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PubMed1999

Constitutive protein-DNA interactions on the abscisic acid-responsive element before and after developmental activation of the rab28 gene.

Transcription of the rab28 gene from maize is induced in late embryo development and in response to abscisic acid. We have studied the regulation of the activity of the rab28 promoter in embryos. Two abscisic acid-responsive elements (ABREs) were necessary for expression in embryos of transgenic Arabidopsis and in transient transformation in maize embryos. In vivo footprinting showed that there was protein binding to the ABREs and to other cis elements in the promoter in young embryos before expression of rab28. This shows that the rab28 promoter is in an open chromatin structure before developmental activation. The ABREs are important for the induction and have protein binding in young embryos. Nuclear proteins extracted from embryos before activation of rab28 bound to the ABREs in band shift assays. A complex with different mobility was formed between nuclear proteins and the ABREs after induction of rab28 suggesting a modification of the ABRE-binding factor or an exchange of proteins. The footprints on the ABREs were unaltered by induction with abscisic acid or during developmental activation of rab28. These results indicate that constitutive binding of transcription factor(s) on the ABRE is central in embryonic regulation of the rab28 gene.

Abscisic Acid↗
PubMed2000

Identification of a novel gp130-responsive site in the vasoactive intestinal peptide cytokine response element.

The neuropoietic cytokine ciliary neurotrophic factor (CNTF) potently induces transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair (bp) cytokine response element (CyRE) in the VIP promoter. We have previously shown that CNTF induction of STAT and AP-1 protein binding within the CyRE is necessary to mediate CNTF induction of VIP gene transcription. We now show that a third, previously uncharacterized site at the 3'-end of the CyRE is also critical to CNTF induction of CyRE transcription. A 4-bp mutation in this 3'-region reduced CNTF-mediated induction of transcription approximately 80%. Whereas mutations in both the STAT and AP-1 sites substantially reduced CNTF induction of transcription, mutations in these sites together with the novel 3'-site completely abolished the ability of CNTF to induce CyRE-mediated transcription. Gel shift analysis indicated that a complex in neuroblastoma cells bound specifically to this 3'-site. This complex was not altered by CNTF treatment. Mutations in an 8-bp sequence (TTACTGGA) eliminated binding of this protein complex and markedly reduced transcriptional activation of the CyRE by CNTF. Thus, we have identified a protein complex binding to a novel DNA sequence that is necessary for full CNTF induction of VIP gene transcription.

Base Sequence↗
PubMed2001

Beta-chemokine induction of activation protein-1 and cyclic AMP responsive element activation in human myeloid cells.

Chemokines effect leukocyte chemotaxis and activation through their binding to specific G protein-coupled receptors. Although early steps in chemokine signal transduction pathways have been characterized, there is relatively limited information available at the transcription factor level. To that end, we have examined the binding activity on activation protein-1 (AP-1) and cyclic AMP responsive element (CRE) target sequences in human THP-1 myeloid cells after treatment with the beta-chemokine macrophage inflammatory protein (MIP-1alpha). MIP-1alpha induced both AP-1 and CRE activation. Although inhibition of protein kinase C blocked the AP-1 activity induced by this chemokine, there was no decrease in CRE activation in the presence of a protein kinase A inhibitor. Using kinase assays, it appeared that mitogen-activated protein kinase pathways were involved in CRE activation. In addition, HIV-1 infection of THP-1 cells resulted in constitutive activation of AP-1 and CRE elements but no further response to MIP-1alpha treatment. These results suggest that beta-chemokines act via protein kinase C-dependent pathways and mitogen-activated protein kinase pathways to modulate the host transcriptional response in myeloid cells, and that this response is altered by HIV infection.

Cell Line↗
PubMed2002

Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes through antioxidant response elements (AREs). Our previous study showed that sulfur amino acid deprivation (SAAD) produces peroxides and induces rat glutathione S-transferase A2 (rGSTA2) through NF-E2-related factor 2 (Nrf2)/ARE activation via the pathway of phosphatidylinositol 3-kinase (PI3-kinase). The current study was designed to investigate the role of peroxynitrite in Nrf2/ARE activation and rGSTA2 induction. L-Arginine deficiency or N(G)-nitro-L-arginine methyl ester (L-NAME) reduced peroxide production induced by SAAD in H4IIE cells. Northern and Western blot analyses revealed that the levels of rGSTA2 mRNA and protein were significantly increased 24h after incubation of the cells in SAAD medium, which was inhibited by L-arginine deficiency or L-NAME. Subcellular fractionation and gel shift analyses revealed that SAAD increased the level of nuclear Nrf2 and activated ARE, which were also blocked by L-arginine deficiency or L-NAME. Whereas the exogenous NO donor S-nitroso-N-acetyl-penicillamine (SNAP) alone failed to significantly induce rGSTA2, SNAP enhanced SAAD-inducible rGSTA2 expression, verifying the notion that peroxynitrite derived from NO contributes to rGSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, increased the rGSTA2 mRNA and protein levels in a dose-dependent manner. SIN-1 increased the level of nuclear Nrf2 and activated Nrf2/ARE, which was supershifted by anti-Nrf2 and anti-Maf antibodies. SIN-1 increased the activity of PI3-kinase, as monitored by phosphorylation of Akt. SIN-1-inducible rGSTA2 expression was inhibited by PI3-kinase inhibitors. These results provide evidence that peroxynitrite plays an essential role in nuclear translocation of Nrf2 and ARE activation through the pathway of PI3-kinase and that nitric oxide synthase is involved in the induction of rGSTA2.

Amino Acids, Sulfur↗
PubMed2001

Interaction of estrogen receptors alpha and beta with estrogen response elements.

To understand how estrogen-responsive genes are regulated, we compared the abilities of estrogen receptors (ERs) alpha and beta to bind to and activate transcription through the consensus vitellogenin A2 ERE and the imperfect pS2, vitellogenin B1, and oxytocin (OT) EREs. Transient transfection experiments demonstrated that ERalpha and ERbeta induced the highest levels of transcription with the A2 ERE, intermediate levels of transcription with the OT ERE, and low levels of transcription with the pS2 and B1 EREs. ERalpha and ERbeta had higher affinities for the A2 ERE than for any of the three imperfect EREs but similar affinities for the pS2, B1, and OT EREs in gel mobility shift assays. ERalpha had a higher affinity and was a more potent activator of transcription than ERbeta. Interestingly, protease sensitivity assays demonstrated that A2, pS2, B1, and OT EREs induced distinct changes in ERalpha and ERbeta conformation thereby providing different functional surfaces for interaction with regulatory proteins involved in control of estrogen-responsive genes.

Animals↗
PubMed2001

Multiple signals required for cyclic AMP-responsive element binding protein (CREB) binding protein interaction induced by CD3/CD28 costimulation.

The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser(133) phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.

Animals↗
PubMed2005

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver.

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2002

Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A.

The effects of E2 on the high-density lipoprotein receptor (HDL-R) scavenger receptor class B type I (SR-BI) gene were examined. Four putative estrogen response element half-site motifs (ERE(1/2)) (-2176, -1726, -1622, and -1211, designated ERE(1/2)-1, 2, 3, and 4, respectively) were identified in the HDL-R SR-BI promoter. Transfection studies and mutation analysis demonstrated that E2 significantly increased HDL-R SR-BI promoter activity and that mutating ERE(1/2)-1, 2, and 4 resulted in a loss of E2 responsiveness. Both ER alpha and ER beta formed specific complexes with ERE(1/2)-1, 2, and 4 but did not bind ERE(1/2)-3 in vitro. Interestingly, ERE(1/2)-3 was the motif shown not to be important for E2-activation of the HDL-R SR-BI promoter in the mutational analysis studies. The influence of SREBP-1a (sterol regulatory element binding protein-1a) on E2 regulation of the HDL-R SR-BI gene was also examined. SREBP-1a was able to bind directly to the ERE(1/2) motifs and enhanced ER binding when both ER subtypes were present. ER alpha and beta also bound to a sterol response element motif, but they did not enhance SREBP-1a binding. Cotransfection studies demonstrated that the presence of the three factors, ER alpha, ER beta, and SREBP-1a, enhanced the overall luciferase activity produced from the HDL-R SR-BI promoter construct in the presence of only one of the factors. Interaction of SREBP-1a with both ERs was demonstrated using a mammalian two-hybrid assay. The data confirmed that E2 through the ERs can positively regulate the HDL-R SR-BI through binding and activation of three ERE(1/2) motifs and identified SREBP-1a as a potential coactivator of the E2-ER-dependent effects on the HDL-R SR-BI gene.

Blotting, Western↗
PubMed1992

Thyrotropin (TSH)-releasing hormone-responsive elements in the rat TSH beta gene have distinct biological and nuclear protein-binding properties.

Two TRH-responsive elements have been identified in the rat TSH beta gene by deletion/mutation analysis of the 5'-flanking region of the gene and transfection of TSH beta-luciferase constructs into the GH3 pituitary cell line. Biological responsiveness was confirmed by inserting synthetic oligonucleotides next to the heterologous viral thymidine kinase (tk) promoter in tk luciferase (tkLUC) constructs. Both DNA regions, termed TSH A (at -274 to -258 bp) and TSH C (-402 to -385 bp), have a high level of sequence similarity to binding sites for the POU domain pituitary transcription factor Pit-1/GHF-1. In transfection assays, the TSH A region had no basal enhancer activity, but did confer 3- to 6-fold TRH- and PMA-stimulated transcriptional responses to the tk promoter. The TSH C region conferred basal enhancer activity (3- to 10-fold above control tkLUC) as well as a 2- to 3-fold TRH or PMA response. Combinations of TSH A and TSH C elements conferred both enhancer activity and a TRH- or PMA-stimulated response, but more than two copies of the regions resulted in no further stimulatory effect. Both TSH beta gene regions bound to nuclear proteins from GH3 cells, as determined by gel retardation analysis. The TSH A region DNA formed three prominent DNA-protein complexes, ranging from slowly to rapidly migrating bands and with calculated affinities of 32, 0.5, and 208 nM, respectively. The TSH C region formed two major complexes, which corresponded on the basis of mobility to the most slowly and rapidly migrating complexes formed by TSH A, but with calculated affinities of 3.1 and 33 nM. TSH C also formed a rapidly migrating minor complex unique for this gene region. The more rapidly migrating complexes appeared to be specific to nuclear proteins from GH3 cells. Treatment of cells with TRH did not significantly alter the affinity of protein binding. Mutation of TSH A and TSH C DNA by T to G substitutions abolished the ability of the DNA to confer a TRH response and severely inhibited the ability of the DNA to bind to GH3 nuclear proteins. Thus, transcriptional regulation of the rat TSH beta gene by TRH is correlated with the ability of the two TRH-sensitive elements to bind nuclear proteins. The differences noted in basal enhancer activity or the degree of TRH responsiveness may be related to some unique proteins bound to each DNA or to the differences in affinity of binding of the proteins common to both elements.

Animals↗
PubMed1992

The LIM family transcription factor Isl-1 requires cAMP response element binding protein to promote somatostatin expression in pancreatic islet cells.

Many eukaryotic genes are regulated by cAMP through a conserved cAMP response element (CRE). Here we show that, in the pancreatic islet cell line Tu6, a well-characterized CRE in the somatostatin gene does not provide cAMP responsiveness but functions as an essential element for its basal activity. DNA-binding and functional analyses indicate that the cAMP-responsive factor CREB regulates somatostatin expression in these cells without requirement for phosphorylation at the protein kinase A-regulated Ser-133 phosphorylation site. In addition to the CRE site, cell-specific expression of the somatostatin gene requires a second promoter element, which binds the recently characterized LIM family protein Isl-1. Thus, Isl-1 and CREB appear to synergize on the somatostatin promoter to stimulate high-level expression in Tu6 cells. The ability of CREB to function in a phosphorylation-independent manner suggests a mechanism by which this protein can regulate gene transcription.

Base Sequence↗
PubMed2000

Mechanical strain activates estrogen response elements in bone cells.

The involvement of the estrogen receptor in the early responses of bone cells to mechanical strain was investigated by subjecting subconfluent monolayer cultures of ROS.SMER #14 cells (ROS 17/2.8 cells stably transfected with additional ER alpha) to 17 beta-estradiol or a single short period of dynamic mechanical strain (600 cycles, 1 Hz). The basal proliferation rate of ROS.SMER #14 cells was similar to ROS 17/2.8 cells, whose proliferative responsiveness to strain and estrogen is similar to that of primary cultures of rat long bone-derived osteoblasts. At peak strains of 3400 mu epsilon, strain-related proliferation in ROS.SMER #14 cells was 1.4 times that of ROS 17/2.8 cells. At 10(-8) mol/L, 17 beta-estradiol-related proliferation was nearly twice greater. The ROS.SMER #14 cells were transiently transfected with an estrogen-responsive reporter, 2ERE-pS2-CAT, containing two consensus estrogen response elements (ERE) linked to a chloroamphenicol acetyl transferase gene. Strain increased normalized ERE-CAT activity threefold and estradiol (10(-8) mol/L) sixfold. Both strain-related and estradiol-related increases in proliferation and ERE-CAT activity were blocked by the estrogen antagonist ICI 182,780 (10(-6) mol/L). These data show that strain as well as estrogen stimulates increased proliferation in ROS 17/2.8 cells and increased ER alpha-related ERE activity in ROS cells transfected with ER alpha. Proliferation is greater in the cells with more estrogen receptors. Both strain- and estrogen-related proliferation and ERE activity are blocked by the estrogen antagonist ICI 182,780. This indicates that ROS cells' early responses to mechanical strain involve ER alpha and estrogen-responsive genes.

Animals↗
PubMed2004

Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult.

Binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) in neural cells results in the induction of a battery of genes that can coordinate a protective response against a variety of oxidative stressors. In this study, tert-butylhydroquinone (tBHQ) and sulforaphane were used as activators of this pathway. Consistent with previous studies, treatment of primary cortical cultures from ARE reporter mice revealed selective promoter activity in astrocytes. This activation protected neurons from hydrogen peroxide and nonexcitotoxic glutamate toxicity. tBHQ treatment of cultures from Nrf2 knock-out animals resulted in neither ARE activation nor neuroprotection. By reintroducing Nrf2 via infection with a replication-deficient adenovirus (ad), both the genetic response and neuroprotection were rescued. Conversely, infection with adenovirus encoding dominant-negative (DN) Nrf2 (ad-DN-Nrf2) or pretreatment with the selective phosphatidylinositol-3 kinase inhibitor LY294002 inhibited the tBHQ-mediated promoter response and corresponding neuroprotection. Interestingly, the adenoviral infection showed a high selectivity for astrocytes over neurons. In an attempt to reveal some of the cell type-specific changes resulting from ARE activation, cultures were infected with adenovirus encoding green fluorescent protein (GFP) (ad-GFP) or ad-DN-Nrf2 (containing GFP) before tBHQ treatment. A glia-enriched population of GFP-infected cells was then isolated from a population of uninfected neurons using cell-sorting technology. Microarray analysis was used to evaluate potential glial versus neuron-specific contributions to the neuroprotective effects of ARE activation and Nrf2 dependence. Strikingly, the change in neuronal gene expression after tBHQ treatment was dependent on Nrf2 activity in the astrocytes. This suggests that Nrf2-dependent genetic changes alter neuron-glia interactions resulting in neuroprotection.

Adenoviridae↗
PubMed1991

Identification of a retinoic acid response element in the human oxytocin promoter.

Retinoids are known to have profound effects on cellular differentiation and embryo pattern formation. In the adult organism, retinoid acid (RA) receptors are present in a large variety of tissues, including brain. However, little is known of the precise roles of RA at these different sites. In the present study we have identified a novel potential target of RA action by identifying an RA response element (RARE) in the human oxytocin (OT) gene promoter. We have used DNA-mediated gene transfer techniques to introduce various portions of the OT 5'-flanking sequences next to the chloramphenicol acetyltransferase (CAT) gene in neuroblastoma cells. RA elicited a marked stimulation of the transcriptional activity of the OT promoter in cells cotransfected with either the human RA receptor alpha, beta, or gamma. In cells cotransfected with the RA receptor alpha, the ED50 of this response was 5 x 10(-10) M. The RA response could also be conferred to a heterologous promoter independent of orientation. 5'-Deletions as well as site-directed mutations demonstrated that four TGACC motifs, located at -162, -156, -103, and -83 in the OT promoter, are necessary for optimal RA induction. Mutation or deletion of any of these elements reduces significantly the RA response. Interestingly, the first two TGACC motifs overlap with the estrogen response element that we have previously characterized in this gene. Furthermore, the TGACC motif located at -83 overlaps with the CCAAT box. We further demonstrate that in neuroblastoma cells transfected with an RAR alpha expression vector expression of the endogenous OT gene is stimulated greater than 4-fold in response to RA. Our studies constitute the first report of a RARE in a neuropeptide gene and define a mechanism by which OT gene expression can be modulated by retinoic acid.

Animals↗
PubMed1998

Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest.

Mice lacking the functional cAMP responsive element modulator (CREM) gene, a component of cAMP-mediated signal transduction, exhibit a specific arrest of round spermatid development although follicle stimulating hormone (FSH) and androgen secretion are not impaired. We studied testicular expression of CREM protein by immunocytochemistry in four patients with complete spermatogenesis (obstructive azoospermia), in 20 infertile patients with round spermatid maturation arrest (n = 10) or mixed atrophy (n = 10) and in six prostate cancer patients undergoing orchidectomy. Concentrations of testosterone were below normal in three patients. Concentrations of luteinizing hormone (LH) were lowered in two patients and elevated in one patient. FSH concentrations were above normal in ten patients. During normal spermatogenesis, CREM was expressed in nuclei of round spermatids in stages I-III of spermatogenesis but not in elongating spermatids. Western blot analysis of testes from prostate cancer patients indicated a major CREM band of approximately 35 kDa. Among patients with predominant round spermatid maturation arrest, CREM expression was significantly reduced (P < 0.05) or undetectable as revealed by quantitative image analysis. CREM-negative spermatids failed to progress beyond stage III of spermatogenesis. Our observations suggest a role for CREM in human spermatid development and raise the possibility that altered CREM expression could be associated with spermatid maturation defects in some cases of idiopathic male infertility.

Adolescent↗
PubMed2002

Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice.

Many phase II protective genes contain a cis -acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis.

Alkaline Phosphatase↗
PubMed1998

An activator protein-1 (AP-1) response element on pro alpha1(l) collagen gene is necessary for thyroid hormone-induced inhibition of promoter activity in cardiac fibroblasts.

Thyroid hormone-induced ventricular hypertrophy is characterized by the absence of fibrosis. Previously, we demonstrated that thyroid hormone inhibits collagen type I gene expression in the myocardium and in cardiac fibroblasts. We also demonstrated that thyroid hormones act as inhibitor of pro alpha1(l) collagen promoter activity. In this study we determined the sequences on pro alpha1(l) collagen gene and transcription factors in cardiac fibroblasts involved in the inhibitory effect of 3,3',5-triiodothyronine (T3). Transient transfection of cells with chloramphenicol acetyl transferase (CAT)-linked deletion mutants of pro alpha1(l) collagen promoter demonstrated that the inhibitory effect of T3 is transmitted via proximal sequences(-225/+115). Gel shift analysis using [32P]-labeled -225/+115 gene fragment and nuclear proteins of cardiac fibroblasts showed T3-induced DNA binding by two proteins. Analysis of non-overlapping restriction sub-fragments by gel shift along with supershift analysis with antibodies to types alpha and beta thyroid hormone receptors identified the lower molecular weight DNA-binding protein as beta receptor and confirmed that the T3-induced protein-DNA binding sites are located at -15/+115. Selective base mutation (C in place of G at +93 and G in place of C at +97) in the activator protein-1 (AP-1) core binding motif(+92/+97) abolished the higher molecular weight T3-induced DNA-protein complex obtained with [32P]-labeled wild type sequences (-225/+115). Additional gel shift analyses using an oligonucleotide containing the AP-1 core binding motif, as an unlabeled competitor and as [32P]-labeled probe, confirmed the T3-induced protein binding to an AP-1 site. Transient transfection with CAT-linked -225/+115 sequences in which the AP-1 site was mutated abolished the T3-induced inhibition of CAT activity. Together, these findings identify sequences necessary for T3-induced inhibition of collagen type I promoter to which thyroid hormone receptor type beta and protein(s) with affinity for AP-1 element bind. They also demonstrate that the AP-1 response element located on these sequences is necessary for T3-induced inhibition of pro alpha1(l) collagen promoter activity. These data identify molecular mechanisms involved in thyroid hormone-induced inhibition of collagen expression in the heart.

Animals↗
PubMed2004

Tissue-selective, bidirectional regulation of PEX11 alpha and perilipin genes through a common peroxisome proliferator response element.

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.

Animals↗
PubMed2005

Luman is capable of binding and activating transcription from the unfolded protein response element.

Luman (or LZIP, CREB3) is a transcription factor with an endoplasmic reticulum (ER)-transmembrane domain. Due to its structural similarities with ATF6, it is thought that Luman might also be involved in cellular stress responses. Here we report that Luman can bind and activate transcription from the consensus unfolded protein response element (UPRE). Mutations that disrupted the binding of Luman to the UPREs impaired its ability to activate transcription from these sites. Overexpression of Luman stimulated transcription of EDEM, a downstream effector of the mammalian unfolded protein response involved in ER-associated degradation (ERAD). Unlike ATF6, however, Luman was not activated by proteolytic cleavage in response to endoplasmic reticulum stressors such as tunicamycin and thapsigargin. These results suggest that the activation of ERAD by Luman is likely through a pathway different from the common ER stress response, and that additional factor(s) are required for the activation of this Luman-mediated pathway.

Binding Sites↗