The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein. However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated. A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli. The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides. The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli. A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity. These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.

The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Id1 expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominantnegative helix-loop-helix proteins.The differentiation of eukaryotic cells is coordinated by changes in the activity and expression of multiple classes of transcription factors. The helix-loop-helix (HLH) family of proteins is one class of transcription factors that has been shown to play a key role in the differentiation processes of a number of cell lineages (reviewed in references 29, 42, 50, and 72). These proteins contain an HLH dimerization domain composed of two conserved amphipathic ␣ helices separated by a loop and an adjacent region that is rich in basic amino acids and that contacts DNA (16,21,43,46,47). Basic HLH (bHLH) proteins bind to a DNA sequence known as an E-box (CANNTG) (24,32,37) or to the related N-box (34, 68), which is found in the promoter-enhancer of genes expressed in a wide variety of cell lineages, such as muscle cells, B lymphocytes, pancreatic ␤ cells, and nerve cell. In general, cell-type-restricted bHLH proteins such as MyoD (17, 67) and achaetescute (70) form heterodimers with the ubiquitously expressed bHLH proteins which are coded for by the E2A gene (E12, E47 [46], and E2-5/ITF1 [27]) and the E2-2 gene (ITF2 [27]) in the mouse and by the daughterless gene (7, 10) in Drosophila melanogaster. The formation of heterodimers is essential for DNA binding and transcriptional activation in vivo (38).The bHLH transcription factors are inhibited by another class of HLH protein, the Id pro...

Approximately 80% of hematopoietic malignancies of the B-cell lineage carry only one or two immunoglobulin heavy chain gene rearrangements indicating their clonal origin. These rearrangements due to the recombination of various variable, diversity, and joining regions of the heavy-chain gene segments during B-cell commitment result in a region called complementarity-determining region m (CDR-III). This region, which encompasses the diversity region of the heavy-chain segment, because of extensive somatic mutations, provides a DNA-encoded signature specific for each B-cell clone. CDR-Ell sequences were obtained from DNA of pre-B-cell acute lymphoblastic leukemia by using suitable primers and the polymerase chain reaction. The sequences were used to generate diagnostic probes that hybridized only to the amplified CDR-HI of leukemic cells from which the sequences were derived. With these probes, leukemic cells could be detected when diluted 1:10,000 with other cells. By cloning the amplified CDR-M into recombinant libraries residual leukemic cells were accurately quantitated in bone-marrow samples from repeated relapses and remissions in one case of acute lymphoblastic leukemia. During a clinical remission lasting >7 mo, malignant cells were present in marrow at >1 per 1000 cells. These findings indicate that custom-made diagnostic probes will be useful in accurate quantitation of malinant cells in acute lymphoblastic leukemia patients in clinicalremission and will allow investigation of the biological cance of low or high numbers of residual leukemic cells in evolution of that disease.When a pluripotent hematopoietic stem cell differentiates into cells of the B-cell lineage, the first detectable genetic event is an immunoglobulin heavy chain (IgH) gene rearrangement involving the variable, diversity, and joining regions of the heavy chain (VH, DH, and JH, respectively), consisting of two successive somatic recombinations, DH-JH and VH-DH-JH (1-4). These changes bring a VH segment next to a JH segment at a distance of <100 bases. The VH segments in the human VH locus probably comprising 100-200 members, have so far been grouped into six families (VH 1-6), and members of each family are highly interspersed, with a distance of at least 3.5-11 kilobases (kb) between two adjacent VH segments (5). The VH locus is ==2500 kb in size (5) and is separated from the JH locus by <100 kb. The JH locus, -3 kb in size, consists of a cluster of six segments and is 6 kb upstream of the constant region ofthe ,u-chain gene (6). The DH regions can be classified into six different DE1 families, with great divergence of nucleotide sequence among different family members (7-9). The six DH genes are multiplied at least five times and are dispersed among VH clusters (9).A diverse and unique VH region is always generated during the formation of a complete VH gene due to the large number of VH, DH, and JH segments, which can be randomly combined, and to the flexibility arising at the recombination sites that can include the insertion...

1 is not only a major calcitrophic hormone that controls systemic calcium metabolism but also a potent modulator of differentiation in several types of cells including osteoblasts (2, 3). Many studies have revealed that the molecular mechanisms of vitamin D actions, including its promotion of cell differentiation, could be explained mainly by its genomic actions via the vitamin D receptor (VDR) as a ligand-dependent transcription factor (4 -7). VDR binds to vitamin D response elements (VDREs) within the promoter regions of the target genes to activate or suppress their expression. Several types of differentiation-related genes are regulated through this type of vitamin D action during cell differentiation. In addition, recent studies also showed the involvement of the nongenomic action of vitamin D in regulation of cell differentiation (9 -11). For instance, monocyte differentiation was reported to be mediated by vitamin D without requiring binding to VDR (9), and keratinocyte differentiationrelated genes were shown to be stimulated by 1,25(OH) 2 D 3 without the presence of VDRE (6, 10, 11). Therefore, vitamin D could promote cell differentiation via both genomic and nongenomic actions (3, 6).We have been interested in the molecular mechanism of the differentiation of osteoblasts as one of the target cells of 1,25(OH) 2 D 3 (3). Similarly to other types of cells, expression of various phenotype-related genes is enhanced by 1,25(OH) 2 D 3 in osteoblasts (11)(12)(13)(14). In parallel to its direct control of the genes encoding phenotype-related proteins in osteoblasts, we hypothesized that vitamin D may regulate higher order regulatory genes to modulate osteoblastic differentiation. We have shown that Id1, a dominant negative regulator of helix-loophelix-type transcription factors (15), is expressed in osteoblasts and that its level is transcriptionally suppressed by 1,25(OH) 2 D 3 (1). Because Id1 has been shown to be a negative modulator of positive regulatory transcription factor(s) that modulate cell differentiation, 1,25(OH) 2 D 3 could exert its effects on osteoblasts by suppressing expression of Id1. In the previous study, we have also shown that the suppression was specific to 1,25(OH) 2 D 3 and was mediated at the level of gene transcription without requiring new protein synthesis (1). However, the mechanism with which 1,25(OH) 2 D 3 suppresses Id1 gene transcription was still unknown.Ligand-dependent or -independent repression by nuclear hormone receptor superfamilies has been investigated (8, 16 -23). However, the molecular mechanisms of transcriptional repression appear to be more complicated than those of transcriptional activation (22,23), and the mechanisms for steroidal or nonsteroidal ligand-dependent repression have also been found to be variable. In some cases, to suppress expression of the target genes, hormones utilize the same or similar response elements as those used for transactivation (18), whereas in other cases, sequences different from the classical hormone response elements are utiliz...