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PubMed1995

Identification of a cis-acting glucocorticoid responsive element in the rat angiotensin II type 1A promoter.

Enhanced vascular responsiveness to angiotensin II at the AT1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (< 12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 mumol/L) and was located at positions -756 to -770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (> 8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro-translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1989

Induction of human growth hormone promoter activity by the adenosine 3',5'-monophosphate pathway involves a novel responsive element.

Regulation of GH gene expression by GRF involves cAMP as a second messenger. We have demonstrated that a 500-basepair fragment of the human GH (hGH) gene 5' flanking region can confer cAMP inducibility upon the chloramphenicol acetyltransferase transcription unit in transient transfections of rat pituitary tumor cells treated with forskolin, an activator of adenyl cyclase. The same hGH construct is not induced by forskolin in nonpituitary-derived cells. Experiments with hGH deletion constructs reveal that binding sites for transcription factor AP-2 and the pituitary-specific factor GHF-1 are not required for forskolin stimulation, but that GHF-1 may potentiate the effect. RNA analyses reveal that forskolin also stimulates accumulation of transcripts initiated at the hGH promoter. Other agents that elevate cAMP levels also stimulate hGH expression. Since the hGH 5' flanking region contains no sequences homologous to the cAMP-responsive element of the somatostatin gene, and the AP-2 sites do not appear to be required for the forskolin response, these results suggest that a novel cAMP-responsive element exists within 82 basepairs upstream from the transcriptional start of the hGH gene and that hGH regulation by GRF may involve interaction between a tissue-specific element and a cAMP-inducible element.

Animals↗
PubMed1996

The rab16B promoter of rice contains two distinct abscisic acid-responsive elements.

To localize abscisic acid (ABA)-inducible gene expression of rab16 genes, rab16A promoter was linked to the gusA reporter gene encoding beta-glucuronidase and introduced into rice (Oryza sativa L.) plants. The activity of rab16A promoter was induced by ABA and osmotic stresses in various tissues of vegetative and floral organs. In anthers and embryos, rab16A promoter was active in the absence of ABA. To elucidate cis-elements of the rab16 promoter that confer ABA-inducible expression, variously modified 40-bp fragments (-264 to -225) of the rab16B promoter were fused to a truncated (-46 bp) cauliflower mosaic virus 35S minimal promoter, and their activities in protoplasts were analyzed. The transient assays revealed that the 40-bp fragment consists of two separate ABA-responsive elements, motif 1 (AGTACGTGGC) and motif III (GCCGCGTGGC). Motif I and motif III are both required for ABA induction; however, each can substitute for the other. Further analyses of these motifs indicated that motif III has a distinct DNA sequence specificity as an ABA-responsive element from motif I, suggesting that the two motifs interact with different transcription factors in vivo.

Abscisic Acid↗
PubMed2004

Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences.

Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human-mouse-rat orthologous genes between -1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse-rat orthologous genes with a DRE between -1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

Animals↗
PubMed2004

Kinetic analysis of RSK2 and Elk-1 interaction on the serum response element and implications for cellular engineering.

Immediate early gene activation upon mitogenic activation occurs through the serum response element (SRE), which makes the delineation of the upstream pathways a powerful means to engineer cellular responses. The malfunctioning of this system leads to a variety of disorders, ranging from neurological disorders such as Coffin-Lowry syndrome (RSK2 mutations) to cancer (c-fos mutations). We therefore investigated the SRE activation mechanism in a typical mammalian cell. Mitogenic signaling uses the mitogen-activated protein kinase (MAPK) module through increased binding of the ternary complex factor (TCF), such as Elk-1, to the promoter DNA (the SRE element) and subsequent transcriptional activation, as well as through activation of a histone kinase, such as the MAPK-activated protein kinase (MAPKAP-K) ribosomal S6 kinase (RSK2). This computational model uses the biochemical simulation environment GEPASI 3.30 to investigate three major models of interaction for Elk-1 and RSK2, and to study the effect of histone acetyl transferase (HAT) recruitment in each of these models on the local chromatin modifications in the presence and absence of MAPK activation. We show that the quickest response on the chromatin can be achieved in the presence of a preformed complex of RSK2, Elk-1 and HAT, with HAT being activated upon dissociation from the complex upon activation of the MAPK cascade. This study presents critical components in the pathway that can be targeted for engineering of specific inhibitors or activators of the system.

Acetyltransferases↗
PubMed2005

Estrogen-related receptor-gamma and peroxisome proliferator-activated receptor-gamma coactivator-1alpha regulate estrogen-related receptor-alpha gene expression via a conserved multi-hormone response element.

The expression of estrogen-related receptor-alpha (ERRalpha) is stimulated by estrogen in selective tissues. Recently, a correlation between ERRalpha expression and the induction of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) in the liver of fasting animals and in cold-stressed brown-fat tissues and skeletal muscle was shown. To explore the molecular mechanisms of ERRalpha regulation by diverse signals, the promoter of the human ERRalpha gene was cloned and characterized. Mutation and deletion analyses revealed that a 53 bp region containing repeated core element AGGTCA motifs of the ERRalpha gene serves as a multi-hormone response element (MHRE) for several nuclear receptors in transient co-transfection studies of human endometrial carcinoma (HEC-1B) cells. Among the nuclear receptors tested, ERRgamma bound to and robustly stimulated the transcription of reporters containing at least two AGGTCA motifs. Ectopic expression of PGC-1alpha in HEC-1B cells strongly activated the reporter containing the MHRE, presumably via the endogenous nuclear receptor binding to the element. Reducing the endogenous level of ERRgamma by small interfering RNA, and increasing the ERRgamma level by ectopic expression, substantially decreased and increased respectively the transactivation capability of PGC-1alpha. The activation function 2 domain of the ERRgamma and the L2 and L3 motifs of PGC-1alpha were essential to transactivate the MHRE. Additionally, PGC-1alpha increases the amount of endogenous ERRgamma bound to the MHRE region as determined by a chromatin immunoprecipitation assay. The present study demonstrates that the MHRE of the ERRalpha gene is a target for ERRgamma transactivation, which is enhanced by PGC-1alpha.

Animals↗
PubMed1998

Mapping of multiple RNA binding sites of human T-cell lymphotropic virus type I rex protein within 5'- and 3'-Rex response elements.

Interaction between the human T-cell lymphotropic virus type I Rex protein and viral transcripts in the nucleus is essential to the cytoplasmic appearance of unspliced and singly spliced viral RNA. Rex has been shown to mediate its function through direct interaction with a highly ordered secondary structure in the 3'-untranslated region of all human T-cell lymphotropic virus type I mRNAs termed the Rex response element (3'-RxRE). Part of the 3'-RxRE sequence is also present in the 5'-end of viral transcripts (5'-RxRE), and we demonstrate that Rex binds to this RNA with essentially the same affinity and specificity as to the 3'-RxRE. We have analyzed the secondary structures and binding sites of Rex within the 5'- and 3'-RxREs by enzymatic probing and chemical modification interference and show that multiple Rex molecules bind within a stem-loop, which is similarly structured in the two RxREs. Our experiments confirm the presence of a previously characterized Rex binding site but also identify a common motif within an extended region that comprises an additional Rex binding site. This suggests that Rex oligomerizes on the RxREs similarly to what has been observed for binding of the human immunodeficiency virus type 1 Rev protein to the Rev response element.

Base Sequence↗
PubMed1993

Intronic androgen response elements of prostatic binding protein genes.

Transcription of the genes that code for the different components of Prostatic Binding Protein is dependent on androgens. DNA-cellulose competition assays have demonstrated the presence of regions with affinity for the androgen receptor located immediately upstream and in the first intron of these genes. DNase I footprinting in vitro now reveals the exact binding site of a receptor dimer in the first intron of the C3(1), C3(2) and C1 genes. Gene transfer experiments resulted in the description of functional androgen response elements in the C3 genes. The C1 intronic binding site, however, has a much weaker affinity for the DNA-binding domain of the androgen receptor, a characteristic that might explain its inability to function as an androgen response element in chimeric gene constructs.

Androgen-Binding Protein↗
PubMed1997

The promoter of the human type I 5'-deiodinase gene--mapping of the transcription start site and identification of a DR+4 thyroid-hormone-responsive element.

The selenoenzyme thyroxine 5'-deiodinase type I deiodinates the prohormone thyroxine to the active thyroid hormone 3,3',5-triiodothyronine. It is thus one of the key enzymes involved in the triiodothyronine-mediated control of growth, differentiation and basal metabolism in vertebrates. We report here the identification of the transcription start site and the cloning of 1500 bases of the upstream regulatory region of the human 5'-deiodinase gene. They contain a complex triiodothyronine-responsive element at nucleotides -696 to -673, consisting of an ideal direct repeat (DR) of two AGGTCA half-sites with a spacing of four nucleotides (DR+4) and a third putative AGTTCA half-site with a spacing of another two nucleotides (DR+2). The whole DR+4+2 specifically bound to thyroid hormone receptor and retinoid X receptor in electrophoretic mobility shift assays. The DR+4+2 mediates triiodothyronine-responsiveness in cotransfection experiments of constructs containing the 5'-deiodinase upstream promoter and enhancer region fused to luciferase or chloramphenicol acetyltransferase reporter genes with expression plasmids of thyroid hormone receptor subtypes. Also, an about 2.5-fold induction of the 5'-deiodinase-promoter-luciferase-reporter construct by all-trans retinoic acid was observed in a cotransfection assay with retinoic acid receptors. Point mutation analysis of the DR+4+2 type hormone-responsive element, however, revealed that it does not alone mediate the retinoic acid effect. The transcription start point of the 5'-deiodinase gene was mapped to nucleotides -23 and -24. No CAAT or TATA box is located within the usual distance to the transcription initiation site. Two GC boxes were found at nucleotides -68 to -63 and -39 to -34. Transfection analysis revealed that the proximal 105 nucleotides in the 5'-flanking region of the 5'-deiodinase gene act as a functional core promoter. This data indicates that triiodothyronine, the end product of thyroid hormone synthesis, positively regulates one of the key enzymes in its production.

Base Sequence↗
PubMed2000

Identification of a membrane Ig-induced p38 mitogen-activated protein kinase module that regulates cAMP response element binding protein phosphorylation and transcriptional activation in CH31 B cell lymphomas.

The cAMP response element (CRE) binding protein (CREB) is emerging as a key regulatory factor of gene transcription in B lymphocytes; however, the postreceptor pathways that regulate CREB activity and CRE-dependent gene transcription remain largely undefined. We investigated B cell Ag receptor (BCR)-mediated phosphorylation and activation of CREB in the surface IgM+ CH31 B cell lymphoma, which undergoes Ag-dependent cell death. The activity of p38 mitogen-activated protein kinase (MAPK) was increased in response to BCR ligation. Phosphorylation of CREB on serine 133, a modification that positively regulates its trans-activation, was concomitantly increased. Inhibition of p38 MAPK by pretreating CH31 B cells with the highly specific bicyclic imidazole inhibitor, SB203580, reduced BCR-induced CREB phosphorylation. BCR cross-linking also led to increased MAPK-activated protein kinase-2 activity, an enzyme that lies immediately downstream from p38 MAPK; MAPK-activated protein kinase-2 immune complexes phosphorylated a peptide substrate containing the CREB serine 133 phosphoacceptor motif. Given the role of CREB in regulating junB gene expression in mature B lymphocytes, we examined whether p38 MAPK activity was necessary for CRE-dependent junB transcription in CH31 B cells. BCR ligation led to increased junB mRNA levels, which were significantly reduced in CH31 B cells pretreated with SB203580. Activation of a CRE-dependent junB promoter/chloramphenicol acetyltransferase (CAT) reporter gene by the BCR was also blocked by SB203580. Similarly, inhibition of p38 MAPK in surface IgM+ WEHI-231 B cell lymphomas resulted in reduced BCR-induced junB mRNA expression and junB promoter activation. The results implicate a p38 MAPK pathway in BCR-mediated CREB phosphorylation and junB transcriptional activation in B cell lymphomas.

Animals↗
PubMed1997

The rat TSHbeta gene contains distinct response elements for regulation by retinoids and thyroid hormone.

We have previously shown that thyroid stimulating hormone-beta (TSHbeta) mRNA levels are modulated by vitamin A status in vivo and using transient transfection, that suppression of rat TSHbeta gene promoter activity by all-trans retinoic acid (RA) requires RA receptor (RAR) and retinoid X receptor (RXR). In this paper we have used deletion analysis to delineate the sequences of the rTSHbeta gene involved in RA regulation, their relationship to the rTSHbeta gene negative thyroid hormone response elements and the retinoid receptor species that interact with these sequences. Using transient transfection in CV-1 cells, we found that the -204/+9 region of the rat TSHbeta gene, when fused to a luciferase reporter, was sufficient for suppression by all-trans-RA in the presence of RAR/RXR. Thus, regulation by RA did not involve the major rTSHbeta negative TRE located between +15 and +43. Mutational analysis also showed that the minor rTSHbeta negative TRE between -11 and +5 was not required by suppression by RA. However, in a heterologous promoter this sequence element acted as a strong positive RARE. The combination of RA and T3 treatment caused synergistic inhibition of rat TSHbeta gene expression in the presence of RAR/RXR and TR. EMSA analysis demonstrated that the -204/-79 sequence binds RAR/RXR heterodimer. Therefore, we conclude that there are separate response elements for RA and T3 on the rat TSHbeta gene, that the RARE binds RAR/RXR heterodimer and that RA and T3 interact functionally via these elements in the negative regulation of rat TSHbeta gene expression.

Animals↗
PubMed2006

Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene.

Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser(133) to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser(133) or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser(133) is necessary for induction of the EGR-1 transcription by exendin-4.

Animals↗
PubMed2006

Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1.

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric à brac/poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase reporter gene constructs, we have identified a p53-responsive element (PRE) 500 bp upstream of the TATA-box containing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53-responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73beta and DeltaNp63alpha, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53.

Amino Acid Sequence↗
PubMed2000

Induction-independent recruitment of CREB-binding protein to the c-fos serum response element through interactions between the bromodomain and Elk-1.

Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100-1286, and Elk-1. DNA binding and glutathione S-transferase pull-down assays demonstrate that binding requires Elk-1(1-212) but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation.

3T3 Cells↗
PubMed2002

Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection.

Ethylene-responsive element binding factors (ERF) proteins are plant-specific transcription factors, many of which have been linked to stress responses. We have identified four Arabidopsis ERF genes whose expression was specifically induced by avirulent and virulent strains of the bacterial pathogen Pseudomonas syringae pv tomato, with overlapping but distinct induction kinetics. However, a delay in ERF mRNA accumulation after infection with the virulent strain was observed when compared with the avirulent strain. The induction of ERF gene expression in most cases preceded the mRNA accumulation of a basic chitinase gene, a potential downstream target for one or more of these ERFs. The expression of the ERF genes was examined among different Arabidopsis tissues, in response to the signaling molecules ethylene, methyl jasmonate, and salicylic acid (SA), and in Arabidopsis mutants with decreased or enhanced susceptibility to pathogens, and significant differences were observed. For example, in seedlings, some of the ERF genes were not induced by SA in the wild-type but were SA responsive in the pad4-1 mutant, suggesting that PAD4-1, which acts upstream of SA accumulation, is also involved in repressing the SA-induced expression of specific ERF genes. The four ERF proteins were shown to contain transcriptional activation domains. These results suggest that transcriptional activation cascades involving ERF proteins may be important for plant defense to pathogen attack and that some ERF family members could be involved in the cross-talk between SA- and jasmonic acid-signaling pathways.

Acetates↗
PubMed2006

9-cis retinoic acid induces insulin-like growth factor binding protein-3 through DR-8 retinoic acid responsive elements.

Retinoic acids, which have shown potential chemopreventive and therapeutic activities for several neoplastic diseases in vitro, modulate the growth-promoting and anti-apoptotic activities of insulin-like growth factors (IGFs), in part by influencing the expression of insulin-like growth factor binding protein-3 (IGFBP-3). This study sought to investigate the effect of 9-cis retinoic acid (9cRA) on the expression of IGFBP-3 and the underlying mechanisms involving retinoic acid receptor-beta (RAR-beta). The pharmacologic activity of 9cRA was characterized by monitoring target modulation as well as by evaluating the underlying mechanisms in NSCLC cells. Treatment of 9cRA inhibited proliferation of a part of NSCLC cell lines including H460 cells in clinically-achievable concentrations and induced IGFBP-3 expression in dose- and time-dependent manners. Transient transfection with a reporter constructs driven by the human IGFBP-3 gene promoter indicated that 9cRA induces gene expression via the -534 to -445 region (relative to translation start site) of the IGFBP-3 promoter. Unilateral deletion and site-directed mutagenesis identified a retinoic acid responsive element (RARE), a direct repeat of two GGGTCA-related hexanucleotides separated by just 8 bp (DR-8-type response element). A cotransfection assay with a RAR-beta expression vector potentiated (and with siRNA for RAR-beta, diminished) the effect of 9cRA on IGFBP-3 expression. IGFBP-3 gene expression by 9cRA is mediated by a distinct DR-8 RARE located in the proximal region of the IGFBP promoter and involves the RAR-beta, a putative tumor suppressor in NSCLC.

Alitretinoin↗
PubMed2005

Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene.

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

Animals↗
PubMed2006

Hemin-mediated regulation of an antioxidant-responsive element of the human ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells.

An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. Here, we have found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5' regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE.

Antioxidants↗