The human ZBP-89 homolog, located at Chromosome 3q21, represses gastrin gene expression

Rat ZBP-89cDNA encodes an 89-kDa, zinc finger protein thatbinds a GC-rich element in the human gastrin promoter. Thiselement modulates both basal- and epidermal growth factor(EGF)-induction of gastrin gene expression (Merchant et al. 1995).Coexpression of ZBP-89 and gastrin reporter constructs blocksEGF induction and represses basal gastrin gene expression (Mer-chant et al. 1996). Rat ZBP-89similarly binds to and represses theactivity of the ornithine decarboxylase (ODC) promoter, concomi-tantly inhibiting cell proliferation (Remington et al. 1997). ZBP-89shares 99% amino acid sequence identity with two closely relatedmouse homologs, G-rich box-binding protein (GRBBP; Passantinoet al. 1996) and BFCOL1 (Hasegawa and de Crombrugghe 1997).Mutations of the BFCOL1 binding site in the pro a2(I) collagenpromoter inhibit BFCOL1 binding and result in increased pro-moter activity (Hasegawa and de Crombrugghe, 1997). Collec-tively, these data show that rodent ZBP-89-related genes act astranscriptional repressors by binding to specific GC-rich promoterelements, and, at least in some cases, this activity exerts a regu-latory effect on cell proliferation.The human cDNA, htb, shares about 90% DNA sequenceidentity with the open reading frames of rodent ZBP-89homologs.However, two single-nucleotide deletions in htb cDNA, 38 of thezinc finger domain, cause premature stop codons. As a result, htbencodes a 494 rather than a 794-amino acid protein (Merchant etal. 1996; Wang et al. 1993). The truncated, 49-kDa, htb proteinacts as a transcriptional activator, moderately activating T-cellreceptor promoters (Wang et al. 1993). The structural and func-tional comparisons of rodent ZBP-89homologs with htb suggestedto us that the point deletions that distinguish htb may have orig-inated as somatic mutations of wild type human ZBP-89 in theJurkat cells from which the htb cDNA was isolated. To test thishypothesis, we isolated a full-length (∼89 kDa) human ZBP-89cDNA from a normal leukocyte library, compared genomic local-ization of the corresponding gene with that of htb, and determinedits effects on gastrin gene expression. We report that full-lengthZBP-89 maps to Chr 3q21 where the htb cDNA was mapped(Schuler et al. 1996). Furthermore, like its rat homolog, humanZBP-89 functions as a repressor of gastrin gene expression.The human ZBP-89 cDNA clone hZBP-89-16 contains a 2.4-kb insert and was isolated from a SuperScript (Life Technologies)leukocyte library by positive selection (GeneTrapper, Life Tech-nologies) with oligonucleotides from htb. The open reading frameof hZBP-89-16 encodes a 794-amino acid protein, similar to the ratand mouse homologs, and lacks the internal stop codons of htb.A600-bp,EcoRV fragment from the 58-UTR of ZBP-89-16 cDNAwas used to screen a human BAC library (Genome Systems, St.Louis, Mo.), and overlapping clones B469N19, B460C18, andB502J22 were isolated (Fig. 1). A subcloned 1.2-kbEcoRI frag-ment from B469N19 was sequenced and used to develop an STS(374F/794R) for PCR-based screening of the human CEPH YACDNA matrix pools (Research Genetics, Huntsville, Ala.). The re-sulting YAC/BAC contig shows that the human ZBP-89 genemaps to chromosome band 3q21 (144–146 cM on the genetic map)and, more precisely, is within 100 kb of D3S1551. This agrees withour FISH data showing hybridization to 3q21 using BACB469N19 as a probe (data not shown). This localization is also thesame as reported for htb (Schuler et al. 1996), suggesting that twoalternative forms of human ZBP-89-related protein are derivedfrom a single locus. To better assess this possibility, we analyzedthe coding sequences contained in the ZBP-89 BAC clones.The ZBP-89 DNA sequence derived from BAC B469N19 wascompared with those of related genes (Fig. 2). The human se-quence shares 97% amino acid sequence identity with correspond-