Negative Regulatory Role of Sp1 in Metal Responsive Element-mediated Transcriptional Activation

Abstract

Transcription of mammalian metallothionein (MT)genes is activated by heavy metals via multiple copies of a cis-acting DNA element, the metal-responsive element (MRE). Our previous studies have shown that certain MREs of the human MT-IIA gene (MREb, MREc, MREd, and MREf) are less active than the others (MREa, MREe, and MREg). Gel shift analysis of HeLa cell nuclear proteins revealed that whereas the active MREs strongly bind the transcription factor MTF-1 essential for metal regulation, the less active MREs bind another distinct protein, MREb-BF. This protein recognizes the GC-rich region of MREb rather than the MRE core required for MTF-1 binding. All the MREs recognized by MREb-BF contain the CGCCC and/or CACCC motif, suggesting that the MREb-BF⅐MRE complex contains Sp1 or related proteins. Supershift analysis using antibodies against Sp1 family proteins as well as gel shift analysis using the recombinant Sp1 demonstrated that Sp1 represents the majority of MREb-BF activity. An MREb mutant with reduced affinity to Sp1 mediated zinc-inducible transcription much more actively than the wild-type MREb. Furthermore, when placed in the native promoter, this mutant MREb raised the overall promoter activity. These results strongly suggest that Sp1 acts as a negative regulator of transcription mediated by specific MREs.Heavy metal-induced transcriptional activation of the genes coding for metallothioneins (MTs) 1 is mediated by multiple copies of a cis-acting DNA element, the metal-responsive element (MRE; Refs. 1-3). It has been shown that the mouse MRE-binding transcription factor-1 (MTF-1) is essential for MRE-mediated transcription from gene knockout (4) and antisense RNA expression studies (5). Nevertheless, it remains possible that metal regulation of MT genes involves additional transcriptional regulators. A number of MRE-binding proteins have been reported (reviewed in Ref. 6), and cDNAs encoding MRE-binding proteins distinct from MTF-1 have been cloned (7,8). However, no protein except MTF-1 has been demonstrated to be functionally relevant to metal regulation.MREs were first identified by deletion analysis in duplicated sites upstream of the mouse MT-I (mMT-I) and human MT-IIA (hMT-IIA) genes (1, 2, 9). After that, a search for MRE-related sequences revealed that there are multiple MRE homologs in the upstream region of all the mammalian MT genes reported so far (3, 10). These facts imply that such a redundancy is probably essential for the high metal inducibility of MT gene promoters. However, MREs are not always functionally equivalent. In transfection assays using reporter genes driven by synthetic MRE-containing promoters, striking differences in zinc-induced transcriptional activity were observed among MREs of the mMT-I (3) and hMT-IIA (11) genes, despite the observation that all these sequences share the highly conserved MRE core (whose functional importance has been demonstrated; Refs. 12, 13) and the flanking semi-conserved GC-rich sequence. In addition, we have observed that the transcriptional acti...

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.…”

“…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).…”

“…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].…”

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).…”

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO4in vivo footprinting on its promoter region

“…34 However, there are several reports that Sp1 serves the unusual role of repressing gene expression and acts as a silencer of gene expression in many cell lines. [35][36][37] Moreover, it was reported that a nuclear protein, p74, interacts directly with the transactivation domain of Sp1 protein and negatively regulates the Sp1- mediated transactivation. 38 These findings suggest that in our present report Sp1 binds gelsolin promoter and directly or indirectly repress expression of gelsolin of human bladder cancer cells.…”