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1.
Cell Mol Life Sci ; 77(9): 1793-1810, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31375868

RESUMO

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica , Íntrons/genética , Fator de Transcrição Sp2/antagonistas & inibidores , Fator de Transcrição Sp2/genética , Valina/metabolismo , Sequência de Aminoácidos , Animais , Duplicação Gênica , Humanos , Filogenia , Homologia de Sequência , Fator de Transcrição Sp2/metabolismo , Ativação Transcricional , Valina/genética
2.
Dig Dis Sci ; 59(8): 1743-53, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24623314

RESUMO

BACKGROUND: MicroRNAs play important roles in the development and progression of various cancers. Recent studies have shown that miR-638 was downregulated in several tumors; however, its role in gastric cancer (GC) has not been investigated in detail. AIMS: The purpose of this study was to determine the role of miR-638 and to elucidate its regulatory mechanism in GC. METHODS: The expression levels of miR-638 and specificity protein 2 (Sp2) were detected by real-time PCR and Western blotting in GC. After pcDNA6.2-GW/EmGFP-miR-638 vector, miR-638 inhibitor and Sp2-siRNA transfection, the AGS cell proliferation was investigated by MTT assay and cell cycle, and apoptosis was detected using the Annexin V/PI. In addition, the regulation of Sp2 by miR-638 was evaluated by real-time RT-PCR, Western blot and luciferase reporter assays; cyclin D1 expression was measured by Western blotting. RESULTS: The expression of miR-638 is dramatically down-regulated and Sp2 expression is remarkably up-regulated in GC tissues. Luciferase assays revealed that miR-638 inhibited Sp2 expression by targeting the 3'-UTR of Sp2 mRNA. Overexpression of miR-638 and Sp2-siRNA reduced Sp2 expression at both the mRNA and protein levels in vitro, and inhibition of miR-638 increased Sp2 expression. Moreover, we found that miR-638 overexpression and Sp2-siRNA markedly suppressed cell proliferation with decreasing expression of cyclin D1 and inducing G1-phase cell-cycle arrest in vitro; inhibition of miR-638 significantly promoted cell proliferation by increasing expression of cyclin D1 and leading more cells into the S and G2/M phase. CONCLUSIONS: Our results demonstrated that miR-638 suppressed GC cell proliferation by targeting Sp2 with influence on the expression of cyclin D1. We suggest that miR-638 might be a candidate predictor or an anticancer therapeutic target for GC patients.


Assuntos
MicroRNAs/metabolismo , Fator de Transcrição Sp2/antagonistas & inibidores , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D2/metabolismo , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Fator de Transcrição Sp2/genética , Neoplasias Gástricas/patologia
3.
Nucleic Acids Res ; 40(16): 7844-57, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22684502

RESUMO

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.


Assuntos
Regulação da Expressão Gênica , Fator de Transcrição Sp2/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proliferação de Células , DNA/química , DNA/metabolismo , Mineração de Dados , Deleção de Genes , Perfilação da Expressão Gênica , Genoma , Células HEK293 , Células HeLa , Humanos , Camundongos , Matrizes de Pontuação de Posição Específica , Regiões Promotoras Genéticas , Interferência de RNA , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp2/antagonistas & inibidores , Fator de Transcrição Sp2/genética
4.
Am J Physiol Heart Circ Physiol ; 291(2): H600-11, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16617124

RESUMO

Combinatorial interactions between cis elements and trans-acting factors are required for regulation of cardiac gene expression during normal cardiac development and pathological cardiac hypertrophy. Sp factors bind GC boxes and are implicated in recruitment and assembly of the basal transcriptional complex. In this study, we show that the cardiac troponin T (cTnT) promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC box in a sequence-specific fashion and consist of Sp1, Sp2, and Sp3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter "in vivo." Cotransfected Sp1 trans-activates the cTnT promoter in cardiomyocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of the cTnT gene in embryonic cardiomyocytes. Sp3 repression of Sp1-mediated cTnT promoter activation is dose dependent, inferring a mechanism of competitive binding/inhibition. To evaluate the role of Sp factors in cardiac gene expression in vivo, we have established a clinically relevant animal model of pathological cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle, and pressure loading of the right ventricle. Sp1 expression is increased in all four hypertrophied cardiac chambers, whereas Sp3 expression is diminished. This observation is consistent with the in vitro activating function of Sp1 and inhibitory effects of Sp3 on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program in vivo.


Assuntos
Cardiomegalia/metabolismo , Coração/fisiologia , Regiões Promotoras Genéticas/fisiologia , Fator de Transcrição Sp2/antagonistas & inibidores , Fator de Transcrição Sp3/biossíntese , Fator de Transcrição Sp3/farmacologia , Troponina T/genética , Animais , Western Blotting , Cardiomegalia/genética , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Cromatina/metabolismo , DNA/biossíntese , DNA/genética , Regulação para Baixo/fisiologia , Drosophila/metabolismo , Elementos E-Box/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Coração/efeitos dos fármacos , Imuno-Histoquímica , Imunoprecipitação , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcômeros/metabolismo , Ovinos , Fator de Transcrição Sp2/farmacologia , Fator de Transcrição Sp3/fisiologia , Técnicas de Cultura de Tecidos , Transfecção
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