In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2

2003 ◽  
Vol 285 (3) ◽  
pp. C633-C641 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Mammalian Cells ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Regulatory Region ◽  
Minimal Promoter ◽  
Human Tissues ◽  
Cis Elements

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5′ regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

2004 ◽  
Vol 287 (4) ◽  
pp. G822-G829 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Hamid M. Said
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Minimal Promoter Region

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

scholarly journals Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

2007 ◽  
Vol 18 (8) ◽  
pp. 2864-2872 ◽  
Author(s):  
Kelly J. Perkins ◽  
Utpal Basu ◽  
Murat T. Budak ◽  
Caroline Ketterer ◽  
Santhosh M. Baby ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Neuromuscular Junctions ◽  
Protein Product ◽  
Mrna Levels ◽  
Laser Capture Microscopy ◽  
Erk Phosphorylation ◽  
Duchenne's Muscular Dystrophy

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

scholarly journals Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 425-438 ◽  
Author(s):  
M P Cooke ◽  
A W Heath ◽  
K M Shokat ◽  
Y Zeng ◽  
F D Finkelman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Transgenic Mice ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Interleukin 4 ◽  
Th Cells

The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.

scholarly journals Coordinate Transcription of the ADAMTS-1 Gene by Luteinizing Hormone and Progesterone Receptor

2004 ◽  
Vol 18 (10) ◽  
pp. 2463-2478 ◽  
Author(s):  
Kari M. H. Doyle ◽  
Darryl L. Russell ◽  
Venkataraman Sriraman ◽  
JoAnne S. Richards
Keyword(s):  
Progesterone Receptor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Nuclear Factor 1

Abstract ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein β, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo

2007 ◽  
Vol 292 (4) ◽  
pp. C1305-C1312 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Hek 293

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5′ regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.

scholarly journals Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model1

2003 ◽  
Vol 51 (7) ◽  
pp. 941-949 ◽  
Author(s):  
Sherry L. Abboud ◽  
Maria Bunegin ◽  
Nandini Ghosh-Choudhury ◽  
Kathleen Woodruff
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Tissue Expression ◽  
Cellular Level ◽  
Liver And Kidney

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a −774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the −774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the −774/+183-bp region driving the E. coli β-galactosidase (lacZ) reporter gene were generated. β-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of β-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the −774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.

UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3288-3293 ◽  
Author(s):  
Sven G. Gehrke ◽  
Hans-Dieter Riedel ◽  
Thomas Herrmann ◽  
Boris Hadaschik ◽  
Karin Bents ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Hereditary Hemochromatosis ◽  
Regulatory Region ◽  
Iron Chelation ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

Abstract SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5′ regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5′ regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

scholarly journals Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice

2021 ◽  
Vol 22 (15) ◽  
pp. 8177
Author(s):  
Rossana Di Martino ◽  
Maria Josè Sisalli ◽  
Rossana Sirabella ◽  
Salvatore Della Notte ◽  
Domenica Borzacchiello ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Fluorescent Dyes ◽  
Neuronal Loss ◽  
Calcium Content ◽  
Alpha Synuclein ◽  
Dopaminergic Neurodegeneration

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.

scholarly journals Identification and characterization of the minimal 5′-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells

2015 ◽  
Vol 308 (2) ◽  
pp. C189-C196 ◽  
Author(s):  
Abhisek Ghosal ◽  
Subrata Sabui ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Intestinal Epithelial ◽  
Identification And Characterization

The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5′-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5′-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between −199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position −74/−71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.

scholarly journals Dock/Nck facilitates PTP61F/PTP1B regulation of insulin signalling

2011 ◽  
Vol 439 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Chia-Lun Wu ◽  
Bree Buszard ◽  
Chun-Hung Teng ◽  
Wei-Lin Chen ◽  
Coral G. Warr ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Insulin Signalling ◽  
Tyrosine Phosphatase ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Negative Regulator ◽  
Stable Complex

PTP1B (protein tyrosine phosphatase 1B) is a negative regulator of IR (insulin receptor) activation and glucose homoeostasis, but the precise molecular mechanisms governing PTP1B substrate selectivity and the regulation of insulin signalling remain unclear. In the present study we have taken advantage of Drosophila as a model organism to establish the role of the SH3 (Src homology 3)/SH2 adaptor protein Dock (Dreadlocks) and its mammalian counterpart Nck in IR regulation by PTPs. We demonstrate that the PTP1B orthologue PTP61F dephosphorylates the Drosophila IR in S2 cells in vitro and attenuates IR-induced eye overgrowth in vivo. Our studies indicate that Dock forms a stable complex with PTP61F and that Dock/PTP61F associate with the IR in response to insulin. We report that Dock is required for effective IR dephosphorylation and inactivation by PTP61F in vitro and in vivo. Furthermore, we demonstrate that Nck interacts with PTP1B and that the Nck/PTP1B complex inducibly associates with the IR for the attenuation of IR activation in mammalian cells. Our studies reveal for the first time that the adaptor protein Dock/Nck attenuates insulin signalling by recruiting PTP61F/PTP1B to its substrate, the IR.

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