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PubMed2002

Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells.

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

CD28 Antigens↗
PubMed1995

Role of specific response elements of the c-fos promoter and involvement of intermediate transcription factor(s) in the induction of Sertoli cell differentiation (transferrin promoter activation) by the testicular paracrine factor PModS.

A mesenchymal-epithelial cell interaction exists in the testis between the Sertoli cells that form the seminiferous tubule and the mesenchymal-derived peritubular myoid cells that surround the tubule. Analysis of the mesenchymal-epithelial interactions between these cells revealed the local production of a mesenchymal factor, PModS. PModS modulates the differentiated functions of Sertoli cells in vitro, including stimulation of the iron-binding protein transferrin (Tf). Previous results have indicated that PModS-induced Tf gene expression involves the activation of immediate early genes. One of the immediate early genes was identified as c-fos. The importance of c-fos was demonstrated in the current study when a c-fos antisense oligonucleotide was found to inhibit the ability of PModS to induce the expression of a Tf promoter-chloramphenicol acetyltransferase (CAT) construct. The regulation of c-fos by PModS was investigated with various CAT constructs containing segments of the c-fos promoter, such as the serum response element (SRE), sis-inducible element (SIE), cAMP response element (CRE), and phorbol ester/TPA response element (TRE), transfected into cultured Sertoli cells. PModS has no effect on cAMP response element-CAT or TRE-CAT, suggesting that PModS does not act through stimulation of cAMP and protein kinase C pathways. PModS was found to activate the c-fos SRE-CAT construct and the SIE-CAT construct. A construct containing both SIE and SRE was stimulated to the same degree as either element alone. Gel mobility shift assays using nuclear extracts from PModS-stimulated Sertoli cells and a radiolabeled SRE oligonucleotide resulted in retarded mobility of a DNA-protein complex. A gel shift with a SRE oligonucleotide containing an ETS domain resulted in a unique shift only detected in PModS stimulated cells. PModS also promoted a gel shift with the SIE that is adjacent to the SRE on the c-fos promoter. The data imply that PModS can activate the c-fos promoter through the SRE and SIE. PModS caused a labeled activating protein 1 (AP1) oligonucleotide to form a DNA-protein complex, indicating activation of the c-fos gene and binding of the c-fos/jun complex. To study the downstream regulation of Sertoli cell differentiation, Tf gene expression was examined. CAT constructs containing deletion mutants of a 3-kilobase (kb) mouse Tf promoter were used. When transfected into Sertoli cells the 581-base pair Tf minimal promoter had only a slight response to PModS, but was activated by FSH. The 2.6-kb Tf promoter construct responded to PModS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Two separate NCoR (nuclear receptor corepressor) interaction domains mediate corepressor action on thyroid hormone response elements.

The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. The interactions between NCoR interaction domains and TR on DNA response elements, however, have not been well characterized. We have found that both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements.

Amino Acid Sequence↗
PubMed1997

Characterization of an intronic hormone response element of the rat liver/skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The glucocorticoid response element of the rat liver/skeletal muscle 6- phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene was characterized. The element is composed of two tandem hormone receptor binding sites separated by 12 base pairs. Addition of dexamethasone to HeLa cells transiently transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid containing the hormone response element and cotransfected with glucocorticoid receptor stimulated transcription 24-fold in an orientation- and position-independent manner. Deletion or mutation of essential G/C pairs of the distal binding site abolished hormone-stimulated CAT activity, whereas deletion or mutation of the proximal binding site decreased the hormone-stimulated response only slightly. Mutation of both distal and proximal binding sites resulted in complete loss of hormone-stimulated CAT activity. Experiments carried out using testosterone and progesterone with their respective receptors revealed qualitatively similar results to those seen with glucocorticoid. Binding of glucocorticoid receptor or androgen receptor DNA binding domains to the hormone response element, visualized by gel mobility shift, was unaffected in the proximal binding site mutant, markedly decreased in the distal binding site mutant, and abolished in the double mutant. In gel mobility shift analysis of separate distal and proximal binding sites, only the native distal site demonstrated high affinity binding to glucocorticoid and androgen receptor DNA binding domains. The results demonstrate that this element is responsible for glucocorticoid, androgen, and progesterone stimulation of transcription of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene and that the distal receptor binding site is dominant.

Animals↗
PubMed2002

Atomic force microscopy reveals kinks in the p53 response element DNA.

p53 is a 53 kDa nuclear phosphoprotein. Its function as a tumor suppressor critically lies in its ability to recognize its target DNA response elements as a tetramer. Here, we report the structural theme intrinsic to the response element DNA that governs this recognition phenomenon. The intrinsic flexibility or dynamic bending between two distinctly different, but naturally occurring p53 response elements has been compared by ring closure. Results show that DNA binding sites containing helically phased d(CATG.CATG) tetra-nucleotide sequences at the centers of quasi-dyad symmetry in each half-response site are more intrinsically flexible (i.e. preferentially bent under axial stress) than their d(CTTG.CTTG) counterparts. Intriguingly, p53 binding sites containing these more flexible d(CATG.CATG) sequence elements also exhibit a stronger tendency for tetrameric binding of the p53 DNA binding domain peptide. Examination of the shapes of DNA microcircles obtained by circularization of oligomers constructed from such flexible p53 target DNA sequences in tandem using MacMode atomic force microscopy directly revealed sequence-specific kinks in solution. The tetra-nucleotide sequence d(CATG.CATG) is highly conserved in most functional p53 response elements. Consequently, we propose that the sequence-specific kinks originating from d(CATG.CATG) sequences could be a common structural theme in p53 response elements and as evident from the results reported here, could be a determinant of binding site recognition by the p53 protein and the subsequent stability of the p53-DNA complex.

Base Sequence↗
PubMed2006

Pescadillo interacts with the cadmium response element of the human heme oxygenase-1 promoter in renal epithelial cells.

Renal tubular cells elicit adaptive responses following exposure to nephrotoxins, such as cadmium. One response is the up-regulation of the 32-kDa redox-sensitive protein, heme oxygenase-1. Exposure of renal proximal tubular epithelial cells to 10 mum cadmium demonstrated induction ( approximately 20-fold) of heme oxygenase-1 mRNA and protein. Using a 4.5-kb human heme oxygenase-1 promoter construct, the importance of a previously identified cadmium response element (TGCTAGAT) in HeLa cells was verified in renal epithelial cells. Specific protein-DNA interaction with this sequence was demonstrated using nuclear extracts from cadmium-treated cells. Yeast one-hybrid screen of a human kidney cDNA library resulted in the identification of pescadillo, a unique nucleolar, developmental protein, as an interacting protein with the cadmium response element and was confirmed by chromatin immunoprecipitation in vivo and gel shift assays with purified glutathione S-transferase-pescadillo protein in vitro. The specificity of the DNA-protein interaction was verified by the absence of a binding complex when the core sequence of the cadmium response element was mutated or deleted. In addition, B23/nucleophosmin, another nucleolar protein, did not interact with the cadmium response sequence. Overexpression of pescadillo resulted in increased activity of the 4.5-kb human heme oxygenase-1 promoter construct but failed to activate this construct when the cadmium response sequence was mutated. The findings demonstrate the important and previously unrecognized role of pescadillo as a DNA-binding protein interacting specifically with the cadmium response element of the human heme oxygenase-1 gene.

Base Sequence↗
PubMed1997

Identification of a strand-specific Egr response element binding complex enriched in rat brain.

Multiple members of the Egr family of transcription regulatory factors are rapidly induced in response to neuronal stimulation and share a common double-stranded DNA binding consensus sequence, referred to as the Egr response element. Recent studies have identified transcription regulatory factors that bind preferentially to short segments of single-stranded DNA, rather than the conventional double-stranded versions of regulatory elements. Accordingly, in the present study, we have investigated whether the Egr response element may also be regulated by trans factors that bind to single-stranded versions of this cis element. Using gel-shift studies, we have identified a protein complex that binds selectively to the G-rich strand of the Egr response element. In competition studies, an RNA oligonucleotide containing the corresponding G-rich sequence is approximately 25-fold less potent than its DNA counterpart. This DNA binding complex, referred to as GS1, is present in several regions of the rat brain with highest levels in cerebellum; negligible binding activity was detected in multiple peripheral tissues surveyed. UV cross-linking studies revealed two major protein bands with estimated molecular masses of 36 and 30 kDa. The highly restricted tissue distribution of this complex and its sequence-specific binding properties indicate that GS1 may be involved in regulating transcription directed by the Egr response element in brain.

Animals↗
PubMed1999

E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the presence of a number of conserved response elements that have previously been shown to regulate cell specific expression of the human transferrin (hTf) promoter. One of these elements is the human PRII region, which is a cAMP response element (CRE)-like element that is more than 80% conserved in the mTf promoter. The activation of the hTf promoter by FSH and cAMP in rat Sertoli cells has been shown to be mediated in part through the CRE-like PRII region and binding of the CRE binding protein (CREB). The present study investigates the role of PRII in the activation of mTf promoter by FSH and cAMP in rat Sertoli cells. Mutations in the PRII of the mTf promoter reduced FSH activation by only 50% and cAMP activation by more than 90%. In contrast, the mutant PRII mTf promoter construct was fully activated by a partially purified testicular paracrine activity PModS(S300). Gel shift experiments demonstrated that proteins that can bind a consensus CRE oligonucleotide also bind the PRII region of the mTf promoter. An immunoblot confirmed that CREB binds the PRII and promotes the gel shift observed. The hypothesis developed was that another cis-acting element in addition to the CRE-like PRII is also involved in FSH actions. A conserved response element in both the mTf and hTf promoters is the basic helix-loop-helix (bHLH) responsive E-box sequence. Both FSH and PModS (S300) activity were found to promote a mTf E-box gel shift that contained the E2A gene product the bHLH protein E47. Interestingly, mutations in the E-box of the mTf promoter completely abolished the PModS(S300) activation and partially (52%) inhibited the activation by FSH. In contrast, the mutant E-box mTf promoter construct was fully activated by cAMP. Finally a double mutation of both the PRII and the E-box completely abolished FSH activation of the mTf promoter. These results suggest that optimal activation of the mouse transferrin promoter by FSH requires both CREB binding to the CRE-like PRII region and bHLH binding to the E-box. Information is provided that indicates a number of Sertoli cell promoters contain a close association of E-box and CRE-like elements. Observations are discussed in regards to the potential interactions of the CRE and E-box response elements in mediating FSH actions in Sertoli cells.

Animals↗
PubMed2001

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

5-Aminolevulinate Synthetase↗
PubMed1999

Salicylic acid and aspirin inhibit the activity of RSK2 kinase and repress RSK2-dependent transcription of cyclic AMP response element binding protein- and NF-kappa B-responsive genes.

Sodium salicylate (NaSal) and other nonsteroidal anti-inflammatory drugs (NSAIDs) coordinately inhibit the activity of NF-kappa B, activate heat shock transcription factor 1 and suppress cytokine gene expression in activated monocytes and macrophages. Because our preliminary studies indicated that these effects could be mimicked by inhibitors of signal transduction, we have studied the effects of NSAIDs on signaling molecules potentially downstream of LPS receptors in activated macrophages. Our findings indicate that ribosomal S6 kinase 2 (RSK2), a 90-kDa ribosomal S6 kinase with a critical role as an effector of the RAS-mitogen-activated protein kinase pathway and a regulator of immediate early gene transcription is a target for inhibition by the NSAIDs. NSAIDs inhibited the activity of purified RSK2 kinase in vitro and of RSK2 in mammalian cells and suppressed the phosphorylation of RSK2 substrates cAMP response element binding protein (CREB) and I-kappa B alpha in vivo. Additionally, NaSal inhibited the phosphorylation by RSK2 of CREB and I-kappa B alpha on residues crucial for their transcriptional activity in vivo and thus repressed CREB and NF-kappa B-dependent transcription. These experiments suggest that RSK2 is a target for NSAIDs in the inhibition of monocyte-specific gene expression and indicate the importance of RSK2 and related kinases in cell regulation, indicating a new area for anti-inflammatory drug discovery.

3T3 Cells↗
PubMed2006

Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

Animals↗
PubMed1996

Regulatable promoters for use in gene therapy applications: modification of the 5'-flanking region of the CFTR gene with multiple cAMP response elements to support basal, low-level gene expression that can be upregulated by exogenous agents that raise intracellular levels of cAMP.

This study focuses on the design, construction, and evaluation of a chimeric promoter for gene therapy applications where it is desirable to have low-level basal expression of the newly transferred gene, which can be induced to higher levels of expression by the administration of pharmacologic agents that can be safely used locally and/or systemically in humans. To achieve this, a chimeric promoter was constructed using fragments of the 5'-flanking region of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, and multiple tandem repeats of the consensus sequence and flanking elements of the cAMP response element (CRE), promoter sequences that support increased transcription in response to elevations in intracellular cAMP levels. Preliminary studies using plasmid vectors demonstrated that: (i) the 5'-flanking sequences from the CFTR gene have low promoter activity in the human airway epithelial cell lines; (ii) chimeras using -718 bp fragment from the 5'-flanking sequence of CFTR gene as the base, with the addition of 4-10 units of a 25-bp sequence containing the CRE consensus sequence, were all inducible by a rise in intracellular cAMP, with the chimera having eight CRE repeats the most responsive; and (iii) a CF126(CRE8) chjimera, consisting of the -126 bp fragment from the 5'-flanking region of CFTR gene together with eight CRE repeats, yielded low-level basal activity but maximal upregulation by cAMP, resulting in expression of the reporter gene that was 51-58% of an RSV-LTR control. On the basis of these observations, replication-deficient adenoviral vectors containing the CF126(CRE8) chimera and the luciferase reporter gene [AdCF126(CRE8).Luc] or the Escherichia coli lacZ (beta-Gal) reporter gene [AdCF126(CRE8). beta gal] were constructed. In several human airway epithelial cell lines, the AdCF126 (CRE). Luc vector provided low basal activity, but was significantly upregulated by agents that increase cAMP levels. Intranasal administration of the beta-Gal-expressing AdCF126(CRE8) beta gal vector into C57B1/6 mice demonstrated cAMP-induced upregulation of the reporter gene in airway epithelial cells. Quantification of the inducibility of the basal promoter activity in the airway epithelium using the AdCF126(CRE8). Luc vector demonstrated an 11-fold upregulation of the basal promoter activity in the lung with the administration of a phosphodiesterase inhibitor and a cAMP analog. These observations demonstrate the feasibility of using a chimeric promoter comprised of a minimal fragment of the CFTR 5'-flanking region, together with added multiple CRE, to control genes delivered in vivo. Importantly, because there are many drugs used in humans that raise cAMP, the concept of using a cAMP-regulatable promoter may also be a useful approach to enhance the safety, efficacy, and feasibility of a variety of human gene therapy strategies.

Animals↗
PubMed1988

The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements.

We report that the thyroid hormone (T3) receptor binds to DNA recognition sequences that are variations of the palindromic motif 5'-TCAGGTCA.TGACCTGA-3', resulting in transcriptional activation in vivo. This sequence is identical to the vitellogenin A2 estrogen response element except for the absence of 3 bp at the center of dyad symmetry, which we have termed a "gap." The T3 receptor binds to estrogen response elements with high affinity in vitro, as well as to variations of the same sequence containing gaps of 1-6 bp. The T3 receptor not only fails to activate transcription from estrogen response elements in vivo, but inhibits estrogen-dependent transactivation. We propose that the T3 receptor binds to estrogen response elements in a transcriptionally inactive form and competes for estrogen receptor binding, resulting in a net decrease in gene expression. These data reveal that only a subset of T3 receptor DNA-binding elements function as T3 response elements.

Animals↗
PubMed1994

The rat and human hemopexin genes contain an identical interleukin-6 response element that is not a target of CAAT enhancer-binding protein isoforms.

Hemopexin (Hx) is an abundant acute-phase protein (APP) that binds heme with high affinity. In rat hepatic cells, the transcription rate of the Hx gene is increased by interleukin (IL)-1 and IL-6. To investigate the cis-acting regulatory elements (REs) responsive to these hormones, chloramphenicol acetyltransferase constructs of rat and human Hx gene sequences were tested in transiently transfected hepatoma cells. An IL-6-RE was identified in the promoter of both rat and human Hx genes, the function of which was dependent on the core sequence (CCGGGAA) common in other APP genes. The previously characterized Hx A element mediated a relatively minor cytokine response as compared with the Hx IL-6-RE. The human Hx A element, in contrast to the rat and human Hx IL-6-REs, was strongly trans-activated by cotransfected CAAT enhancer-binding proteins (C/EBP)-beta and -delta. The rat gene homolog of the human Hx A element was inactive as a cytokine RE and was minimally trans-activated by C/EBP isoforms. Results of electrophoretic mobility shift assays indicated that the Hx IL-6-RE is a binding site for the IL-6-inducible nuclear protein IL-6 RE-BP, which also binds to the conserved IL-6-REs of other APP genes and is distinct from C/EBP beta.

Animals↗
PubMed2000

Phosphorylated cAMP response element binding protein increases in neurokinin-1 receptor-immunoreactive neurons in rat spinal cord in response to formalin-induced nociception.

The rat neurokinin-1 (NK1) receptor gene contains a cyclic adenosine monophosphate (cAMP) response element, and gene transcription may be activated upon binding of phosphorylated cAMP response element binding protein (pCREB). If pCREB contributes to increased expression of NK1 receptors, pCREB should increase in neurons that express NK1 receptors under conditions that increase NK1 receptor mRNA. Evidence for this relationship was found following injection of formalin into one hindpaw of rats. Immunohistochemistry was employed to visualize NK1 receptors and pCREB in spinal cord sections. Formalin injection produced an increase in pCREB-immunofluorescence within NK1 receptor-immunoreactive neurons from segments L4 and L5. No change occurred in pCREB-immunofluorescence within NK1 receptor-immunoreactive neurons from segment T11. These data support the hypothesis that transcription factor pCREB contributes to increased expression of spinal NK1 receptors during persistent pain.

Animals↗
PubMed1994

A novel protein interacts with the major transforming growth factor-beta responsive element in the plasminogen activator inhibitor type-1 gene.

Multiple transforming growth factor-beta (TGF-beta) responsive elements have been identified within the 5'-flanking region of the plasminogen activator inhibitor type-1 (PAI-1) gene. This study was designed to characterize the major TGF-beta responsive element (-804 to -546). DNA footprint assays showed that the region of protein contact (-726 to -703) did not include consensus sequences for any known transacting factors. The results of UV cross-linking and Southwestern blot experiments showed that a protein of M(r) 100,000 specifically binds to the TGF-beta responsive element and that this protein undergoes post-transcriptional activation within 5 min after stimulation of Hep G2 cells by TGF-beta resulting in a marked increase in affinity for the target DNA sequence. These results show that stimulation of Hep G2 cells with TGF-beta increases the affinity of a novel 100-kDa protein for the major TGF-beta responsive element within the PAI-1 gene.

Base Sequence↗
PubMed1996

Juvenile hormone regulation of an insect gene: a specific transcription factor and a DNA response element.

We have used locust fat body nuclear protein extracts and upstream DNA of the juvenile hormone (JH)-inducible locust gene, jhp21, to examine the regulation of specific transcription by JH. Promoter activity was assayed with G-free cassette reporter constructs. Nuclear extracts from adult female fat body, previously exposed to JH or an analog, actively transcribe from the jhp21 promoter and a control adenovirus major late (AdML) promoter, whereas extracts from JH-deprived female fat body, or other tissues, transcribe strongly from the AdML promoter but weakly or not at all from the jhp21 promoter. Transcription is enhanced by sequences between -140 and -211 nt from the jhp21 transcription start point (tsp), which include a CAAT box, and also by sequences between -1056 and -1200. A 15-nt partially palindromic sequence element found at -1152, resembling known hormone response elements, was shown to stimulate transcription when restored to truncated jhp21 DNA. Two very similar sequences occur further upstream. In electrophoretic mobility shift assays (EMSA), the same sequence element was shown to specifically bind a protein that was present in nuclear extracts from JH-exposed, but not from JH-deprived, fat body. Several lines of evidence suggest that the DNA element may be a JH response element (JHRE). The JH-induced protein that binds to it appears to be a transcription factor that activates the initiation of JH target gene (jhp21) transcription, and could be a JH receptor.

Animals↗
PubMed2006

In silico modelling of hormone response elements.

BACKGROUND: An important step in understanding the conditions that specify gene expression is the recognition of gene regulatory elements. Due to high diversity of different types of transcription factors and their DNA binding preferences, it is a challenging problem to establish an accurate model for recognition of functional regulatory elements in promoters of eukaryotic genes. RESULTS: We present a method for precise prediction of a large group of transcription factor binding sites - steroid hormone response elements. We use a large training set of experimentally confirmed steroid hormone response elements, and adapt a sequence-based statistic method of position weight matrix, for identification of the binding sites in the query sequences. To estimate the accuracy level, a table of correspondence of sensitivity vs. specificity values is constructed from a number of independent tests. Furthermore, feed-forward neural network is used for cross-verification of the predicted response elements on genomic sequences. CONCLUSION: The proposed method demonstrates high accuracy level, and therefore can be used for prediction of hormone response elements de novo. Experimental results support our analysis by showing significant improvement of the proposed method over previous HRE recognition methods.

Binding Sites↗