Activate Reporter Gene Expression Research Articles

Functional Characterization of and Cooperation between the Double-stranded RNA-binding Motifs of the Protein Kinase PKR

The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.

Downregulation of Fasting-Induced cAMP Response Element-Mediated Gene Induction by Leptin in Neuropeptide Y Neurons of the Arcuate Nucleus

States of increased metabolic demand such as fasting modulate hypothalamic neuropeptide gene expression and decrease circulating leptin levels. This study tested the hypotheses that fasting stimulates gene induction mediated by cAMP response element (CRE)-dependent increases in gene transcription and that fasting-induced decreases in leptin can regulate this CRE-mediated gene induction. Using C57BL/6J mice transgenic for a CRE-lacZ construct, an immunocytochemical study showed that fasting activated reporter gene expression in the hypothalamic arcuate nucleus (Arc) in a small subset of neurons and increased phosphorylation of CRE binding protein. The increase of beta-galactosidase expression caused by fasting was inhibited by a protein kinase A inhibitor, Rp-8-Br-cAMPS, when the compound was microinjected into the medial basal hypothalamus, and enhanced by intraperitoneal injection of selective phosphodiesterase inhibitors. In situ hybridization studies showed that neuropeptide Y (NPY) mRNA levels increased in the Arc during fasting, whereas proopiomelanocortin (POMC) mRNA levels decreased. Double labeling of mRNA and beta-galactosidase immunoreactivity in the fasted brain indicated that the subpopulation of the neurons expressing beta-galactosidase all produced NPY but not POMC. To study the possible involvement of decreased circulating leptin during starvation on CRE-mediated gene induction, leptin was administered intraperitoneally to fasted mice. Leptin significantly attenuated both beta-galactosidase expression and NPY gene expression stimulated by fasting, suggesting that leptin inhibits fasting-stimulated NPY gene expression at least in part through downregulation of CRE-mediated gene induction in the Arc. Leptin-induced modification of CRE-mediated gene induction in the Arc may play an essential role in the central regulation of feeding behavior and energy expenditure.

Identification of a S-ribonuclease-binding protein in Petunia hybrida.

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBPI may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.

Cell signaling switches HOX-PBX complexes from repressors to activators of transcription mediated by histone deacetylases and histone acetyltransferases.

The Hoxb1 autoregulatory element comprises three HOX-PBX binding sites. Despite the presence of HOXB1 and PBX1, this enhancer fails to activate reporter gene expression in retinoic acid-treated P19 cell monolayers. Activation requires cell aggregation in addition to RA. This suggests that HOX-PBX complexes may repress transcription under some conditions. Consistent with this, multimerized HOX-PBX binding sites repress reporter gene expression in HEK293 cells. We provide a mechanistic basis for repressor function by demonstrating that a corepressor complex, including histone deacetylases (HDACs) 1 and 3, mSIN3B, and N-CoR/SMRT, interacts with PBX1A. We map a site of interaction with HDAC1 to the PBX1 N terminus and show that the PBX partner is required for repression by the HOX-PBX complex. Treatment with the deacetylase inhibitor trichostatin A not only relieves repression but also converts the HOX-PBX complex to a net activator of transcription. We show that this activation function is mediated by the recruitment of the coactivator CREB-binding protein by the HOX partner. Interestingly, HOX-PBX complexes are switched from transcriptional repressors to activators in response to protein kinase A signaling or cell aggregation. Together, our results suggest a model whereby the HOX-PBX complex can act as a repressor or activator of transcription via association with corepressors and coactivators. The model implies that cell signaling is a direct determinant of HOX-PBX function in the patterning of the animal embryo.

The Splicing Factor U1C Represses EWS/FLI-mediated Transactivation

EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.

A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay.

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10

In Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-related genes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one of these genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function early in development. Here, we report the characterization of a novel tinman-related gene , XNkx2-10, which is expressed during early heart development in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previously shown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 and SRF transcription factors to activate reporter gene expression.

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene.

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

Characterization and properties of heteromeric plant protein complexes that interact with tef cis-acting elements in both RNA polymerase II-dependent promoters and rDNA spacer sequences.

The tef box, a cis-acting element identified in promoters of several plant genes encoding components of the translation apparatus, is involved in the activation of gene expression in cycling cells. In vitro, this element mediates the formation of two protein complexes called C1 and C2. A tef-like box is also found within the intergenic transcribed spacer of several plant rRNA genes. In radish this sequence has already been described as a protein-binding site putatively involved in the regulation of rDNA expression and is sufficient for formation of C1 complexes. By using mutated tef boxes, we show that tef-dependent activation of transcription is correlated with formation of both C1 and C2 complexes in a context-dependent manner. In transient expression experiments, the activation of a minimal promoter-GUS gene fusion is associated with the formation of C2 complexes. In contrast, the ability to form C1 complexes appears to allow activation of reporter gene expression in root meristems of transgenic Arabidopsis. SDS-PAGE analysis of purified protein fractions containing either the C1 or the C2 activity indicates a complex heteromeric structure for these potential regulators. Thus, the tef box seems to be a central component of the regulation of gene transcription in distinct and overlapping developmental programs, and could be involved in co-regulation of transcription by RNA polymerases I and II.

Nuclear localization and formation of beta-catenin-lymphoid enhancer factor 1 complexes are not sufficient for activation of gene expression.

In response to activation of the Wnt signaling pathway, beta-catenin accumulates in the nucleus, where it cooperates with LEF/TCF (for lymphoid enhancer factor and T-cell factor) transcription factors to activate gene expression. The mechanisms by which beta-catenin undergoes this shift in location and participates in activation of gene transcription are unknown. We demonstrate here that beta-catenin can be imported into the nucleus independently of LEF/TCF binding, and it may also be exported from nuclei. We have introduced a small deletion within beta-catenin (Delta19) that disrupts binding to LEF-1, E-cadherin, and APC but not axin. This Delta19 beta-catenin mutant localizes to the nucleus because it may not be efficiently sequestered in the cytoplasm. The nuclear localization of Delta19 definitively demonstrates that the mechanisms by which beta-catenin localizes in the nucleus are completely independent of LEF/TCF factors. beta-Catenin and LEF-1 complexes can activate reporter gene expression in a transformed T-lymphocyte cell line (Jurkat) but not in normal T lymphocytes, even though both factors are nuclear. Thus, localization of both factors to the nucleus is not sufficient for activation of gene expression. Excess beta-catenin can squelch reporter gene activation by LEF-1-beta-catenin complexes but not activation by the transcription factor VP16. Taken together, these data suggest that a third component is necessary for gene activation and that this third component may vary with cell type.

Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression

Estradiol (E 2)-liganded estrogen receptor (ER) bound to three or four tandem copies of a consensus ERE (EREc38) in a cooperative manner. E 2-ER binding to one or two EREs was non-cooperative. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), ER-ERE binding was not cooperative, regardless of the number of EREs. Here we evaluated how binding to EREc38 affects ER conformation in the ligand binding domain (LBD) as reflected in the dissociation kinetics of [ 3H]ligand from the ER. Binding of ER to EREc38 slowed the rate of dissociation of either E 2 or 4-OHT, indicating that DNA allosterically modulates the LBD conformation creating a tighter fit between the ligand and the ER. Conformational differences in ER induced by E 2 versus antiestrogen were not reflected in differences in E 2 or 4-OHT dissociation parameters under these conditions. No difference in the association rate of E 2- versus 4-OHT-liganded ER binding to EREc38 was detected in electrophoretic mobility shift assay (EMSA). Synergistic, E 2-dependent activation of a reporter gene was detected from three and four, but not one or two, tandem copies of EREc38. These observations suggest that cooperative binding of E 2-ER to multiple copies of EREc38 is likely responsible for transcriptional synergy and that cooperativity may not involve direct interaction between the LBDs of ERE-bound ER. Since the number of copies of EREc38 did not alter E 2 dissociation kinetics, functional synergy must involve cellular factors in addition to the ER ligand.

M phase‐specific activation of theNicotiana sylvestris Cyclin B1promoter involves multiple regulatory elements

Summary B‐type cyclins are cell cycle regulatory proteins specifically expressed in G2‐M phases. To understand the mechanisms that regulate their expression, it is important to identify the required promoter element(s). By using synchronized BY‐2 cell cultures stably transformed with chimeric genes composed of sequential Nicsy; CycB1 ; 1 promoter deletions fused to the β‐glucuronidase reporter gene ( gus ), we show that at least five distinct promoter regions are required for maximal M‐phase expression. Furthermore, two distinct promoter regions contain sufficient element(s) to induce M‐phase‐specific expression. In one of these regions, a 23 bp promoter element is able to activate reporter gene expression in cells induced to divide in an orientation‐independent manner but without an M‐phase specific expression. Therefore, this 23 bp element, which contains a 5 bp element identical to the MYB binding core, is a good upstream activating sequence (UAS) candidate. Moreover, electrophoretic mobility shift assays show that this putative UAS specifically binds protein complexes that appear to differ whether cells are cycling or not. The constitutive transcriptional activation mediated by this UAS would suggest that the Nicsy; CycB1 ; 1 gene promoter is regulated through an activation/repression mechanism.

A positive GATA element and a negative vitamin D receptor-like element control atrial chamber-specific expression of a slow myosin heavy-chain gene during cardiac morphogenesis.

We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif, control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity were found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increased in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form.

Regulation of rat hepatic cytochrome P450 expression by sterol biosynthesis inhibition: inhibitors of squalene synthase are potent inducers of CYP2B expression in primary cultured rat hepatocytes and rat liver.

The effects of treatment with squalestatin 1, a potent inhibitor of squalene synthase, the first committed enzyme of sterol biosynthesis, were examined on cytochrome P450 expression in primary cultured rat hepatocytes and rat liver. Incubation of cultured hepatocytes with squalestatin 1 caused marked accumulations (maximal elevations that were approximately 25-100% of phenobarbital-elicited increases) of CYP2B mRNA and immunoreactive protein but not of CYP1A, CYP3A, or CYP4A. Squalestatin 1 treatment increased CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA content in hepatocyte cultures with comparable potencies (ED50 = 5.0 and 18 nM, respectively), and significantly induced CYP2B (mRNA, immunoreactive protein, and pentoxyresorufin O-dealkylase activity) in the livers of treated rats, producing maximal increases at a dose of 25 mg/kg/day that were approximately 32-87% of phenobarbital-induced increases. Squalestatin 1 treatment induced both CYP2B1 and CYP2B2 and activated reporter gene expression in cultured hepatocytes transiently transfected with a plasmid containing approximately 2.4 kb of CYP2B1 gene 5'-flanking region or containing a previously described phenobarbital-responsive region. Coincubation of cultured hepatocytes with 25-hydroxycholesterol suppressed squalestatin 1-mediated CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A mRNA induction with approximately the same potency. Treatment of cultures with SQ-34919, a structurally distinct squalene synthase inhibitor, produced the same selective CYP2B mRNA induction as did squalestatin 1. These results suggest that inhibition of hepatic sterol synthesis activates processes that culminate in increased CYP2B gene transcription.

Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene.

A functionally important cis-acting element termed P2 was identified in the liver promoter of the glucokinase gene. Element P2 was delineated by footprinting in vitro with nuclear proteins from rat liver and spleen. Its core sequence in the rat gene is a canonical CACGTG E-box. In the electrophoretic mobility-shift assay with nuclear proteins from rat liver, hepatocytes and hepatoma cells, an oligonucleotide with P2 in the context of the glucokinase promoter sequence gave rise to a DNA-protein complex shown to contain the upstream stimulatory factor (USF) by specific competition experiments and by reactivity with anti-USF antibodies. Transient transfection of hepatoma HepG2 cells, combined with site-directed mutagenesis, demonstrated that the P2 element was important for liver glucokinase promoter activity. Co-transfection of an expression plasmid coding for USF1 activated reporter gene expression in a manner dependent on an intact P2 element, whereas an expression plasmid for c-Myc was ineffective. Expression of a truncated form of USF1 lacking the transcription activation domain and the basic region decreased reporter activity by a dominant-negative effect. The functional significance of the P2 element was also demonstrated in transient transfection of primary hepatocytes.

DNA regions supporting hippocalcin gene expression in cell lines

The rat hippocalcin gene −3.2 to +0.6 kb region activates reporter gene expression in the NG108-15 and PC12 neuronal cell lines, but not in NIH3T3 or HEK-293 cells. Three fragments (−3.2 to −2.6, −2.6 to −2.3 and −2.3 to −1.8 kb) weakly activate transcription, and "−1.8 to −1.5" kb is a strong activator. Thus cell type-specific expression of the rat hippocalcin gene is regulated by distributed elements in the −3.2 to −1.5 kb region.

Elements Regulating Cardiomyocyte Expression of the Human Sarcomeric Mitochondrial Creatine Kinase Gene in Transgenic Mice

Sarcomeric mitochondrial creatine kinase (sMtCK) is one component of a multiprotein, high energy channeling complex consisting of porin, mitochondrial creatine kinase, and adenine nucleotide translocase. To study the transcriptional mechanisms specifying sMtCK gene expression to the heart, transgenic mice were created carrying the 5'-flanking sequences of the human sMtCK gene ligated upstream of the human growth hormone (hGH) reporter gene. RNA blot hybridization demonstrated that the human sMtCK sequence, -485 to +6 base pair (bp), did not activate reporter gene expression to a detectable level. However, the human sMtCK sequence, -921 to +6 bp, expressed the hGH reporter gene at a high level in heart and skeletal muscle and at a very low level in esophagus and kidney, and it did not express the hGH gene in other organs tested (brain, lung, liver, spleen, bladder, uterus, and stomach). In situ hybridization revealed that reporter gene transcription was specified to cardiac and skeletal myocytes, recapitulating precisely the expression pattern of the endogenous gene. Sequence analysis identified several consensus binding sites between -921 and -757 bp, including four GATT motifs, one E box, and one MEF2 site. Further analysis of a third transgenic mouse strain demonstrated that the human sMtCK sequence, -757 to +6 bp, did not direct detectable expression of the hGH reporter gene. We conclude that this 160-bp genomic sequence, from -921 to -757 bp, is necessary in specifying expression of the human sMtCK gene to the oxidative and highly metabolically active heart tissue.

POU Domain Factors of the Brn-3 Class Recognize Functional DNA Elements Which Are Distinctive, Symmetrical, and Highly Conserved in Evolution

To better understand the diversity of function within the POU domain class of transcriptional regulators, we have determined the optimal DNA recognition site of several proteins of the POU-IV (Brn-3) subclass by random oligonucleotide selection. The consensus recognition element derived in this study, ATAATTAAT, is clearly distinct from octamer sites described for the POU factor Oct-1. The optimal POU-IV site determined here also binds Brn-3.0 with significantly higher affinity than consensus recognition sites previously proposed for this POU subclass. The binding affinity of Brn-3.0 on its optimal site, several variants of this site, and several naturally occurring POU recognition elements is highly correlated with the activation of reporter gene expression by Brn-3.0 in transfection assays. The preferred DNA recognition site of Brn-3.0 resembles strongly the optimal sites of another mammalian POU-IV class protein, Brn-3.2, and of the Caenorhabditis elegans Brn-3.0 homolog Unc-86, demonstrating that the site-specific DNA recognition properties of these factors are highly conserved between widely divergent species.

Cooperative regulation of the human aromatase cytochrome P450 gene transcription by placenta-specific cis-acting elements

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between −242 and −166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between −238 and −200), and element II (located between −196 and −176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein β), which is located between −2141 and −2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.

Cooperative Regulation of the Human Aromatase Cytochrome P450 Gene Transcription by Placenta-specific cis-acting Elements

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between −242 and −166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between −238 and −200), and element II (located between −196 and −176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein β), which is located between −2141 and −2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.