scholarly journals Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro

2010 ◽  
Vol 298 (1) ◽  
pp. R198-R210 ◽  
Author(s):  
David L. Allen ◽  
Jill J. Uyenishi ◽  
Allison S. Cleary ◽  
Ryan S. Mehan ◽  
Sarah F. Lindsay ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Endurance Exercise ◽  
Binding Sites ◽  
Promoter Activity ◽  
Wheel Running ◽  
Mrna Levels ◽  
Dna Binding Sites ◽  
Single Bout

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2–10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C2C12 myotubes with the calcium ionophore A23187. Cotransfection of C2C12 myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C2C12 myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

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 Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

scholarly journals Properties of initiator-associated transcription mediated by GAL4-VP16.

1993 ◽  
Vol 13 (12) ◽  
pp. 7469-7475 ◽  
Author(s):  
C Chang ◽  
J D Gralla
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Dna Binding Sites ◽  
Terminal Deoxynucleotide Transferase ◽  
Initiator Element ◽  
High Level ◽  
Primary Activation ◽  
Initial Assembly

Transcription associated with a terminal deoxynucleotide transferase gene initiator element is shown to respond to the transcription factor GAL4-VP16 both in vivo and in vitro. High-level transcription requires both an intact initiator element and bound activator. Transcription from this initiator-directed promoter is synergistic in vivo in that five GAL4 DNA binding sites yield 36 times the expression of a single site. Promoters dominated by initiator and TATA elements respond similarly to several GAL4-based activators, including GAL4-Sp1, GAL4-CTF, GAL4(1-147), GAL4-p53, GAL4-C/EBP, and GAL4-ER(EF), as well as GAL4-VP16 and Sp1. These and other similarities suggest that primary activation of TATA- and initiator-dominated promoters occurs at common steps. Since the initial assembly steps do not appear to be common for the two promoter types, the results place interesting constraints on models for how activation occurs.

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.

scholarly journals Lactobacillus acidophilus stimulates the expression of SLC26A3 via a transcriptional mechanism

2010 ◽  
Vol 298 (3) ◽  
pp. G395-G401 ◽  
Author(s):  
Geetu Raheja ◽  
Varsha Singh ◽  
Ke Ma ◽  
Redouane Boumendjel ◽  
Alip Borthakur ◽  
...  
Keyword(s):  
Western Blot ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Long Term Effects ◽  
Exchange Activity

Clinical efficacy of probiotics in treating various forms of diarrhea has been clearly established. However, mechanisms underlying antidiarrheal effects of probiotics are not completely defined. Diarrhea is caused either by decreased absorption or increased secretion of electrolytes and solutes in the intestine. In this regard, the electroneutral absorption of two major electrolytes, Na+ and Cl−, occurs mainly through the coupled operation of Na+/H+ exchangers and Cl−/OH− exchangers. Previous studies from our laboratory have shown that Lactobacillus acidophilus (LA) acutely stimulated Cl−/OH− exchange activity via an increase in the surface levels of the apical anion exchanger SLC26A3 (DRA). However, whether probiotics influence SLC26A3 expression and promoter activity has not been examined. The present studies were, therefore, undertaken to investigate the long-term effects of LA on SLC26A3 expression and promoter activity. Treatment of Caco-2 cells with LA for 6–24 h resulted in a significant increase in Cl−/OH− exchange activity. DRA mRNA levels were also significantly elevated in response to LA treatment starting as early as 8 h. Additionally, the promoter activity of DRA was increased by more than twofold following 8 h LA treatment of Caco-2 cells. Similar to the in vitro studies, in vivo studies using mice gavaged with LA also showed significantly increased DRA mRNA (∼4-fold) and protein expression in the colonic regions as assessed by Western blot analysis and immunofluorescence. In conclusion, increase in DRA promoter activity and expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of LA.

scholarly journals An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

2009 ◽  
Vol 191 (17) ◽  
pp. 5489-5498 ◽  
Author(s):  
Steve D. Seredick ◽  
Barbara M. Seredick ◽  
David Baker ◽  
George B. Spiegelman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Molecular Basis ◽  
Response Regulator ◽  
Mutant Protein ◽  
Wild Type ◽  
Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

scholarly journals A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

2021 ◽  
Author(s):  
Soumen Khan ◽  
Saurabh J. Pradhan ◽  
Guillaume Giraud ◽  
Françoise Bleicher ◽  
Rachel Paul ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Morphological Changes ◽  
Binding Motif ◽  
Core Sequence ◽  
Dna Binding Sites ◽  
Target Binding ◽  
Hox Protein

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter

1994 ◽  
Vol 14 (10) ◽  
pp. 6607-6615
Author(s):  
E Neuman ◽  
E K Flemington ◽  
W R Sellers ◽  
W G Kaelin
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Mrna Abundance ◽  
Dna Binding Sites ◽  
Cell Cycle Dependence ◽  
Transcription Factor E2f ◽  
Cycle Dependent

The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance. The E2F-1 promoter contains four potential E2F-binding sites organized as two imperfect palindromes. Gel shift and transactivation studies suggested that these sites can bind to E2F in vitro and in vivo. Mutation of the two E2F palindromes abolished the cell cycle dependence of the E2F-1 promoter. Thus, E2F-1 appears to be regulated at the level of transcription, and this regulation is due, at least in part, to binding of one or more E2F family members to the E2F-1 promoter.

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