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

Abstract

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C 2 H 2 -type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GCbox recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.Transcriptional regulation of the eukaryotic gene is a complicated process, involving a series of complex molecular interactions among regulatory and transcription factors. Specificity protein 1 (Sp1) 1 is probably one of the best characterized sequence-specific transcription factors, and has numerous functions in the transcription of many cellular and viral genes harboring GC boxes in their promoters (1, 2). Sp1 and its family of proteins have been implicated in a host of essential biological processes, and have been proven important in apoptosis, cell growth inhibition, differentiation, and carcinogenesis (Refs. 3-5 and references therein).Sp1 is a member of the Sp-multigene family, which also includes Sp2, Sp3, Sp4, Sp5, Sp6, Sp7, and Sp8 (3-5). The Sp-family proteins exhibit similar domain structures and are evolutionarily closely related. All of these proteins possess highly conserved C 2 H 2 -type zinc finger DNA binding domains at their C termini, and all belong to the Krü ppel-like zinc finger superfamily (3-5). In addition, the Sp-family proteins have been demonstrated to undergo post-translational modifications as the result of diverse mechanisms. For example, Sp1 is glycosylated and phosphorylated by Erk2, protein kinase C, casein kinase II, and cAMP-dependent protein kinase; Sp3 is acetylated and SUMOylated by the protein inhibitor of activated STAT (PIAS1, Ref. 3 and references therein, Refs. 6 -10).The Sp1 protein co...

A Novel POK Family Transcription Factor, ZBTB5, Represses Transcription of p21CIP1 Gene

“…The various pGL2-p21-Luc, pGL2-p53-Luc, pGL2-ARF-Luc, pGL2-HDM2-Luc, pcDNA3.1-p53, pcDNA3.1-Sp1, pG5-5x(GC-box)-Luc, corepressor expression vectors, and VP16-corepressors we used have been reported elsewhere or were prepared by us (20,23,25). The pcDNA3-ZBTB5 plasmid was prepared by cloning a cDNA fragment (KIAA0354) into pcDNA3.0 (Invitrogen).…”

“…The molecular interaction between ZBTB5 and p53 or Sp1 on the endogenous p21 promoter and histone modification at the p21 proximal promoter in the cells was analyzed by the standard qChIP assay protocol, as described elsewhere (20,23,25).…”

“…Electromobility Shift Assay (EMSA)-EMSAs were carried out as described previously (20,23,25). The probe sequences of Sp1 response elements on the p21 proximal promoter or the sequences of p53 response elements on the p21 distal promoter ZBTB5, Transcription Repressor of p21CIP1 used in EMSA were as follows (only top strands are shown): GC-box 1, 5Ј-GATCGGGAGGGCGGTCCCG-3Ј; GC-box 2, 5Ј-GATCTCCCGGGCGGCGCG-3Ј; GC-box 3, 5Ј-GATCCG-AGCGCGGGTCCCGCCTC-3Ј; GC-box 4, 5Ј-GATCCTTGA-GGCGGGCCCG-3Ј; GC-box 5/6, 5Ј-GATCGGGCGGGGCG-GTTGTATATCA-3Ј; p53RE-1, 5Ј-GATCCGTTAG-AGGAAGAAGACTGGGCATGTCTG-3Ј; p53RE-2, 5Ј-GATCCATCAGGAACATGTCCCAACATGTTGAGCTC-3Ј.…”

HDACs and HDAC inhibitors in colon cancer

“…18 NCoR and SMRT also serve as the principal contact between the co-repressor complex and its transcription factor partners, which among others, include nuclear hormone receptors (thyroid, retinoic acid and retinoid X receptors), NFκB, AP-1, SMAD proteins, c-Myb, PLZF, Bcl-6, MyoD and Sp1. [19][20][21] The interaction of HDAC3 with the N-terminal DAD (deacetylase activating domain) domain of NCoR/SMRT, has also been shown to be essential for activation of HDAC3 deacetylase activity. 22 Finally, the class II HDACs 4, 5 and 7 interact with the B-CoR, 23 BCoRL1 24 and CtBP co-repressors.…”

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).…”