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Stability of the Sp3-DNA complex is promoter-specific: Sp3 efficiently competes with Sp1 for binding to promoters containing multiple Sp-sites

Center for Molecular Biology of Oral Diseases, College of Dentistry (M/C 860), University of Illinois at Chicago, 801 South Paulina Street, Chicago, IL 60612, USA.
Nucleic Acids Research (Impact Factor: 9.11). 10/2003; 31(18):5368-76. DOI: 10.1093/nar/gkg706
Source: PubMed

ABSTRACT The transcription regulatory protein Sp3 shares more than 90% sequence homology with Sp1 in the DNA-binding domain and they bind to the same cognate DNA-element. However, the transcriptional activities of these two Sp-family factors are not equivalent. While Sp1 functions strictly as a transcriptional activator, Sp3 has been shown to be transcriptionally inactive for promoters containing multiple Sp-binding sites. In the present study, we show that the DNA-binding property of Sp3 is promoter dependent and is different from Sp1. The 116 kDa Sp3 polypeptide binds as a monomer to a single Sp-binding site but readily forms slower migrating complexes with adjacent Sp-binding sites. The slower migrating Sp3-DNA complexes are significantly more stable than monomeric Sp3-DNA complexes or multimeric Sp1-DNA complexes. As a consequence, Sp3 can efficiently compete with Sp1 for binding to regions containing multiple Sp sites. The transcription regulatory function of Sp3 is also significantly different from Sp1. Unlike Sp1, Sp3 does not synergistically activate transcription of promoters containing multiple Sp-binding sites. Therefore, although Sp3 is a transcription activator, Sp3 reduces Sp1-dependent transcription of promoters containing adjacent Sp-binding sites by competing with Sp1 for promoter occupancy and thereby blocking the synergistic transactivation function of Sp1. Taken together, this study provides a possible mechanism of the promoter-specific transcription repression function of Sp3.

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    • "Whereas Sp1 is usually an activator of transcription, Sp3 functions either as an activator or as a repressor [12] [13] [14]. Although Sp3 lacks the ability of Sp1 to bind to DNA as a multimer, it can antagonize Sp1 by blocking its synergistic transactivation function [15]. Sp1 and Sp3 generally compete for the same binding sites on a variety of promoters and regulate the target gene expression [16] [17] [18] [19] [20] [21]. "
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    ABSTRACT: Ascent to high altitude is associated with tissue hypoxia resulting from the decrease in partial pressure of atmospheric oxygen. The hippocampus, in particular, is highly vulnerable to hypoxic insult, which at least in part can be attributed to the occurrence of glutamate excitotoxicity. Although this excitotoxic damage is often related to increased NMDA receptor activation and subsequent calcium-mediated free radical generation, the mechanisms involving the transcriptional regulation of NMDA receptor subunit expression by hypoxic stress remains to be explored. Our study reveals a novel mechanism for the regulation of expression of the NR1 subunit of NMDA receptors by the Sp family of transcription factors through an oxidative-stress-mediated mechanism that also involves the molecular chaperone Hsp90. The findings not only show the occurrence of lipid peroxidation and DNA damage in hippocampal cells exposed to hypoxia but also reveal a calcium-independent mechanism of selective oxidation and degradation of Sp3 by the 20S proteasome. This along with increased DNA binding activity of Sp1 leads to NR1 upregulation in the hippocampus during hypoxic stress. The study therefore provides evidence for free radical-mediated regulation of gene expression in hypoxia and the scope of the use of antioxidants in preventing excitotoxic neuronal damage during hypoxia.
    Free Radical Biology and Medicine 04/2010; 49(2):178-91. DOI:10.1016/j.freeradbiomed.2010.03.027 · 5.71 Impact Factor
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    • "Although Sp1 and Sp3 share more than 90% sequence homology in their DNA-binding domain and although they bind to the same cognate DNA element with similar affinity, they have strikingly different functions (for review, see Li et al., 2004). Sp3 has been reported to act both as a positive or negative regulator of transcription, depending on promoter and cell type (Hagen et al., 1994; Yu et al., 2003). It has been suggested that Sp3 displays repression of Sp1-mediated activation in promoters with multiple functional Sp-binding sites, whereas promoters with a single Sp-binding site are not responsive to Sp3-mediated repression (Suske, 1999). "
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    ABSTRACT: The molecular mechanism regulating serotonin 5-HT(7) receptor expression is still unclear. In this study we provide evidence that transcription of the 5-HT(7) gene is at least partly regulated by Sp1 and Sp3. We isolated and sequenced >3000 bp of the upstream sequences and identified by RACE a number of transcriptional initiation sites over a region of 300 bp upstream of the coding region. This region has a high GC content, but contains no obvious TATA or a CAAT box. Besides a Sp1/Sp3 consensus motif, regulatory elements for AP2, Egr-1 and MAZ are also present. Transient transfection assays using deletion variants indicated that the GC-rich region is essential for full promoter activity. The role of Sp1 in this was confirmed by transient overexpression of both wild type Sp1 or dominant-negative forms. By gel shift and supershift analyses, targeting the Sp1 consensus sequence and the GC-rich region just upstream of the transcription initiation sites, binding of Sp1 and Sp3 was demonstrated. Both in vitro as well as in vivo experiments, using a cell line which endogenously expresses the 5-HT(7) receptor, indicated that mithramycin A, an inhibitor of Sp1/3 transcription factor binding, was able to inhibit 5-HT(7) promoter activity. Taken together these results support the essential role of Sp factors in regulating 5-HT(7) promoter activity.
    Gene 05/2007; 391(1-2):252-63. DOI:10.1016/j.gene.2007.01.003 · 2.08 Impact Factor
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    • "The Sp family members are characterized by a motif of three conserved Cys2His2 zinc fingers, which form the DNA-binding domain (Suske, 1999). Sp1 and Sp3 share more than 90% sequence homology in the DNA-binding domain and bind to the same GC-rich DNA domains, which have been hypothesized to lead to either activation or repression of gene activity based on the promoter context or cellular background (Majello et al., 1997; Bouwman and Philipsen, 2002; Ammanamanchi et al., 2003; Yu et al., 2003). "
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    ABSTRACT: Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.
    Molecular Biology of the Cell 03/2006; 17(2):585-97. DOI:10.1091/mbc.E05-08-0775 · 4.55 Impact Factor
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