Sp1 In Cells Research Articles

MED27 promotes malignant behavior of cells by affecting Sp1 in breast cancer.

The aim of this study was to investigate the expression of mediator complex subunit 27 (MED27) in breast cancer (BC) and explore its effects on the proliferation and apoptosis of BC cells. The expression of MED27 in 60 BC tissues and para-cancer tissues was detected. Based on the significantly high expression level of MED27 in tumors, the tumor samples were divided into high-expression group and low-expression group according to the median standard, with 30 samples in each group. Then, the association between MED27 expression and clinicopathological features of patients was analyzed. The correlation between MED27 expression and survival time of patients was estimated using the Kaplan-Meier method. Next, the expression level of MED27 in cells was also measured using qRT-PCR assay. In vitro study, si-MED27 was designed to interfere with the expression of MED27 in MDA-MB-231 cells. To further explore the mechanism of MED27 in BC, the expression level of SP1 in cells was examined after different treatments. In quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay, MED27 was found to be highly expressed in both BC tissues and cells. Then, the relationship between MED27 expression and clinical pathological data was statistically analyzed, and it was found that MED27 expression was correlated with tumor size and grade. In the 60-month follow-up and Kaplan-Meier analysis, patients with high expression of MED27 had a poor prognosis. In in vitro study, MED27 expression in cells was down-regulated by transfection with si-MED27. Western blot (WB) analysis suggested that si-MED27 could effectively reduce the protein expression level of MED27 in cells, and the specificity protein 1 (Sp1) expression was also limited. In CCK-8, clone formation and flow cytometry experiments, the proliferation of cells with low MED27/Sp1 expression was suppressed, while cell apoptosis was promoted. MED27 acted as an oncogene in BC. By affecting Sp1, MED27 could be a new therapeutic target for the treatment of BC.

Context-dependent Dysregulation of Transcription by Mutant Huntingtin

Huntington disease (HD) is an adult-onset neurodegenerative disease caused by expansion of a polyglutamine (poly(Q) tract in the N-terminal region of huntingtin (htt). Although the precise mechanisms leading to neurodegeneration in HD have not been fully elucidated, transcriptional dysregulation has been implicated in disease pathogenesis. In HD, multiple N-terminal mutant htt fragments smaller than the first 500 amino acids have been found to accumulate in the nucleus and adversely affect gene transcription. It is unknown whether different htt fragments in the nucleus can differentially bind transcription factors and affect transcription. Here, we report that shorter N-terminal htt fragments, which are more prone to misfolding and aggregation, are more competent to bind Sp1 and inhibit its activity. These effects can be reversed by Hsp40, a molecular chaperone that reduces the misfolding of mutant htt. Our results provide insight into the beneficial effects of molecular chaperones and suggest that context dependent transcriptional dysregulation may contribute to differential toxicity of various N-terminal htt fragments.

Insulin Dynamically Regulates Calmodulin Gene Expression by Sequential O-Glycosylation and Phosphorylation of Sp1 and Its Subcellular Compartmentalization in Liver Cells

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Vascular smooth muscle cell-specific regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1) transcription by Sp1 is mediated via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.

Smooth muscle cells (SMC) play a central role in common vascular pathologies such as atherosclerosis and restenosis. Understanding the molecular regulation of SMC proliferation at a transcriptional level may provide important clues for the targeted control of vascular hyperplasia. We recently reported the capacity of the transcription factor Sp1 to down-regulate p21(WAF1/Cip1) production thereby reducing p21(WAF1/Cip1)-cyclin D1-Cdk4 complex formation and inhibiting vascular SMC proliferation (Kavurma and Khachigian [2003] J. Biol. Chem. 278, 32537-32543). We have now localized the Sp1-response elements in the p21(WAF1/Cip1) promoter responsible for p21(WAF1/Cip1) repression in WKY12-22 SMCs. The proximal region of the p21(WAF1/Cip1) promoter contains five distinct Sp1-binding elements that we have termed A, B, C, D, and E. Electrophoretic mobility shift analysis revealed that SMC nuclear Sp1 interacts with all five Sp1-binding sites, and each of these sites is critical for Sp1 repression of the p21(WAF1/Cip1) promoter, since mutation in any one element ablates repression, and in some cases results in activation. In contrast, only elements C, D, and E are bound by Sp1 in endothelial cells. Sp1 overexpression activates the p21(WAF1/Cip1) promoter in this cell type. Furthermore, mutation in any of these five elements is not sufficient to prevent activation of the p21(WAF1/Cip1) promoter by Sp1 in endothelial cells. Surprisingly, double mutations of elements C and E facilitates superactivation by Sp1 in both cell types, whereas triple mutations of C, D, and E inactivate the promoter. These findings demonstrate cell type-specific regulation of p21(WAF1/Cip1) transcription by Sp1 via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.

O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability.

The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.

The Sp1 Transcription Factor Regulates Cell Type-Specific Transcription of MUC1

Because the MUC1 mucin is highly expressed in breast and other carcinomas, interest is focused on the MUC1 promoter, particularly in the context of the delivery of genes to carcinomas. Earlier in vitro studies showed that the region between -152 and -66 of the MUC1 promoter is required for transcriptional activity in MUC1-expressing cells. Experiments reported here showed that sequences -119/-62 within this region are able to modulate transcription of the heterologous constitutively active herpes simplex virus thymidine kinase promoter in a pattern consistent with MUC1 expression. Band-shift experiments showed that although several factors (including Sp1 and Sp3) bind to these sequences, the element important in directing this MUC1 pattern of expression was an Sp1 GC box at -97. The data also show that the positioning or phase of the GC box was crucial for directing expression. The importance of the Sp1 transcription factor was confirmed by demonstrating that overexpression of Sp1 in MUC1-nonexpressing cells increased, not only the expression of a reporter gene driven by the 1.4-kb MUC1 promoter, but also the expression of MUC1 from the endogenous gene. Together, these data define an important role for Sp1 in the cell type-specific transcription of MUC1.

Cooperative Actions of HIV-1 Vpr and p53 Modulate Viral Gene Transcription

Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have examined the capacity of the viral auxiliary protein, Vpr, to modulate transcriptional activity of the HIV-1 promoter sequence located within the long terminal repeat (LTR). We demonstrate that ectopic expression of Vpr in human astrocytic cells, U-87MG, enhances the basal activity of the viral promoter in transfected cells and that the GC-rich sequences, spanning nucleotides -80 to -43, are important for this activity. Since this region serves as the target for p53-induced suppression of LTR activity and interacts with the ubiquitous transcription factor, Sp1, we examined the cooperative activity of Vpr, p53, and Sp1 upon LTR transcription. Results from co-transfection studies indicated that overexpression of wild type p53, but not mutant p53, decreases the level of activation of the LTR by Vpr. Transcriptional activation of the LTR by Vpr required the presence of Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed to augment LTR activity. Results from protein-protein interaction studies indicated that Vpr is associated with both p53 and Sp1 in cells with ectopic expression of these proteins. Moreover, it was evident that p53 and Sp1 interact with each other in these cells. These functional and structural studies provided a working model on the cooperative interaction of Vpr with cellular proteins Sp1 and p53 and control of viral gene transcription at immediate early stage of infection prior to the participation of other viral regulatory proteins.

Increased DNA binding and transcriptional activity associated with transcription factor Sp1 in K562 cells transfected with the myeloid-specific c-fes tyrosine kinase gene

Myeloblast cell line K562, when stably transfected with the human genomic c-fes sequence encoding a proto-oncogene tyrosine-protein kinase, acquires the characteristics of more mature granulocytic cells (WS-1 cells) and the ability to undergo differentiation (Yu, G., Smithgall, T. E., and Glazer, R. I. (1989) J. Biol. Chem. 264, 10276-10281). To explore the role of transcription factors in the differentiation process, WS-1 cells were analyzed for the presence of DNA-binding proteins capable of interacting with the 5'-long terminal repeat (LTR) region of human immunodeficiency virus (HIV)-1, that contains the binding sequences for transcription factors Sp1 and NFKB. Southwestern blotting and mobility shift assays revealed the presence of Sp1 in K562 and WS-1 cells. The DNA-binding activity of Sp1 was significantly greater in WS-1 cells than in K562 cells, despite the detection by immuno-blotting of equivalent quantities and degrees of heterogeneity of Sp1 in both cell lines. DNA footprinting of the HIV-1 5'-LTR demonstrated that two of the three Sp1-binding sites and both NFKB binding sequences were protected by nuclear extracts from WS-1 cells, while no protection was afforded by nuclear extracts from K562 cells. Analysis of transcription in vitro by primer extension revealed enhanced initiation of transcription from the HIV-1 5'-LTR by nuclear extracts from WS-1 cells, but not from K562 cells. These data indicate that the response evoked by the c-fes tyrosine-protein kinase leads to enhanced DNA binding activity of Sp1 and NFKB, that results in the activation of transcription from the HIV-1 5'-LTR.