Sp3 is a member of the Sp family of transcription factors and binds to DNA with affinity and specificity comparable to that of Sp1. We demonstrate that Sp3 is a bifunctional transcription factor that can both activate and repress transcription. Gene fusion experiments in mammalian cells demonstrate that the Sp3 activation potential is distributed over an extensive glutamine-rich N-terminal region, whereas the repressor activity has been mapped in a 72-amino acid region located at the 5' of the zinc finger DNA-binding domain. We demonstrated that the repression activity is strictly dependent on the context of the DNA-binding sites bound by Sp3. We found that Sp3 represses transcription of promoters bearing multiple GAL4 DNA-binding sites, whereas it activates isogenic reporters containing a single GAL4-binding site. Transfection experiments in Drosophila cells that lack endogenous Sp activity demonstrated that Sp3 does not possess an active repression domain that can function in insect cells, rather it is a weak transcriptional activator of the c-myc promoter. Our results strongly suggest that Sp3 is a dual-function regulator whose activity is dependent upon both the promoter and the cellular context.

Recently, a family of transcription factors structurally related to Sp1 has been described; thus, more than one activator may bind to the GC boxes present in a number of viral and cellular promoters. We have compared the transactivation potentials of Sp1, Sp3 and Sp4 proteins on the human immunodeficiency virus type 1 (HIV-1) promoter. The long terminal repeat (LTR) of HIV-1 contains three binding sites for the transcription factor Sp1 (GC boxes) which are involved in both basal and Tat-mediated transcriptional activation. Moreover, a cooperative interaction between NF-kappa B and Sp1 is required for HIV enhancer activation. We now demonstrate that Sp4 is an activator, while the Sp3 protein represses basal expression of HIV promoter. Remarkably, we found that over-expression of the transcription factor Sp3 was able to suppress Tat-mediated transactivation. These inhibitory effects of Sp3 correlate with its DNA binding activity, suggesting that Sp3 inhibition involves competition with Sp1 for occupancy of the GC boxes. Next, we have analyzed the role of different Sp1-related proteins in the stimulation of HIV-1 promoter in response to mitogens. We found that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, an interaction between NF-kappa B and the trans-acting domain (A domain) of Sp1 bound to an adjacent site must occur. We found that the cooperative interaction between NF-kappa B and Sp1 is highly specific, since neither Sp3 nor Sp4 is capable of cooperating with NF-kappa B.

MiR-21 is a microRNA (miRNA) frequently overexpressed in human cancers. Here we show that miR-21 is upregulated both in vitro and in vivo by oncogenic Ras, thus linking this miRNA to one of the most frequently activated oncogenes in human cancers. Ras regulation of miR-21 occurs with a delayed kinetic and requires at least two Ras downstream pathways. A screen of human thyroid cancers and non-small-cell lung cancers for the expression of miR-21 reveals that it is overexpressed mainly in anaplastic thyroid carcinomas, the most aggressive form of thyroid cancer, whereas in lung its overexpression appears to be inversely correlated with tumor progression. We also show that a LNA directed against miR-21 slows down tumor growth in mice. Consistently, a search for mRNAs downregulated by miR-21 shows an enrichment for mRNAs encoding cell cycle checkpoints regulators, suggesting an important role for miR-21 in oncogenic Ras-induced cell proliferation.