Insulin-like growth factor binding protein-3. Organization of the human chromosomal gene and demonstration of promoter activity.

Insulin-like growth factor binding protein-3 (IGFBP-3) can modulate the mitogenic and metabolic effects of the insulin-like growth factors (IGFs). IGFBP-3 protein levels are developmentally regulated and influenced by a number of hormonal stimuli both in vitro and in vivo. As a first step toward understanding how hormonal and developmental factors regulate IGFBP-3 production, we are characterizing the human IGFBP-3 chromosomal gene and promoter. Southern analysis demonstrates a single copy of the IGFBP-3 gene in the human genome. This gene spans 8.9 kilobases; the protein-coding region is divided into four exons while a fifth exon contains the 3'-untranslated region. Primer extension studies locate the IGFBP-3 mRNA cap site 132 base pairs 5' to the ATG translation initiation codon. On the chromosomal gene, this cap site is located 30 base pairs 3' to the start of a TATA box and 97 base pairs 3' to a consensus GC upstream promoter element, an organization common to many eukaryotic promoters. When this potential IGFBP-3 promoter region is placed upstream to the chloramphenicol acetyltransferase reporter gene, it directs high-level production of chloramphenicol acetyltransferase in transfected COS-1 cells. These observations suggest an uncomplicated organization for the IGFBP-3 chromosomal gene and promoter in the human genome.     

Characterization and chromosomal localization of the human insulin-like growth factor-binding protein 6 gene

Insulin-like growth factor-binding protein 6 (IGFBP6), an extracellular protein with preferential affinity for insulin-like growth factor (IGF) II, belongs to a family of binding proteins with at least six members. We have characterized the genomic structure and the chromosomal location of the human IGFBP6, which is present in the human genome as a single-copy gene spanning 4.7 kb. It consists of four exons, encoding the translated regions, with sizes of 334, 146, 120, and 123 bp, while the intervening introns are 2661, 182, and 844 bp. Three mRNA cap sites were localized 101, 100, and 96 bp upstream of the ATG translation start codon as determined by S1 nuclease analysis. The proximal 5′-flanking region does not have any TATA or CAAT consensus sequences. The IGFBP6 was localized to Chr 12 by analysis of somatic cell hybrids and regionalized to 12q13 by fluorescence DNA in situ hybridization. Received: 14 October 1998 / Accepted: 1 December 1998     

Identification and characterization of a novel repressor site in the human tumor necrosis factor alpha gene.

In human monocytic cell lines, tumor necrosis factor alpha (TNF alpha) expression is induced by phorbol myristate acetate (PMA). We have identified positive and negative cis-acting elements in the TNF alpha promoter by deletion analysis. Here we present the initial characterization of the repressor element. The repressor element was shown to function in either orientation and at various distances upstream from the positive element of the TNF alpha promoter. The TNF alpha repressor site (TRS) has been localized to a 25 bp region between base pairs -254 and -230 in the promoter. This region contains a 10 bp sequence with homology to the binding site of the activator protein AP-2. Mutation of the 6 C's of this 10 bp AP-2-like site abolish TRS repressor function. However, this AP-2-like site is not a binding site for AP-2 protein based on gel retardation analysis. In addition, a well-characterized AP-2-binding site placed upstream of the positive element of the TNF alpha gene did not cause repression. Therefore, this repression is very likely mediated by a novel protein(s) which interacts with the AP-2 consensus site in the TRS.     

The promoter of the human gene for insulin-like growth factor binding protein-1. Basal promoter activity in HEP G2 cells depends upon liver factor B1.

Characterization of the human insulin-like growth factor binding protein-1 (IGFBP-1) promoter was initiated to facilitate study of developmental and hormonal factors regulating IGFBP-1 production. The region immediately 5' to the IGFBP-1 mRNA capsite is typical of a eukaryotic promoter, with a TATA sequence beginning 28 base pairs (bp) and a CCAAT promoter element beginning 72 bp upstream from this capsite. A 1.3-kilobase insert containing the IGFBP-1 capsite and 1205 bp of this putative IGFBP-1 promoter region directs expression of the reporter gene chloramphenicol acetyltransferase (CAT) in an orientation-specific manner in transfected HEP G2 cells, and the capsite identified for the CAT mRNA is identical to that identified for native IGFBP-1 mRNA. These observations suggest that the 1.3-kilobase insert contains the IGFBP-1 promoter. This promoter was further characterized by deletion analysis, site-directed mutagenesis, gel mobility shift assays, and DNaseI protection assays. These studies identify the CCAAT box region as the major cis element involved in basal IGFBP-1 promoter activity in HEP G2 cells, demonstrate that increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region, and indicate that the DNA binding factor(s) responsible for increased basal promoter activity is related to liver factor B1. These observations suggest that liver B1 is the major trans-acting factor stimulating basal IGFBP-1 promoter activity in HEP G2 cells.     

Positive and negative regulatory DNA elements including a CCArGG box are involved in the cell type-specific expression of the human muscle dystrophin gene.

The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.     

Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes

Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin.     

Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5' promoter region.

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. Computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5' promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.