Positive Regulatory Sequences Research Articles

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5′-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the −134 to −19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT)

Human epithelial, endothelial and PMA-differentiated THP-1 cell lines were used as model systems to study the transcriptional regulation of the human FCGRT gene encoding the alpha chain of hFcRn. The data obtained from site-directed mutagenesis in transient transfection experiments indicate that the Sp1 sites at positions -641, -635, and -313, CF1/YY1 elements at positions -586 and -357, and the AP-1 motif at -276 within the-660/-233 fragment of the human FCGRT promoter (hFCGRT) participate in the regulation of human FCGRT in all selected cell lines. However, their individual contribution to promoter activity is not equivalent. The Sp1 binding site at -313 and the AP-1 site at -276 are critical for the activity of the hFCGRT promoter in epithelial and endothelial cells. Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter. In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation. EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

Eukaryotic enhancers: common features, regulation, and participation in diseases.

Enhancers are positive DNA regulatory sequences controlling temporal and tissue-specific gene expression. These elements act independently of their orientation and distance relative to the promoters of target genes. Enhancers act through a variety of transcription factors that ensure their correct match with target promoters and consequent gene activation. There is a growing body of evidence on association of enhancers with transcription factors, co-activators, histone chromatin marks, and lncRNAs. Alterations in enhancers lead to misregulation of gene expression, causing a number of human diseases. In this review, we focus on the common characteristics of enhancers required for transcription stimulation.

Expression of rod-derived cone viability factor: dual role of CRX in regulating promoter activity and cell-type specificity.

BackgroundRdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina.Methodology/Principal FindingsIn order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5′-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5′-deletion analysis identified the human −205/+57 bp and murine −351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human −393/+27 bp and murine −195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (−393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (−77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role.Conclusions/SignificanceThe regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity.

HTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

BackgroundActivation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.MethodsUsing luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.ResultsWe found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.ConclusionsMethylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.

Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

Functional analysis on the 5′-flanking region of human FXR gene in HepG2 cells

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Although modulation of FXR expression has been reported, the mechanisms underlying the regulation of human FXR are yet unclear. Functional assays showed that the − 150/+ 29 nucleotides region from the first nucleotide at the Exon I is the minimal promoter of the human FXR gene by the technique of serial deletion and point mutants of the 5′-flanking region. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assay revealed that hepatic nuclear factor 1α (HNF1α) interacted with the region. Co-transfection of the promoter with HNF1α expression vectors enhanced promoter activity of FXR gene. Over-expression of HNF1α up-regulated FXR expression in HepG2 cells. These data indicate that (a) the identified HNF1α binding site serves as a positive regulatory sequence, (b) the binding site is functionally active both in vivo and in vitro, and (c) the transcription factor HNF1α that binds to this site plays an important role in the regulation of human FXR promoter activity.

Structural analysis and promoter characterization of the human membrane-type matrix metalloproteinase-1 (MT1-MMP) gene

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades extracellular matrix components directly and indirectly by activation of other matrix metalloproteinases (MMPs). In the present study, we have isolated and characterized the human MT1-MMP gene and its promoter. The gene consists of 10 exons and nine introns spanning more than 10 kilobases (kb). The locations of two exon–intron splicing sites are distinct from the preserved positions among other known MMP genes. Primer extension and RNAse and S1 nuclease protection analyses indicated that there are four major and several minor transcription start sites. The 5′-flanking sequence of the gene contains putative regulatory elements, including one Sp-1 site and four CCAAT-boxes, whereas there is no TATA-box. The Sp-1 binding site was functional, as shown by gel shift and supershift analyses. Transfection studies with promoter constructs containing 0.1 to 7.2 kb of 5′-flanking sequence coupled to a luciferase reporter gene indicated that the promoter contains additional positive and negative regulatory sequences. Deletion of the Sp-1 binding site by site-directed mutagenesis reduced luciferase activity by about 90%, demonstrating the crucial role of this element in maintaining MT1-MMP transcription. Our findings indicate that the human MT1-MMP promoter has distinctive structural and functional features compared with other MMP genes, which may lead to a unique expression pattern and regulation during physiological and pathological processes.

An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1.

The Pbx/Exd family of homeodomain (HD) proteins contribute to the transcriptional and developmental roles of other Hox and Meis/Prep1/Hth HD proteins through heterodimer formation. E2a-Pbx1 is an oncogenic derrivative of Pbx1 produced by the t(1;19) translocation in pediatric pre-B cell acute lymphoblastic leukemia. E2a-Pbx1 heterodimerizes with Hox but not with Meis/Prep1 proteins, produces acute myeloid leukemia in mice, and blocks differentiation of cultured murine myeloid progenitors. Here, we characterize negative and positive regulatory sequences that flank the Pbx1 HD and determine their importance for myeloid immortalization by E2a-Pbx1. A 25 residue predicted alpha helix preceding the Pbx1 HD bound the HD and prevented both its binding to DNA and its ability to heterodimerize with Hox proteins. Addition of 39 residues N-terminal to this inhibitory helix exposed a Pbx dimerization interface that orchestrated cooperative DNA-binding of E2a-Pbx1 and all Pbx proteins as homodimers and heterdimers. Sequences inhibiting DNA-binding and mediating Pbx dimerization coincided with those reported to have nuclear export function. An additional 103 residues N-terminal to the Pbx dimerization interface restored heterodimerization with Hox and Meis1/Prep1 proteins. This negative switch domain - comprised of the inhibitory helix and N-terminal regions required for its partner-mediated derepression - was dispensable for myeloid immortalization by E2a-Pbx1. While stabilizing the heterodimer, the 310 helix C-terminal to the Pbx1 HD was also dispensable for the ability of E2a-Pbx1 to heterodimerize with Hox proteins and immortalize myeloblasts. Retention of myeloid immortalization by E2a-Pbx1 proteins lacking all Pbx1 sequences N- or C-terminal to the HD indicates that Hox proteins, or a yet undefined factor that binds the Pbx1 HD and derepresses DNA-binding by the HD, cooperate with E2a-Pbx1 in myeloid immortalization.

Human Integrin β3 Gene Expression: Evidence for a Megakaryocytic Cell-Specific cis-Acting Element

The human integrin β3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for β3 expression, a number of upstream regulatory regions in the β3 gene were identified. (1) The three regions from −1159 to −584, −290 to −146, and −126 to −115 demonstrated positive, negative, and negative activity, respectively. (2) The region from −115 to +29 of the β3 gene was sufficient for cell-specific activity. Deletion of the sequence from −115 to −89 produced a 6- to 40-fold reduction in reporter gene activity in β3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in β3-expressing endothelial and melanoma cell lines, and 1.3- and 2.8-fold reduction, respectively, in non–β3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions −113 to −108) is a megakaryocytic cell line-specificcis-acting element. (3) The region from −89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position −70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5′ region of the β3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment β3 gene expression.© 1998 by The American Society of Hematology.

Human Integrin β3 Gene Expression: Evidence for a Megakaryocytic Cell-Specific cis-Acting Element

The human integrin beta3 participates in a wide range of adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for beta3 expression, a number of upstream regulatory regions in the beta3 gene were identified. (1) The three regions from -1159 to -584, -290 to -146, and -126 to -115 demonstrated positive, negative, and negative activity, respectively. (2) The region from -115 to +29 of the beta3 gene was sufficient for cell-specific activity. Deletion of the sequence from -115 to -89 produced a 6- to 40-fold reduction in reporter gene activity in beta3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and 2.7-fold reduction, respectively, in beta3-expressing endothelial and melanoma cell lines, and 1.3- and 2. 8-fold reduction, respectively, in non-beta3-expressing Chinese hamster ovary and 293 cell lines. This sequence also bound nuclear proteins in a cell-specific manner in electrophoretic mobility shift assays. Mutational analysis indicated that the sequence GAGGGG (positions -113 to -108) is a megakaryocytic cell line-specific cis-acting element. (3) The region from -89 to +29 promoted lower activity in all cell lines. We also provide evidence that a CCCACCC sequence at position -70 has transcriptional activity, most likely through the Sp1 transcription factor. These data supply the first detailed map of the transcriptional regulatory elements of the 5' region of the beta3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment beta3 gene expression.

Identification of Regulatory Elements of Human α6 Integrin Subunit Gene

The integrin α6 subunit associates with either the β1 or β4 subunit to form receptors for laminin, a major component of the basement membrane. Here, we characterized basal promoter of the human integrin α6 subunit gene. The transcription start site, mapped by primer extension analysis, was 208 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes, but did contain a TATA-like sequence (GATAAA) 23 bp upstream of the major transcription start site. Consensus binding sites for Sp1 and the NF-κB complex were also present in the promoter region. A putative glucocorticoid/progesterone receptor responsive element (GRE/PRE), together with the Ap1 and c-myc binding sites located around 350-360 bp upstream of the transcription start site, represented positive regulatory sequences. Our current study showed a molecular model by which progesterone promotes tumor cell invasion through the basement membrane by up-regulating the laminin receptor.

Genomic Organization, Chromosomal Localization, and Expression of the Murine Thromboxane Synthase Gene (Tbxas1)

Thromboxane synthase (TS) is a membrane-bound cytochrome P450 enzyme catalyzing the synthesis of TxA2, a potent modulator of vascular smooth muscle contraction and platelet aggregation. TS plays an important role in hemostasis and may be intimately involved in the etiology of cardiovascular, renal, and immune diseases. Restriction enzyme mapping, subcloning, and DNA sequencing analysis of recombinant phage λ and P1 clones revealed that exons encoding the 1.9-kb mouse TS mRNA are dispersed over >150 kb genomic DNA. Determination of the intron–exon splicing junctions established that the mouse TS gene (Tbxas1) is encoded by 13 exons ranging in size from 53 (exon III) to 315 bp (exon IX). Genomic Southern analysis and fluorescencein situhybridization suggested that the gene is a single-copy gene, located on chromosome 6 near the midpoint between the centromere and the Igκ gene. An alternatively spliced variant of theTbxas1transcript, lacking the exon XII-encoded sequence, has been detected in normal mouse tissues. Ribonuclease protection and 5′-RACE assays identified at least five major transcription start sites clustered within 31 bp of theTbxas1promoter. The 5′-most start site is not preceded by a TATA box, suggesting transcription can be initiated in a TATA-independent manner. Transfection analyses indicated that the expression ofTbxas1is controlled by a short (70-bp) positive regulatory sequence and several upstream repressive elements. Mutational studies further demonstrated that NF-E2/AP-1 and Sp1 exerted activating and repressive, respectively, effects on the promoter. These studies provide the genetic tools and information for TS research in mice, which should expedite understanding of the genetic contribution of TS in normal physiology as well as in disease states.

Further Characterization of the Major and Minor RabbitFMO1Promoters and Identification of both Positive and Negative Distal Regulatory Elements

Previously, two promoters were identified for the rabbitFMO1gene: a major, upstream promoter (P0) that initiates transcription from exon 0 and a second, minor promoter (P1) located approximately 200 bp downstream and initiating transcription from exon 1. Transcription initiation from the P0promoter results in elimination of the exon 1 leader sequence from the mature transcript. In this report, we further define the major promoter and identify several positive and negative upstream regulatory domains employing deletion analysis and transient expression in HepG2 cells. Of interest, P0and P1were equally active in these assays. A 49-bp fragment spanning position −41 to +8 was found essential for the activity of P0and also capable of basal transcriptional activity. Interestingly, this same 49-bp region was found necessary for P1activity. Upstream of P0, three positive regulatory regions (positions −348 to −176, −757 to −584, and −1196 to −829) and two negative regulatory regions (positions −2120 to −1724 and −829 to −757) were identified using deletion mutants. Both P0and P1share the most proximate, positive regulatory domain but were regulated differentially by more distal 5′ sequences. In addition to the upstream regulatory sequences, a potent negatively acting element was observed within intron 1. Using DNA fragments representing the most potent positive (position −348 to −176) and negative (position −829 to −757) regulatory sequences as probes, we demonstrate the formation of multiple specific DNA/protein complexes with protein factor(s) present in HepG2 nuclear extract.

Novel Testis-specific Protein-DNA Interactions Activate Transcription of the Mouse Protamine 2 Gene during Spermatogenesis

The mouse protamines are expressed exclusively in postmeiotic male germ cells and are crucial for the compaction of chromatin during the late stages of spermatogenesis. The temporal expression of the two mouse protamines is transcriptionally regulated in the testis. Recent studies have demonstrated that ubiquitous and testis-specific proteins bind to the promoter of the mouse protamine 2 (mP2) gene. We have performed in vitro transcription and mobility shift assays to characterize the functional significance of the protein-DNA interactions within 180 base pairs upstream of the mP2 transcription start site. Deletion and mutational analyses reveal two positive regulatory sequences for mP2 transcription at positions -59/-47 and -83/-72 of the mP2 promoter. The proximal element at -59/-47 binds to a novel testis-specific protein we name protamine-activating factor 1 (PAF-1). PAF-1 reaches high levels in round spermatids at the time of mP2 transcription. Deletion of the -59/-47 sequence results in about a 3-fold reduction of mP2 transcription in vitro. Although the PAF-1 binding site (PAF-responsive element, PAF-RE), contains the sequence GTCA present in the cAMP-responsive element and is very similar to the estrogen-responsive element, mobility shift assays revealed that neither the cAMP-responsive element modulator nor the estrogen receptor is the protein(s) binding to PAF-RE. Competition mobility shift assays reveal that the second positive regulatory element at -83/-72 binds a Y-box-binding protein. Using in vitro transcription assays, a 5-fold decrease in mP2 transcription is seen when both the PAF-RE and this Y-box are deleted. These data suggest that the testis-specific PAF-1 and a Y-box-binding protein are needed to activate mP2 transcription in postmeiotic male germ cells.

The transcription factor E2F-1 modulates TGF-beta1 RNA expression in glial cells.

The cell type specificity of the regulation of expression of the potent growth inhibitory cytokine transforming growth factor-beta (TGF-beta), prompted our analyses of the regulation of TGF-beta1 gene expression in glial cells by viral and cellular oncoproteins. We have shown that SV40 T-antigen diminished TGF-beta1 expression in glial cells and this repression was dependent on the ability of T-antigen to interact with the tumor suppressor protein, pRb, and two structurally related proteins, p107 and p130. The cellular transcription factor E2F-1, which is a downstream effector of T-antigen, was unable to influence expression from the TGF-beta1 promoter by itself. Interestingly, E2F-1 could overcome viral T-antigen-mediated repression of the TGF-beta1 promoter, suggesting potential feedback loop between TGF-beta and E2F in virally transformed glial cells. Using deletion analyses, we have mapped two E2F-1-responsive regions on the TGF-beta1 promoter: a T-antigen-dependent negative regulatory sequence (TdNRS) between -323 and -175, and a T-antigen-independent positive regulatory sequence (TiPRS) between -34 and +10 on the TGF-beta1 promoter. Further examination of TiPRS revealed the presence of a functional E2F binding site. Interestingly, the amino terminus of E2F-1 was required for its activation of TGF-beta1 expression, as mutations in that domain abolished the ability of E2F-1 to increase TGF-beta1 expression. These data suggest that yet-uncharacterized interaction between the amino terminus of E2F-1 and cellular proteins regulates TGF-beta1 expression. The mechanism for E2F-1-mediated T-antigen-dependent regulation of TGF-beta1 expression from TdNRS awaits further characterization.

The bZIP Transcription Factor Nrl Stimulates Rhodopsin Promoter Activity in Primary Retinal Cell Cultures

In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp. Activity of the -40 to -84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.

Multiple factors regulating the expression of human thromboxane synthase gene.

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

Analysis of the rat clusterin gene promoter and cyclic AMP-regulated mRNA stability in testicular cells.

Clusterin, also known as SGP-2 or TRPM-2, is expressed in the male reproductive tissues at different levels. The genomic structure of the rat clusterin gene was recently reported by our laboratory and others. In this study, we have determined the promoter responsible for the basal expression of the rat clusterin gene in testicular cells by analyzing the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in MA-10 cells driven by different segments of the 5'-flanking region and the first intron of the clusterin gene. The region required for maximal basal expression was identified at -266 to +54. Addition of DNA fragments of the rat clusterin gene from -1298 to -266 bp, or from +54 to +1153 to (-266/+54)CAT resulted in a 87% decrease in CAT activity, suggesting the presence of inhibitory DNA elements in both the 5'-flanking region and the first intron. When DNA fragment in the first intron, +1153 to +2874, was included, CAT activity in the (-266/+2874)CAT construct increased to 70% of the clusterin promoter (-266/+54)CAT, indicating that stimulatory DNA elements may be present in this region of the first intron. Treatment of MA-10 cells with cyclic AMP (cAMP) neither decreased CAT activity driven by any of the clusterin/CAT chimeric plasmids examined in transient transfection studies, nor reduced the synthesis of nuclear clusterin RNA in nuclear run-on assays, indicating that the reduction of clusterin mRNA levels by cAMP previously reported in our laboratory is not exerted at the transcriptional level. Furthermore, addition of transcriptional or translational inhibitors (actinomycin D and cycloheximide respectively) abolished the cAMP effect observed in MA-10 cells. In summary, we have demonstrated that the basal transcription of the rat clusterin gene in testicular cells is under the control of both positive and negative regulatory sequences at the 5'-flanking region as well as in the first intron. The reduction of clusterin mRNA after exposure of MA-10 cells to cAMP is not due to a decrease in its transcriptional activity, but rather to an increase in the degradation of this mRNA through synthesis of a destabilizing protein(s) and its mRNA.

Structural and functional analysis of the 5′-flanking region of the rat β2-adrenergic receptor gene

We have analyzed 3.4 kb of DNA from the 5′-flanking region of the rat β 2-adrenergic receptor gene and assessed its promoter activity in A549 cells, a human lung adenocarcinoma cell line. A single transcription start site was identified approx. 223 bp upstream of the ATG start codon. A549 cells were transfected with luciferase reporter plasmids containing segments of the rat β 2-adrenergic receptor 5′-flanking region. Our results suggest that both positive and negative cis-acting regulatory sequences are present in the 5′-flanking region of the rat β 2-adrenergic receptor gene.