Galactosiltransferases/metabolismo , Neuroblastoma/metabolismo , Transdução de Sinais , Fator de Transcrição Sp3/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Regiões Promotoras Genéticas , Fator de Transcrição Sp3/genética , Ativação TranscricionalRESUMO
Specificity proteins (SPs) have pro-oncogenic functions in cancer cells, ranging from cancer cell proliferation, migration, invasion, and angiogenesis. There is strong evidence that several antineoplastic drugs target depletion of SP proteins via different pathways. However, the mode of action of SP3 and the underlying consequences of its depletion are not well understood. Here, we demonstrate that SP3 is overexpressed in invasive breast cancer cells vs normal counterparts. The gene expression analysis from The Cancer Genome Atlas datasets indicated that SP3 is strongly correlated with Akt signalling-related proteins, G protein subunit alpha 13, and RAB33B (RAB33B, member RAS oncogene family). RNA interference of SP3 decreased active phosphorylation of Akt at serine and threonine sites. These findings indicate that SP3 exhibits a pro-oncogenic function, which clearly fits the description of an nononcogene addiction gene. Future analyses are prompted to uncover the SP3 gene regulation function and to reveal downstream targets of SP3 in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Transcrição Sp3/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Transcrição Sp3/genéticaRESUMO
AIMS: Estrogen can induce inhibition of colonic smooth muscle contraction in male and female mice, which may lead to constipation; however, the mechanisms of inhibition are poorly understood. Hence, this study investigated the effect of estrogen on rat colonic smooth muscle contraction and role of small-conductance Ca2+-activated K+ 3 (SK3) and transcription factors (Sp1 and Sp3) in the underlying mechanisms. MAIN METHODS: The experiment included 24 female Sprague-Dawley (SD) rats divided into 4 groups. The rats were oophorectomized surgically, and a silicone tube containing blank solvent, 0.3 mg/mL estrogen (E2), equal-concentration of estrogen and estrogen receptor antagonist (EI), and bovine serum albumin-E2 (BSA-E2) was implanted. The rats were sacrificed on day 14. The molecular insights were confirmed using real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analyses to determine the effect of estrogenic stimulation on gene and protein expression analyses, respectively. KEY FINDINGS: The E2 group showed significantly greater SK3 expression (P < .005) compared with other groups and significantly lowers smooth muscle cell (SMC) contractility (P < .005). Estrogen stimulation and SK3 overexpression resulted in a significant decrease (P < .05) in Ca2+ mobilization in the E2 group versus the control group. Further, the E2 group showed significantly higher Sp1 mRNA (P < .05) but lower Sp3 mRNA expression (P < .05) and protein expression (P < .001) compared with other groups. SIGNIFICANCE: E2 may promote SK3 expression by its genomic effect and inhibit colonic contraction by affecting SK3 expression via an interaction between Sp1 and Sp3.
Assuntos
Cálcio/metabolismo , Colo/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Contração Muscular , Músculo Liso/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Feminino , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismoRESUMO
Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.
Assuntos
Dexametasona/efeitos adversos , Fêmur , Desenvolvimento Fetal/efeitos dos fármacos , Lâmina de Crescimento , Exposição Materna/efeitos adversos , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Células Cultivadas , Feminino , Fêmur/efeitos dos fármacos , Fêmur/embriologia , Fêmur/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/patologia , Histona Desacetilase 1/metabolismo , Masculino , Troca Materno-Fetal , Células-Tronco Mesenquimais/citologia , Gravidez , Ratos , Ratos Wistar , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Fator de Transcrição Sp3/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismoRESUMO
The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.
Assuntos
Herpesvirus Humano 8/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Transativadores/genética , Transcrição Gênica , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Células Clonais , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Herpesvirus Humano 8/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Linfócitos/virologia , Camundongos , Ligação Proteica , Elementos de Resposta , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Algoritmos , Animais , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Edição de Genes , Camundongos , Fator de Transcrição Sp3/metabolismo , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
BACKGROUND: Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. RESULTS: Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. CONCLUSIONS: Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
This study aims to investigate whether terminal differentiation-induced ncRNA (TINCR) has an effect on apoptosis and autophagy induced by ALA-PDT in cutaneous squamous cell carcinoma (CSCC). A431 cells were treated with 5-aminolevulinic acid (ALA) solution at different concentrations and for different duration time. A431 cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, relative TINCR messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). A431 cell apoptosis was examined by flow cytometry. Relative apoptosis/autophagy-related protein expression was analyzed by Western blot analysis. The effect of TINCR on cell autophagy was detected by RFP-LC3 immunofluorescence assay. Reactive oxygen species concentration was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Relative expressions of ERK1/2 and specificity protein 3 (Sp3) in A43 cells were detected by Western blot analysis and qRT-PCR. Sp3 binding sites were analyzed by ChIP-qPCR. The relative transcription activity was measured with luciferase reporter assay. ALA-PDT treatment at 3.2 mmol/L for 120 minutes significantly promoted TINCR expression in CSCC A431 cells, and TINCR promoted ALA-PDT-induced apoptosis and cell autophagy. Furthermore, ALA-PDT promoted TINCR expression through ERK1/2-SP3 pathway. Sp3 promoted TINCR transcription by binding TINCR promoters. Our data indicated that TINCR involves in ALA-PDT-induced apoptosis and autophagy in CSCC.