Identification of a novel suppressive vitamin D response sequence in the 5'-flanking region of the murine Id1 gene.

Vitamin D promotes differentiation of cells either by simply enhancing phenotypic gene expression and/or by suppressing expression of inhibitors of differentiation. Previously, we reported that expression of a gene encoding Id1, a negative type helix-loop-helix transcription factor, was transcriptionally suppressed by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (1). To identify the sequence required for the negative regulation by 1, 25(OH)2D3, a 1.5-kilobase 5'-flanking region of murine Id1 gene was examined by transiently transfecting luciferase reporter constructs into ROS17/2.8 osteoblastic cells. The transcriptional activity of this construct was repressed by 10(-8) M 1,25(OH)2D3. Deletion analysis revealed that a 57-base pair (bp) upstream response sequence (URS) (-1146/-1090) was required for the suppression by 1,25(OH)2D3. This sequence conferred negative responsiveness to 1,25(OH)2D3 to a heterologous SV40 promoter. The 57-bp URS contained not only Egr-1 consensus sequence (2) but also four direct repeats of a heptamer sequence (C/A)CAGCCC. Electrophoresis mobility shift assay revealed that the 57-bp URS formed specific nuclear protein-DNA complexes, which were neither competed by previously known positive and negative vitamin D response elements nor supershifted by anti-vitamin D receptor antibody, suggesting the absence of vitamin D receptor in these complexes. These results indicate the involvement of the novel 57-bp sequence in the vitamin D suppression of Id1 gene transcription.

Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1.

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 Idl 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 dominant-negative helix-loop-helix proteins.

Characterization of the zinc finger protein encoded by the WT1 Wilms' tumor locus.

We analysed the biochemical properties of the transcription factor encoded by the putative tumor-suppressor gene present at the WT1 Wilms' tumor locus. A gene containing the full-length amino acid coding sequence of human wt1 was reconstructed from synthetic oligonucleotides and cloned into expression vectors for in vitro and in vivo protein synthesis. Polyclonal rabbit antibodies specific for the WT1 protein were raised to an Escherichia coli-produced 91 amino acid N-terminal segment and to a 136 amino acid C-terminal segment, which contains the zinc finger domain. WT1 produced by in vitro translation migrated as a 52 kDa protein on sodium dodecylsulfate-polyacrylamide gels and bound to the EGR consensus sequence in gel-retardation assays. Expression of the wt1 gene via transient transfection in COS-1 cells revealed a 52 kDa protein which was immunoprecipitated by both the N-terminal- and C-terminal-specific antisera. Immunofluorescence studies of wt1-transfected COS-1 cells revealed that the WT1 protein was localized to the nucleus. Metabolic labeling with [32P]orthophosphate failed to reveal significant phosphorylation of the WT1 protein in COS-1 cells. Two immunoreactive WT polypeptides of 52 and 54 kDa were observed in murine embryonic stem cells and COS-1 kidney cells and may represent previously identified splicing variants of WT1. These antisera should be useful in characterizing the structure and function of the WT1 protein in human Wilms' tumor specimens.

Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence.

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.

Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third-complementarity-determining region (CDR-III)-specific probes.

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 III (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-III 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-III 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-III 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 greater than 7 mo, malignant cells were present in marrow at greater than 1 per 1000 cells. These findings indicate that custom-made diagnostic probes will be useful in accurate quantitation of malignant cells in acute lymphoblastic leukemia patients in clinical remission and will allow investigation of the biological significance of low or high numbers of residual leukemic cells in evolution of that disease.

Point mutations in both transforming and non-transforming codons of the N-ras proto-oncogene of Ph+ leukemias.

The distribution and frequency of point mutations in the first and second coding exons of the N-ras proto-oncogene was examined in 6 cases of Philadelphia positive (Ph+) hemopoietic malignancies. To increase the detection sensitivity of the mutations and to estimate more accurately the frequency of abnormal alleles in the hemopoietic cell population, a polymerase chain reaction (PCR)/shotgun cloning/double stranded DNA sequencing method was used. Mutations activating the ras oncogenes involving codon 61 were observed in 5 out of 6 cases; in one of these cases (CML3), mutation at codon 61 involved a two base transition. Mutations involving codon 59 were also observed in one case (CML1). In longitudinal studies of 3 cases of chronic myelogenous leukemia samples obtained at the time of initial diagnosis and 5 to 7 years later, a multiplicity of mutations were detected at the time of initial diagnosis prior to any therapy. In one case (CML3), a mutation in codon 61 detected at diagnosis was still present 5 years later, in a second case (CML1) a mutation in codon 61 appeared during the course of the disease and persisted for at least one year, and in the third case (CML2) a mutation in codon 61 was present at diagnosis but absent 5 years later. In one instance (CML1) a mutation in codon 59 was present at the time of initial diagnosis but was not detectable in later samples. Several other point mutations leading to aminoacid changes were scattered predominately through the second exon but were not consistently detected in longitudinal studies on cells from the same patient. The data suggest that there is considerable genetic instability in the 2nd exon of N-ras in the myeloid leukemias but in every case a small subset of cells contains the mutations and these cells do not have a proliferative advantage.