We have cloned and characterized the genomic structure of the human gene for Myc-associated zinc finger protein (MAZ), which is located on chromosome 16p11.2. This gene is transcribed as an mRNA of 2.7 kilobases (kb) that encodes a 60-kDa MAZ protein. A 40-kb cosmid clone was isolated that includes the promoter, five exons, four introns, and one 3-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. The promoter region has features typical of a housekeeping gene: a high G ؉ C content (88.4%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. An S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 174 nucleotides (nt) upstream of the ATG codon and such expression was reflected by the promoter activity of a MAZ promoter/CAT (chloramphenicol acetyltransferase) reporter gene. Cis-acting positive and negative elements controlling basal transcription of the human MAZ gene were found from nucleotides (nt) ؊383 to ؊248 and nt ؊2500 to ؊948. Moreover, positive and negative autoregulatory elements were also identified in the regions from nt ؊248 to ؊189 and from nt ؊383 to ؊248 after co-transfection of HeLa cells with plasmids that carried the MAZ promoter/CAT construct and the MAZ-expression vector. Our results indicate that the 5-end flanking sequences are responsible for the promoter activities of the MAZ gene.The c-myc protooncogene is a member of a family of genes that encode DNA sequence-specific transcription factors with basic, helix-loop-helix, and leucine zipper domains. The Myc protein binds to DNA as heterodimers with a related polypeptide, Max (1-4). Appropriate regulation of expression of the human c-myc gene is necessary for the proliferation and differentiation of cells and for progression of the cell cycle, and deregulation of the expression of c-myc is associated with tumorigenesis and apoptosis (1, 4). Regulation of the expression of the human c-myc gene occurs at multiple levels, which include the initiation, the termination, and the attenuation of transcription (2, 4). In proliferating cells, the initiation of transcription of the c-myc gene is controlled by two major promoters, P1 and P2, and the RNA initiated from the P2 promoter accounts for 80 -90% of the total RNA initiated from the P0, P1, and P2 promoters (4, 5). Initiation of transcription from the P2 promoter requires at least three cis-elements: ME1a2, E2F, and ME1a1 (6, 7). Several transcription factors, including Sp1 (5, 8), the Myc-associated zinc finger protein (MAZ) 1 (9, 10), Pur-1 (11), and E2F (12) bind to these elements in vitro and in vivo.The MAZ protein was identified as a transcription factor that binds to a GA box (GGGAGGG) at the ME1a1 site, to the attenuator region of P2 within the first exon of the c-myc gene, and to a related sequence that is involved in the termination of transcription of the gene for ...

No abstract

No abstract

A detailed analysis is reported of the binding of the zinc finger protein THZif-1 to the nuclease-hypersensitive element (NHE) in the promoter region of the c-MYC gene using the electrophoretic mobility shift assay and a series of mutants of a fusion protein composed of glutathione S-transferase and THZif-1. The THZif-1 protein bound specifically to the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NHE-C) with an apparent dissociation constant (K d (app) ) of 0.077 M. By contrast, no binding to the single-stranded purine-rich DNA of the NHE (ss c-myc NHE-G) was detected. Moreover, the binding affinity of THZif-1 protein was 2-fold higher for the single-stranded 5-methyl-2-deoxycytidine derivative of NHE (ss c-myc NHE-me 5 C) than for the unmethylated NHE. In the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHEme 5 CG), no significant binding to the DNA was observed. The decrease in binding to DNA of THZif-1 was significant in the case of mutated ds c-myc NHE, in which more than two sites of deoxycytidine residues were methylated. However, the binding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical. Moreover, the domain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me 5 C corresponded to the amino-terminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity with ss c-myc NHE-C, ds c-myc NHE-CG, and ts c-myc NHE but not with ss c-myc NHE-G and ds c-myc NHE-me 5 CG in vitro.

No abstract