Spl Binding Sites Research Articles

Analysis of Human Kl-67 Gene Promoter and Identification of the Spl Binding Sites Crucial for the Gene transcription

Objective To investigate the regulation difference of Sp1 binding sites ( - 170 ～- 145 nt) in the human Ki-67 gene promoter which contains two coupled Spl binding sites and further illuminate the potential cause of Ki-67 high expression in the tumor cells.Methods Site-directed mutagenesis and firefly luciferase reporter gene assay were used to detect the contribution of these Sp1 binding sites mutations Mut A ( - 170/- 160 nt) and Mut B ( - 159/- 145 nt) on transcriptional activation of the Ki-67 gene in Hela cells,OS-RC-2 cells and A549 cells,respectively.Results The transcriptional activity of the two mutant plasmids were decreased in these three cancer cells,and the decline of the transcriptional activity of mutant B is greater.In Hela cells,OS-RC-2 cells,A549 cells the relative dual luciferase reporter gene of mutant B exhibited transcriptional activities respectively equivalent to 36.9%,26.9% and 28.6% of plasmid pGLBK235.Conclusion Spl binding sites ( - 159/- 145 nt) in the human Ki-67 gene promoter is essential for basal transcription activation of the human Ki-67 gene. Key words: Ki-67 promoter; Deletion mutagenesis; Transcription factors Sp1; Cis-acting element

Structural and functional analysis of human Ki-67 gene promoter

Objective To investigate the transcriptional regulatory elements of the core promoter of Ki-67 gene. Methods The truncated fragment of Ki-67 core promoter BK235 ( -223 to + 12) was directly cloned into pGL3-Basic vector, then transfected into Hela cells, SW1990 cells, SGC-7901 cells and A549 cells. The transcriptional activity of BK235 in each carcinoma cell line was identified using the dualluciferase reporter assay system. Four internally deleted vectors of Ki-67 promoter BK235 (-223 to + 12)were prepared, whose Sp1 binding site including (- 198 to - 172), (- 170 to - 144), (-63 to -37)and (-14 to + 12) was deleted respectively, and their transcriptional activity was tested by dual-luciferase reporter assay system to locate the core regulatory element. Results The transcriptional activity of Ki-67 core promoter BK235 (-223 to + 12) in Hela cells, SW1990 cells, SGC-7901 cells and A549cells was respectively 1.2-fold, 2.0-fold, 1.7-fold and 1.4-fold of Ki-67 core promoter BK2 + (-223 to + 30). Deletion of the putative Spl binding site within BK235 resulted in the decreases of promoter activities, especially deletion of (- 170 to - 144) yielded lowest transcriptional activity, equivalent to 16. 2%,10.4%, 6.7% and 12. 2% of BK2 + (-223 to +30) promoter in Hela cells, SW1990 cells, SGC-7901cells and A549 cells, respectively. Conclusion A new core promoter BK235 ( -223 to + 12) of Ki-67gene was cloned and It had a better transcriptional performance than BK2 + (-223 to + 12). All of four Spl binding sites of Ki-67 promoter had a positive effect on transcriptional regulation function and - 170 to -144 was the core regulatory sequence of Ki-67 promoter. Key words: Ki-67 gene; Promoter; Ttranscriptional regulation

Interaction of Actinomycin D with Promoter Element of c-met and Its Inhibitory Effect on the Expression of c-Met

c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at −142 to −119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4–5 × 105 M−1 with ΔΔG of −47 ± 1.5 Kcalmol−1; (ii) marked increase by + 10 °C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Spl binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC50) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.

Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc

The goal of this investigation was to study the expression and regulation of beta1,3-Glucuronosyltransferase-I (GlcAT-I), a key enzyme regulating GAG synthesis in cells of the intervertebral disc. There was a robust expression of GlcAT-I in the nucleus pulposus in vivo. Treatment with the calcium ionophore ionomycin resulted in increased GlcAT-I expression, whereas GlcAT-I promoter constructs lacking TonE site or a mutant TonE were unresponsive to the ionophore. Experiments using TonEBP and DN-TonEBP constructs showed that TonEBP positively regulated GlcAT-I promoter activity. ChIP analysis confirmed binding of TonEBP to the promoter. We further validated the role of TonEBP in controlling GlcAT-I expression using mouse embryo fibroblasts from TonEBP null mice. GlcAT-I promoter activity in null cells was significantly lower than the wild type cells. In contrast to wild type cells, treatment with ionomycin failed to increase GlcAT-I promoter activity in null cells. We then investigated if calcineurin (Cn)-NFAT signaling played a regulatory role in GlcAT-I expression. Inhibition of Cn following ionomycin treatment did not block GlcAT-I and tauT, a TonEBP-responsive reporter activity. GlcAT-I promoter activity was suppressed by co-expression of Cn, NFAT2, NFAT3, and NFAT4. Moreover, following ionomycin treatment, fibroblasts from CnAalpha and CnAbeta null mice exhibited robust induction in GlcAT-I promoter activity compared with wild type cells. Results of these studies demonstrate that calcium regulates GlcAT-I expression in cells of the nucleus pulposus through a signaling network comprising both activator and suppressor molecules. The results suggest that by controlling both GAG and aggrecan synthesis, disc cells can autoregulate their osmotic environment and accommodate mechanical loading.

Spinophilin inhibits the binding of RGS8 to M1-mAChR but enhances the regulatory function of RGS8

We showed previously that RGS8 directly binds to the third intracellular loop (i3L) of the M1 muscarinic acetylcholine receptor using the sequence MPRR at the N-terminus of RGS8 and specifically inhibits signal transduction. Here, we identified spinophilin (SPL) as an RGS8-interacting protein. We found that the SPL-binding site of RGS8 is the MPRR sequence, and the M1 receptor and SPL compete for binding to RGS8. However, we also observed that the expression of SPL significantly enhances the inhibitory function of RGS8, and that SPL can bind to the M1 receptor, demonstrating the indirect binding of RGS8 to the M1 receptor through SPL for an efficient regulatory function.

The nuclear factor kappa B and Spl binding sites do not appear to be involved in virus suppression by CD8 T lymphocytes.

CD8 T lymphocytes suppress primate lentivirus replication in a non-cytotoxic manner. This antiviral activity, mediated by a CD8 cell antiviral factor (CAF), involves an arrest in viral transcription. Present studies indicate that the CD8 T cell non-cytotoxic antiviral activity and CAF inhibit the replication of an SIV mutant virus lacking the nuclear factor kappa B (NF-kappaB) and Spl binding domains. The results strongly suggest that the NF-B and Spl binding sites are not involved in virus suppression by CD8 T lymphocytes.

COL1A1 Sp1 Polymorphism Predicts Perimenopausal and Early Postmenopausal Spinal Bone Loss

Genetic factors play an important role in the pathogenesis of osteoporosis but the genes that determine susceptibility to poor bone health are defined incompletely. Previous work has shown that a polymorphism that affects an Spl binding site in the COLIA1 gene is associated with reduced bone mineral density (BMD) and an increased risk of osteoporotic fracture in several populations. Data from cross-sectional studies have indicated that COLIA1 Sp1 alleles also may be associated with increased rates of bone loss with age, but longitudinal studies, which have examined bone loss in relation to COLIA1 genotype, have yielded conflicting results. In this study, we examined the relationship between COLIA1 Sp1 alleles and early postmenopausal bone loss measured by dual-energy X-ray absorptiometry (DXA) in a population-based cohort of 734 Scottish women who were followed up over a 5- to 7-year period. The distribution of genotypes was as expected in a white population with 484 "SS" homozygotes (65.9%); 225 "Ss" heterozygotes (30.7%), and 25 "ss" homozygotes (3.4%). Women taking hormone-replacement therapy (HRT; n = 239) had considerably reduced rates of bone loss at the spine (-0.40 +/- 0.06%/year) and hip (-0.56 +/- 0.06%/year) when compared with non-HRT users (n = 352; spine, -1.36 +/- 0.06%/year; hip, -1.21 +/- 0.05%/year; p < 0.001 for both sites). There was no significant difference in baseline BMD values at the lumbar spine (LS) or femoral neck (FN) between genotypes or in the rates of bone loss between genotypes in HRT users. However, in non-HRT users (n = 352), we found that ss homozygotes (n = 12) lost significantly more bone at the lumbar site than the other genotype groups in which ss = -2.26 +/- 0.31%/year compared with SS = -1.38 +/- 0.07%/year and Ss = -1.22 +/- 0.10%/year (p = 0.004; analysis of variance [ANOVA]) and a similar trend was observed at the FN in which ss = -1.78 +/- 0.19%/year compared with SS = -1.21 +/- 0.06%/year and Ss = -1.16 +/- 0.08%/year (p = 0.06; ANOVA). The differences in spine BMD loss remained significant after correcting for confounding factors. Stepwise multiple regression analysis showed that COLIA1 genotype independently accounted for a further 3.0% of the variation in spine BMD change after age (4.0%), weight (5.0%), and baseline BMD (2.8%). We conclude that women homozygous for the Sp1 polymorphism are at significantly increased risk of excess rates of bone loss at the spine, but this effect may be nullified by the use of HRT.

Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.

Collagen alpha1(I) gene contains an element responsive to tumor necrosis factor-alpha located in the 5' untranslated region of its first exon.

The aims of the present study were to identify the cis-acting element through which tumor necrosis factor-alpha (TNFalpha) inhibits collagen alpha1(I) gene transcription and the trans-acting factors involved in this effect in cultured hepatic stellate cells. Deletion analysis of the collagen alpha1(I) promoter demonstrated that TNFalpha inhibited gene expression through an element located between -59 and + 116 bp relative to the transcription start site. DNase I protection assays revealed a footprint between +68 and +86 bp of the collagen first exon, the intensity of which decreased when the DNA probe was incubated with nuclear protein from TNFalpha-treated hepatic stellate cells. This footprint contained a G+C-rich box. Transfection experiments demonstrated that mutations in this G+C-rich element abrogated the inhibitory effect of TNFalpha on the collagen alpha1(I) promoter. Gel retardation experiments using a radiolabeled oligonucleotide containing sequences of this region confirmed that TNFalpha treatment decreased the formation of two complexes between nuclear proteins and DNA. These complexes were efficiently blocked with an oligonucleotide containing an Spl-binding site and were supershifted with specific Spl and Sp3 antibodies. These results suggest that TNFalpha inhibits collagen alpha1(I) gene expression by decreasing the binding of Spl to a G+C-rich box in the 5' untranslated region of its first exon.

Transcription of human cathepsin B is mediated by Sp1 and Ets family factors in glioma.

Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5'-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneider's Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.

Cis-elements required for expression of human protein C inhibitor gene in HepG2 cells and its androgen-dependent expression in rat reproductive organs.

Protein C inhibitor (PCI) is a plasma serine protease inhibitor of activated protein C, which is the main protease of the anticoagulant protein C pathway. Human PCI is synthesized in the liver, kidney, and several reproductive organs (testis, seminal vesicle, and prostate). In the present study, we characterized cis-elements of the human PCI gene required for expression in the hepatoma-derived cell line, HepG2 cells, and also evaluated rat PCI mRNA expression, particularly on the effect of androgen in rat reproductive tissues. On the PCI gene expression in HepG2 cells, transient expression assays using several deletion mutants and site-directed mutants of the human PCI gene and gel mobility shift assays using several synthetic oligonucleotides showed that the Spl-binding site (residues -302 to -294) and the upstream AP2-binding site (residues -350 to -343) play roles as the promoter and the enhancer, respectively. Both the A-activator-binding site (residues -422 to -414) and the interferon-gamma response element (residues -164 to -157) serve as the silencer. In the study on PCI mRNA expression in the reproductive organs, we first cloned rat PCI cDNA and then evaluated the effect of androgen on the PCI mRNA expression. The isolated rat PCI cDNA contained a 1,218-bp coding region of a 406-amino acid precursor protein. The deduced amino acid sequence of rat PCI showed an 85.7% and 62.2% homology with that of mouse and human PCIs, respectively. Northern blot analysis showed that rat PCI mRNA was strongly expressed in the seminal vesicles, moderately in the testes, but not in the liver. PCI mRNA expression in seminal vesicles and testes increased during the process of development. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or after 17beta-estradiol treatment. Treatment with testosterone in the castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is under androgen control.

NF-kappaB inhibits expression of the alpha1(I) collagen gene.

Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene.

Regulation of avian fibroblast growth factor receptor 1 (FGFR-1) gene expression during skeletal muscle differentiation.

Myogenic cell proliferation and differentiation are regulated by a fibroblast growth factor (FGF) signal transduction cascade mediated by a high-affinity fibroblast growth factor receptor (FGFR). Exogenous FGF added to myogenic cultures has a mitogenic effect promoting myoblast proliferation while repressing differentiation. We have examined the regulation of the FGFR-1 gene (cek-1) in avian myogenic cultures by immunocytochemistry and Northern blot analysis. FGFR-1 protein was readily detected in undifferentiated myoblast cultures and was significantly reduced in differentiated muscle fiber cultures. Similarly, FGFR-1 mRNA was 2.5-fold more abundant in myoblast cultures than in differentiated cultures. To define the molecular mechanism regulating FGFR-1 gene expression in proliferating myoblasts and post-mitotic muscle fibers, we have isolated and partially characterized the avian FGFR-1 gene promoter. Transfection of FGFR-1 promoter-chloramphenicol acetyltransferase gene constructs into myogenic cultures identified two regions regulating expression of this gene in myoblasts. A distal region of 2226 bp conferred a high level of expression in myoblasts. This region functioned in an orientation-dependent manner and interacted with a promoter element(s) in a proximal 1058 bp promoter region to direct transcription. Deletion analysis revealed a 78 bp region that confers a high level of cek1 promoter activity in myoblasts. This DNA segment also contains Spl binding sites and interacts with a component in myoblast nuclear protein extracts. The proximal promoter region alone demonstrated no activity in directing transcription in either myoblasts or muscle fibers. Using the full-length promoter, gene expression was significantly decreased in differentiated muscle fibers relative to undifferentiated myoblasts indicating that the promoter-reporter gene constructs contain elements regulating expression of the endogenous FGFR-1 gene in both myoblasts and muscle fibers.

The human c-Src proto-oncogene promoter contains multiple targets for triplex-forming oligonucleotides.

The overexpression and activation of the human c-Src proto-oncogene is closely associated with cancer of the colon and breast. Characterization of the 5' region of the c-Src gene revealed that the promoter is very GC rich, regulated by the Sp family of transcription factors, and contains four perfect homopolypurine/homopolypyrimidine tracts (Pu:Py tracts). These Pu:Py tracts (TC1, TC1.1, TC2, and TC3) are located near or overlap critical Spl binding sites required for full activation of the gene. Triplex-forming oligonucleotides (TFOs) can be targeted to such sequences with high affinity to form intermolecular triple-helical DNA and modulate transcriptional activity. We therefore designed a series of antiparallel purine-based TFOs and measured their ability to form triplexes with the c-Src promoter Pu:Py tracts using comigration, bandshift, and chemical footprint techniques. With one interesting exception, all of the TFOs were found to bind with specificity and high affinity (67 nM-28 nM) to their target sequences at physiologic pH. These results indicate that the c-Src gene can successfully form stable triplexes under physiologic conditions and is, therefore, an excellent candidate for triplex-mediated transcriptional downregulation.

Involvement of AP-2 in regulation of the R-FABP gene in the developing chick retina.

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5' flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5' flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5' flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.

Structural organization and expression of the gene for the mouse GM2 activator protein.

The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region.Gm2a is about 14 kb, has four exons, and the 5' flanking region contains a CAAT box, Spl binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.

CD11c Integrin Gene Promoter Activity During Myeloid Differentiation

The integrin CD11c/CD18 functions as a cell surface receptor for numerous soluble factors and proteins (LPS, fibrinogen, iC3b), mediates leukocyte interactions with other cell types and is a signal transducing receptor. CD11c/CD18 is found primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages. To determine the transcription factors and cis-acting elements driving the developmentally-regulated expression of CD11c/CD18 the proximal regulatory region of the CD11c gene has been structurally and functionally characterized using the U937 and HL-60 cell lines as myeloid differentiation models. The tissue-specific activity of the CD11c promoter is conferred by two Spl-binding sites and an adjacent C/EBP-binding element, with a likely contribution from other transcription factors with a more limited tissue distribution (PU. 1. Oct-2, Myb). The participation of Sp1 in the transcription of the CD11c gene strongly suggests that CD11c/CD18 expression is dependent on the proliferative state of the cell, thus establishing a first level of control for the regulated expression of CD11c/CD18 during myeloid differentiation. The differentiation responsiveness of the CD11c promoter has been mapped to an AP-1-binding site whose mutation greatly decreases the inducibility of the promoter during the PMA-triggered differentiation of U937 cells. Although AP-1 mediates the responsiveness to several other differentiating agents including GM-CSF, additional elements are required for induction of the CD11c promoter activity upon Sodium Butyrate-triggered differentiation. In fact, the Sodium Butyrate-responsiveness and the presence of both AP-I- and C/EBP-binding sites suggests that the proximal regulatory region of the CD11c promoter might include an extracellular matrix-response element. As a whole, the transcription of the CD11c gene appears to be controlled by the proliferative state of the cell and is tightly coupled to progression along the myeloid differentiation pathway. The differentiation inducibility of the CD11c promoter has been further demonstrated after stable transfection into U937 cells, where the -361/+43 fragment retains the capacity to drive luciferase expression upon PMA-, GM-CSF- or Sodium Butyrate-triggered myeloid differentiation. Thus, while the characterization of the transcription factors regulating CD11c expression is still in progress, the CD11c promoter has been shown to constitute a very useful tool for the identification of myeloid-differenting agents which might be of potential therapeutical interest.

Regulation of the Hypoxanthine Phosphoribosyltransferase Gene: In Vitro and In Vivo Approaches

The hypoxanthine phosphoribosyltransferase (HPRT) locus is a constitutively expressed housekeeping gene characterized by a notably higher level of expression in the mammalian brain. The enzyme it encodes is key to purine salvage in humans and is the basis for the X-linked recessive disorder, Lesch-Nyhan syndrome (LNS). Methylation in the promoter plays a critical, if not fully understood, role in transcriptional silencing of the locus on the inactive chromosome, possibly by conferring structural stability. In vivo footprinting assays of the promoter region have shown protein interaction with multiple Spl-binding sites, a possible AP2 site, and a potentially novel binding site. In vitro studies of HPRT promoter deletion constructs have identified a minimal promoter element necessary for maximal transcription and a position-dependent, orientation-independent repressor element (HPRT-NE) that functions on heterologous promoters. Regulatory intron elements have also been observed. Studies on transgenic mice bearing HPRT promoter constructs have shown that the minimal promoter element is insufficient for in vivo expression and that HPRT-NE is responsible for conferring neuronal specificity. HPRT-mice possess metabolic defects similar to LNS patients, but fail to develop human behavioral abnormalities, perhaps because of species differences in purine metabolism. A neuronal-specific protein complex appears to be necessary for activator function of HPRT-NE, while a ubiquitously expressed complex may be responsible for repression. Sequence analysis Indicates that the latter complex may depend on the multifunctional transcription factor YY1 for binding. A fuller understanding of HPRT gene regulation will hopefully provide insight into the transcriptional mechanisms controlling the expression of housekeeping and brain-specific genes.

Cloning and transcription factor-binding sites of the human c- rel proto-oncogene promoter

We report here the cloning, sequencing, functional analysis and DNase I footprinting of the human c- rel promoter region. The results revealed an 824-bp BsaAI-StuI minimal promoter region with a large number of NF-κB, Ap2 and Spl-binding sites, some of them variants of known consensus sequences. This is the first promoter in the Rel/NF-κB/IκB family to be subjected to a detailed footprinting analysis for the binding of transcription activator proteins. Our finding of 14 Ap2-binding sites may indicate why the human c- rel promoter, unlike the chicken c- rel promoter, has a strong function and is highly responsive to phorbol esters. The presence of five NF-κB and six Spl-binding sites in turn adds to growing evidence that, in mammals, the promoter of the Rel/NF-κB/IκB family may utilize multiple NF-κB- and Spl-binding sites for their interactive regulation. Furthermore, there are putative binding sites for the PU.1 and Oct 1 2 transcription activator proteins, also present in the mouse c- rel promoter, which may help explain the preferential transcription of the c- rel gene in B- and T-lymphoid cells.

The bovine DNA polymerase β promoter: cloning, characterization and comparison with the human core promoter

The core promoter of the human DNA polymerase β (βPol)-encoding gene (POLβ) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755–12763]. To study the homologous promoter (ppolβ) in a bovine system, we cloned and characterized the 5′-flanking region of the bovine gene (polβ). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5′-flanking region was isolated from a testis library in bacteriophage λEMBL3. S1 nuclease mapping and primer extension analysis of the 5′-end of the polβ mRNA identified the major transcription start point (tsp), which is located 142-bp 5′ of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5′-deletion mutants demonstrated that a fragment of only 91-bp 5′ of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOLβ). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Spl-binding sites, and the spacing separating them.