Sp1 acts as a repressor of the human adenine nucleotide translocase-2 (ANT2) promoter

Abstract

The human adenine nucleotide translocator-2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides 279 and 268 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1-mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1-mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox-mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox-bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species.Keywords: mitochondrial promoter; transcription repression; adenine nucleotide translocator; Sp1.Sp1 is the prototype member of a family of four [1,2] [6,7] that accounts at least partially for the observations that, unlike Sp1 and Sp4, Sp3 functions as a repressor [4,8].Although Sp1 bears no repression domain or motifs, Sp1 protein or Sp1 binding elements are often involved in the negative regulation of specific genes. Recently, it was shown that Sp1 can direct repression of specific promoters by targeting corepressor (histone deactylase) to chromatin [9]. However, in most cases such negative regulation can be accounted for by binding-site competition and Sp1 replacement by Sp3 [4,10,11] or other Sp-like inhibitor proteins [12][13][14][15]. Inhibitory complex formation has also been reported between Sp1 and other transfactors [16 -19]. These presumably mask the Sp1 activation domain sites. However, the results of several experiments suggest that negative regulation can also be exerted between separate GC elements that bind Sp proteins [20 -25]. The mechanism of such repression is not yet clear.In our studies on the promoter region of the human adenine nucleotide translocator-2 (ANT2) gene [25,26], we identified a unique Sp1-dependent repressor site (the Cbox) located at the transcription start site that appears to prevent activation by Sp1 bound to the major activating elements (the Abox and Bbox). Because of the location of the Cbox, we proposed that Sp1 might act sterically to prevent assembly of the preinitiation complex or recruitment of the polymerase complex to initiation start. In the present study, we provide evidence that this is not the case. Rather, our data support a mechanism of repression in which Sp1 bound to the repressor element near the transcription start site is able to attenuate activation by Sp1 bound to upstream activation elements. E X P E R I M E N T ...

Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

“…In addition, the molecular interactions between Sp1ID and the corepressors can explain how Sp1 can function as a transcription repressor in certain Sp1-bound promoters (23)(24)(25)(26)(27), even though Sp1 is known to be a transcription activator, in general (Refs. 3-5 and references therein) (Fig.…”

“…Indeed, many reports have implied that Sp1 and the Sp1-like transcription factors exhibit a bipolar character; i.e. they function as either transcription activators or repressors, depending on the promoter to which they bind and the coregulators with which they interact (1, 2, 9, 12-14, [23][24][25][26][27].…”

“…In addition, these molecular interactions with the corepressors may constitute a key factor in the transcriptional regulation of the Sp-family, via the modulation of the target GC-box binding of Sp1ZFDBD. Moreover, the molecular interactions occurring between Sp1ID and the corepressors may explain the transcription repression by the Sp1 bound to the enhancer or to the proximal promoter GC-box (23)(24)(25)(26)(27).…”

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…Sp1 is ubiquitously expressed in human tissues serving mainly as an activator of transcription for housekeeping genes and genes involved in growth regulation, but it can also act as a repressor in certain circumstances (Armstrong et al, 1997;Briggs et al, 1986;Dennig et al, 1995;Hagen et al, 1994;Kadonaga et al, 1987;Kwon et al, 1999;Lee et al, 2005;Li et al, 1998;Murata et al, 1994;Ogra et al, 2001;Shou et al, 1998;Suzuki et al, 2000;Zaid et al, 2001). Containing three C2H2 domains, Sp1 binds to the consensus sequence 5'-(G/t)GGGCGG(G/A)(G/A)(C/T)-3' in GC-rich promoters found in many genes (Armstrong et al, 1997;Berg, 1992;Bucher, 1990;Kadonaga and Tjian, 1986).…”

Transcriptional regulation of the Drosophila ANT gene by the DRE/DREF system

“…It has been reported that ANT2 mRNA levels were increased significantly in the patient liver biopsies (Bonod-Bidaud et al 2001) and in cases of renal oncocytoma (Heddi et al 1996). Although it has been reported that Sp1 functions as a repressor of the ANT2 promoter (Zaid et al 2001), and that Ant1 is regulated specifically by transforming growth factor-β1 (TGF-β1) in astrocytes (Law et al 2004), the regulatory mechanisms of ANT gene expression have yet to be thoroughly elucidated. Drosophila harbors two ANT genes, sesB and ANT2, which share with one another 72% of the nucleotide sequence and an amino acid sequence identity of 78% (Zhang et al 1999).…”