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The transcription factors Sp1 and MAZ (Myc-associated zinc finger protein) contain several zinc finger motifs, and each functions as both a positive and a negative regulator of gene expression. In this study, we characterized the extremely GC-rich promoter of the human gene for MAZ, which is known as a housekeeping gene. Unique symmetrical motifs in the promoter region (nucleotides -383 to -334) were essential for the expression of the gene for MAZ, whereas an upstream silencer element (nucleotides -784 to -612) was found to act in a position-dependent but orientation-independent manner. Sp1 and MAZ bound to the same cis-elements in the GC-rich promoter, apparently sharing DNA-binding sites. The relative extent of binding of Sp1 and MAZ to these cis-elements corresponded to the extent of negative regulation of the expression of the gene for MAZ in various lines of cells. Furthermore, novel repressive domains in both Sp1 (amino acids 622-788) and MAZ (amino acids 127-292) were identified. Suppression by Sp1 and suppression by MAZ were independent phenomena; histone deacetylases were involved in the autorepression by MAZ itself, whereas DNA methyltransferase 1 was associated with suppression by Sp1. Our results indicate that both deacetylation and methylation might be involved in the regulation of expression of a single gene via the actions of different zinc finger proteins that bind to the same cis-elements.

A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.