David J. Law

Holt-oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family

Holt-Oram syndrome is a developmental disorder affecting the heart and upper limb, the gene for which was mapped to chromosome 12 two years ago. We have now identified a gene for this disorder (HOS1). The gene (TBX5) is a member of the Brachyury (T) family corresponding to the mouse TbxS gene. We have identified six mutations, three in HOS families and three in sporadic HOS cases. Each of the mutations introduces a premature stop codon in the TBXS gene product. Tissue in situ hybridization studies on human embryos from days 26 to 52 of gestation reveal expression of TBXS in heart and limb, consistent with a role in human embryonic development.

MULTIPLE GENETIC ALTERATIONS IN DISTAL AND PROXIMAL COLORECTAL CANCER

Multiple genetic alterations were investigated in colorectal cancer, including changes in DNA content, mutations in ras oncogenes, and deletions involving chromosomes 5, 17, and 18. A non-random association of deletions and mitotic abnormalities by site was seen, with both types of alterations occurring significantly more frequently in distal tumours. In contrast, the frequency of c-Ki-ras mutations did not differ between proximal and distal cancers. In addition, deletions were significantly associated with each other and with change in DNA content. The data provide strong support for the hypothesis that proximal and distal colon carcinoma might differ in the genetic mechanisms in their initiation and/or progression.

Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter

Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, andin vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider’sDrosophila line 2, consistent with properties previously attributed to NF-ODC1.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of gene whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair (bp) product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% petide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, D17S794, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone λcTBX2-1 confirmed homology to Mm-Tbx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 15F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 transcripts of 3.5 and 2.8kb, with high levels of TBX2 expression in fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.

Mutation of p53 in Recurrent Hepatocellular Carcinoma and Its Association with the Expression of ZBP-89

p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.

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-

Detailed deletion analysis of sporadic breast tumors defines an interstitial region of allelic loss on l7q25

Whole genome analyses of breast tumors with polymorphic markers have detected nonrandom loss of heterozygosity on multiple chromosomes, providing clues to the locations of suspected tumor suppressor genes. Tumors are thought to initiate, progress, and metastasize as mutations accumulate in multiple growth‐regulatory genes; thus, identification and characterization of these genes are critical to understanding and controlling breast tumorigenesis. To map more precisely a novel breast tumor suppressor gene that has been localized previously to distal 17q, we constructed a detailed deletion map of 17q25 by analyzing eight microsatellite markers on 39 sporadic primary breast tumors. The smallest overlapping region of interstitial loss was narrowed to approximately 3 cM and included D17S937/AFM107ye3, which showed the highest percentage of allelic loss (41%). These results provide a framework from which a genomic contig will be constructed and candidate transcripts will be analyzed. Genes Chromosom Cancer 17:64–68 (1996).

FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors

Soluble thymidine kinase (TK1) is an important 17q marker in somatic cell genetics. Its activity is increased in many malignancies, including breast cancer. Through somatic cell hybrid and fluorescence in situ hybridization studies, we mapped TK1 to 17q25.2-->25.3, in region demonstrating loss of heterozygosity in primary breast tumors. It lies near D17S836 and is proximal to the avian erythroblastic leukemia viral oncogene homolog 2-like gene (ERBA2L).

Intestinal overexpression of ZNF148 suppresses ApcMin/+ neoplasia

ZNF148 (ZBP-89, Zfp148) is a multifunctional transcription factor expressed at low levels in most tissues. When overexpressed in gastrointestinal cancer cell lines, ZNF148 inhibits cellular proliferation and induces apoptosis. We sought to determine whether intestinal ZNF148 overexpression would abrogate adenoma development in the ApcMin/+ mouse, i.e., whether ZNF148 is a tumor suppressor. The 13-kb villin promoter was spliced upstream of the ZNF148 cDNA to generate transgenic villin-ZNF148 (ZNF148TgVZ) mice. Intestinal mucosal ZNF148 expression was elevated in four of five ZNF148TgVZ lineages and correlated with increased caspase-3 expression and activation. In addition, DNA fragmentation was increased in ZNF148TgVZ mice relative to wild-type littermates. These results suggested that increased intestinal ZNF148 expression induces apoptosis. ZNF148TgVZ mice were crossed with ApcMin/+ mice to assess the biological significance of intestinal ZNF148 overexpression. The presence of the ZNF148TgVZ allele in ApcMin/+ mice correlated with reduced gastrointestinal bleeding at 5 weeks, a 50% reduction in adenoma burden at 20–22 weeks, and prolonged survival (median survival of 33.5 days vs. 21.5 days), relative to nontransgenic littermates. These data suggest that enhanced ZNF148 expression activates intestinal apoptosis and thereby mitigates disease burden in ApcMin/+ mice. They also suggest that ZNF148 is a therapeutic target to inhibit colon cancer development.

An isoform of ZBP-89 predisposes the colon to colitis

Alternative splicing enables expression of functionally diverse protein isoforms. The structural and functional complexity of zinc-finger transcription factor ZBP-89 suggests that it may be among the class of alternatively spliced genes. We identified a human ZBP-89 splice isoform (ZBP-89ΔN), which lacks amino terminal residues 1–127 of the full-length protein (ZBP-89FL). ZBP-89ΔN mRNA was co-expressed with its ZBP-89FL cognate in gastrointestinal cell lines and tissues. Similarly, ZBP-89ΔN protein was expressed. To define its function in vivo, we generated ZBP-89ΔN knock-in mice by targeting exon 4 that encodes the amino terminus. Homozygous ZBP-89ΔN mice, expressing only ZBP-89ΔN protein, experienced growth delay, reduced viability and increased susceptibility to dextran sodium sulfate colitis. We conclude that ZBP-89ΔN antagonizes ZBP-89FL function and that over-expression of the truncated isoform disrupts gastrointestinal homeostasis.