Proximal Promoter Activity Research Articles

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity.

The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes, and the majority of CD4(-)CD8(+) and CD4(-)CD8(-) [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP(+) cells was detected in non-T lineage subsets, including mature and immature B cells, CD5(+) B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP(+) cells detected were found in the CD44(+)CD25(-) DN thymocyte subpopulation. The developmental potential of GFP(-) and GFP(+) cells in the CD44(+)CD25(-) DN fraction was examined using in vitro culture systems. The generation of substantial numbers of alphabeta and gammadelta T cells as well as NK cells was demonstrated from both GFP(-) and GFP(+) cells. However, no development of B cells or dendritic cells was detected from GFP(+) CD44(+)CD25(-) DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44(+)CD25(-) DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.

CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Igamma1 proximal promoter.

To understand the underlying basis for the strong IL-4- and CD154-mediated Igamma1 promoter activity in Ramos 2G6 B cells, we carried out transient transfection assays with luciferase-based constructs containing approximately 2.2 kb and 500 bp of the human Igamma1 proximal promoter region. As a comparison, the corresponding regions of the human Igamma3 promoter were tested under identical conditions. We found that both Igamma1 and Igamma3 promoter constructs were activated upon transfection into Ramos B cells and that activity was significantly up-regulated by CD154 and IL-4 signals. However, the Igamma1 promoter was measurably stronger than the Igamma3 promoter with respect to both basal and induced responses. Sequence comparison revealed a divergent 36-bp region containing multiple putative transcription factor binding sites in the Igamma1 but not the Igamma3 promoter. A mutational "swap" of this sequence resulted in a marked decrease and increase in Igamma1 and Igamma3 basal and induced promoter activity, respectively. Gel retardation assays with Igamma1-specific probes revealed CREB-containing complexes that were not observed with the corresponding Igamma3 probes. Mutation of a single nucleotide in overlapping CREB sites in the Igamma1 sequence resulted in a significant decrease in basal activity with a corresponding reduction in the level of IL-4- and CD154-mediated transcription.

Single-stranded DNA-binding Complex Involved in Transcriptional Regulation of Mouse μ-Opioid Receptor Gene

Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is approximately 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.

Characterization of an enhancer element in the proximal promoter of the mouse glucagon receptor gene

The 5′-flanking region of the mouse glucagon receptor has been previously cloned and two promoter regions were characterized. Functional analysis of the proximal promoter was now performed to characterize cis-acting element(s) regulating basal gene expression. Promoter analysis using deletion constructs in a rat cell line (CA-77) expressing the glucagon receptor, showed that the region from −64 to +127 relative to the proximal transcription start site was sufficient for maximal proximal promoter activity. A DNA sequence spanning the −28 to −16 region organized as an imperfect palindrome was demonstrated to be functional as a cis-acting enhancer. Constructs including several copies of this motif strongly increased activity of the heterologous thymidine kinase promoter. Gel mobility shift assays performed with different DNA fragments spanning this region confirmed that it specifically bound nuclear protein(s) from CA-77 cells, mouse MIN-6 cells or mouse liver. Mutations in the core sequence of this site impaired both reporter gene activity and nuclear protein binding. The palindrome is a novel DNA sequence with no homology to existing transcription factor binding site database. This is the first characterization of a functional cis-acting sequence into the proximal promoter of the mouse glucagon receptor that may support constitutive expression of the gene.

Transcriptional regulation of the human FTZ-F1 gene encoding Ad4BP/SF-1.

Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene encoding Ad4BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was found to be unlikely in the hFTZ-F1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad4BP had little effect on hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such Ad4BP-independent regulation of the hFTZ-F1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which the proximal promoter activity is Ad4BP-dependent and the transactivation by Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be independent of both Ad4BP and DAX-1.

Structural and Functional Characterizations of the 5′-Flanking Region of the Mouse Glucagon Receptor Gene: Comparison with the Rat Gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5′-rapid amplification of cDNA ends from mouse liver tissue. The 5′-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5′ untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5′ region of the rat GR gene. The mouse distal promoter region, extending up to −1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5′-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.

PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proα2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proα2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position −45 to −38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

An Upstream Regulator of the Glycoprotein Hormone α-Subunit Gene Mediates Pituitary Cell Type Activation and Repression by Different Mechanisms

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.

Retinoic Acid Synergizes with Cyclic AMP to Enhance MMP-2 Basal Promoter Activity

Matrix metalloproteinase-2 (MMP-2) degrades basement membrane collagen and its abnormal expression is associated with the enhanced malignancy of metastasizing cancer cells. Retinoids and cyclic AMP analogs have been shown to affect MMP-2 production. Here we demonstrate that the expression of the human MMP-2 gene is enhanced by a synergistic action of retinoic acid (RA) and dibutyryl cyclic AMP (Bt2cAMP). RA also synergizes with Bt2cAMP in enhancing the basal promoter activity when the MMP-2 proximal promoter activity is induced by transient transfection and RA/Bt2cAMP treatment in human fibrosarcoma HT1080 cells. Deletions beyond −315 bp from the transcription initiation site drastically reduce the synergistic enhancement. A search for acis-element in the MMP-2 proximal promoter shows the presence of a CRE-like sequence (TGACGTCCC) at position −292 bp in the opposite strand. The CRE-like sequence makes complexes with two DNA binding proteins. The results demonstrate that the RA/Bt2cAMP-enhanced transcription of the MMP-2 gene is dependent on the general transcription machinery and suggest that the basal promoter may be a potential target for gene-specific activators.

The THCRE2 site in the rat tyrosine hydroxylase gene promoter is responsive to phorbol ester

Recently, our laboratory has identified a tyrosine hydroxylase (TH) gene promoter element, THCRE2 (located at −97 to −90), that is required for maximal response to cyclic AMP. In this study we test whether the THCRE2 is responsive to phorbol ester. Rat PC12 cells were transfected with a reporter gene construct, TH(−272/+27)-CAT, which is driven by the first 272 bp of the rat TH gene 5′ flanking region. Treatment of transfected cells with 0.1 μM 12- O-tetradecanoylphorbol 13-acetate (TPA) elicited a 5–6-fold increase in TH gene proximal promoter activity. Mutagenesis of the THCRE2 sequence diminished TPA-responsiveness of the TH gene promoter by approximately 50%. Minimal promoter constructs driven by a single copy of the region of the TH gene that encodes the THCRE2 (from −117 to −59) were also responsive to TPA. Our results suggest that the THCRE2 is a phorbol ester-responsive element, as well as a cyclic AMP-responsive element.

Functional interactions of an upstream enhancer of the mouse glycoprotein hormone α-subunit gene with proximal promoter sequences

Transcription of the glycoprotein hormone α-subunit gene in the pituitary is governed by different promoter elements in thyrotropes and gonadotropes. We recently identified an upstream enhancer that directs a high level of cell type specific expression in transgenic mice and stimulates proximal promoter activity in cultured αTSH and αT3 cells. To assess the contribution of promoter sequences that functionally interact with the enhancer, we mutated two proximal elements shown to be important in both thyrotrope and gonadotrope cells. Disruption of the pituitary glycoprotein hormone basal element (PGBE), which binds a LIM homeodomain protein, resulted in a decrease in basal promoter activity in both αTSH and αT3 cells. Enhancer function was completely abolished by the PGBE site mutation in αT3 gonadotropes, whereas some stimulatory activity remained in αTSH thyrotropes. Mutation of the gonadotrope specific element (GSE), which binds SF1 and is important for basal activity in gonadotropes and TRH response in thyrotropes, resulted in declines in basal and enhanced promoter activity only in αT3 cells and not in αTSH cells. Despite this decrease in enhanced activity, the GSE mutated promoter still retained some enhancer stimulated activity, suggesting that the PGBE site still functionally interacts in the absence of an intact GSE. This mutation had no effect in αTSH cells. These data suggest that although the enhancer works in both cell types it exhibits cell type specific functional characteristics.

Isolation and characterization of the 5'-flanking regulatory region of the human natriuretic peptide receptor C gene.

Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a central role in the pathogenesis of atherosclerosis. Natriuretic peptide receptor-C (NPR-C) is highly expressed in vascular SMCs in the experimental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to the phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene expression, a genomic clone containing over 8 kilobases of the 5'-flanking region of the human NPR-C gene has been isolated. Sequence analysis revealed that a number of putative regulatory elements including unusual tandem repeated AP-2-like sequences were observed in the 5'-flanking region. Primer extension and ribonuclease protection analyses revealed that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, highly producing NPR-C transcripts, showing that the region (from - 33 to + 13 relative to the transcription start point) had a potential promoter activity suggested that the region from -33 to + 13, containing a pyrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinct DNA sequences surrounding the transcription start site (P1, from -60 to -33; P2, from + 14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s) from the human cells was demonstrated to specifically bind to the sequences, respectively. However, promoter analysis has shown that the P2 and P3 sequences could not activate the human NPR-C promoter in a synergistic manner. On the basis of deoxyribonuclease I footprinting analysis showing that a DNA element from +48 to +60 within the P3 sequence is preferentially protected, the P3 sequence appears to contain a potential regulatory element involved in NPR-C gene expression. The present study demonstrated the structure of the 5'-regulatory region of the human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role in regulation of human NPR-C gene expression.

Human S mu binding protein-2 binds to the drug response element and transactivates the human apoA-I promoter: role of gemfibrozil

Previously, we demonstrated that protein–DNA interactions at the drug response element (DRE) in the human apoA-I promoter were important for the induction of apoA-I gene expression by gemfibrozil. We now report the cloning and characterization of a DRE transactivating factor. The cloned protein is identical to the putative helicase and potential transcription factor human S mu binding protein-2 (HSμBP2). It is also related to glial factor-1 (GF1), an incomplete version of HSμBP2 lacking the first 494 and the last 128 amino acids. Gel mobility shift assays demonstrated that HSμBP2 binds apoA-I DRE oligomers and forms a specific protein–DNA complex. Northern blot analysis showed that HSμBP2 mRNA is expressed at various levels in a wide range of human tissues. Transient cotransfection experiments performed in HepG2 cells demonstrated that overexpression of HSμBP2 or GF1 induced apoA-I proximal promoter activity by 3-fold and that the apoA-I DRE was necessary for transactivation. Additionally, we demonstrated that transactivation was increased a further 2- to 3-fold by exposing the cells to gemfibrozil. Together these observations indicate that HSμBP2 acts as a transcription factor that regulates apoA-I gene expression in hepatoma cells and whose activity may be stimulated by gemfibrozil treatment.—Mohan, W. S., Z-Q. Chen, X. Zhang, K. Khalili, T. Honjo, R. G. Deeley, and S-P. Tam. Human S mu binding protein-2 binds to the drug response element and transactivates the human apoA-I promoter: role of gemfibrozil.

Osteogenic Protein-1 Stimulates Production of Insulin-Like Growth Factor Binding Protein-3 Nuclear Transcripts in Human Osteosarcoma Cells

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3–36 h; treatment× time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or β-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1–24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5′-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5′-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

Osteogenic protein-1 stimulates production of insulin-like growth factor binding protein-3 nuclear transcripts in human osteosarcoma cells.

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3-36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or beta-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1-24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

Characterization of the rat insulin-like growth factor I gene promoters and identification of a minimal exon 2 promoter.

Insulin-like growth factor I (IGF-I) promoter activity was characterized in C6, GH3, OVCAR-3, and Chinese hamster ovary (CHO) cells. Maximal exon 1 promoter activity was present in the region extending from -133 to +362 (where +1 is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 transcription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity was also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstream of -133 inhibited exon 1 proximal promoter activity in a cell type-specific manner. Exon 2 promoter activity was observed in all cell lines with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region. A putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell type-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon 1 promoter was used with nuclear extracts from C6 and GH3 cells. Multiple specifically bound bands with slower mobility were observed when the -236/+44 exon 2 promoter fragment was incubated with C6, GH3, CHO, and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter regions were able to inhibit each other's binding in electrophoretic mobility shift assay using GH3 cell and OVCAR-3 cell nuclear extracts, respectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH3 cell nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter binding. Our data suggest that the sequence surrounding and including start site 3 in exon 1 functions as a minimal independent promoter. The minimal exon 2 promoter is contained within the 73 bp upstream and 44 bp downstream of the transcription start site cluster. These minimal promoters contain similar and distinct elements that are important for basal transcription. Upstream sequences may contain cell type-specific silencer elements.

Characterization of the Rat Insulin-Like Growth Factor I Gene Promoters and Identification of a Minimal Exon 2 Promoter

Insulin-like growth factor I (IGF-I) promoter activity was characterized in C6, GH3, OVCAR-3, and Chinese hamster ovary (CHO) cells. Maximal exon 1 promoter activity was present in the region extending from -133 to +362 (where +1 is the first transcription start site). Promoter activity was higher in the +75/+362 fragment, which contains exon 1 transcription start sites 3 and 4, than in the -133/+74 fragment, which contains exon 1 transcription start sites 1 and 2. Promoter activity was also observed in constructs containing sequences from -133 to +192, which includes start sites 1, 2, and 3. Inclusion of sequences upstream of -133 inhibited exon 1 proximal promoter activity in a cell type-specific manner. Exon 2 promoter activity was observed in all cell lines with a construct containing 73 bp of 5'-flanking sequence and 44 bp of exon 2. Exon 2 promoter activity was abolished when only 36 bp of 5'-flanking sequence and 44 bp of exon 2 were present, suggesting that an essential minimal promoter element(s) is contained within the -73 to -36 region. A putative CACCC box was observed within this region at -53. Upstream sequence regulated exon 2 promoter activity in a cell type-specific manner. Electrophoretic mobility shift assays revealed a single specifically bound band when the +75/+362 fragment of the exon 1 promoter was used with nuclear extracts from C6 and GH3 cells. Multiple specifically bound bands with slower mobility were observed when the -236/+44 exon 2 promoter fragment was incubated with C6, GH3, CHO, and OVCAR-3 cell nuclear extracts. The exon 1 and exon 2 promoter regions were able to inhibit each other's binding in electrophoretic mobility shift assay using GH3 cell and OVCAR-3 cell nuclear extracts, respectively. Oligonucleotides containing consensus activating protein-1 (AP-1) and AP-3 sequences inhibited exon 1 promoter binding by GH3 cell nuclear extracts. AP-2 and AP-3 sites inhibited exon 2 promoter binding. Our data suggest that the sequence surrounding and including start site 3 in exon 1 functions as a minimal independent promoter. The minimal exon 2 promoter is contained within the 73 bp upstream and 44 bp downstream of the transcription start site cluster. These minimal promoters contain similar and distinct elements that are important for basal transcription. Upstream sequences may contain cell type-specific silencer elements.

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression.

The pituitary-specific, POU-homeodomain factor GHF-1/Pit-1 is necessary, but not sufficient, for cell-specific expression of prolactin (PRL), growth hormone (GH), and thyrotropin. Combinatorial interactions of GHF-1 with other factors are likely to be required; however, such factors and their mechanisms of action remain to be elucidated. Here we identify Ets-1 as a factor that functionally and physically interacts with GHF-1 to fully reconstitute proximal PRL promoter activity. In contrast, Ets-2 has no effect, and the alternatively spliced GHF-2/Pit-1beta variant fails to synergize with Ets-1. The Ets-1-GHF-1 synergy requires a composite Ets-1-GHF-1 cis element and is dependent on an Ets-1-specific protein domain. Furthermore, the ancestrally related and GHF-1-dependent GH promoter, which lacks this composite element, does not exhibit this response. Finally, Ets-1, but not Ets-2, binds directly to GHF-1 and GHF-2. These data show that a functional interaction of GHF-1 and Ets-1, acting via a composite DNA element, is required to establish lactotroph-specific PRL gene expression, thus providing a molecular mechanism by which GHF-1 can discriminate between the GH and PRL genes. These results underscore the importance of transcription factors that are distinct from, but interact with, homeobox proteins to establish lineage-specific gene expression.