bFGF induces BCK promoter-driven expression in muscle via increased binding of a nuclear protein

1997 ◽  
Vol 273 (1) ◽  
pp. C223-C229 ◽  
Author(s):  
L. Kim ◽  
A. Steves ◽  
M. Collins ◽  
J. Fu ◽  
M. E. Ritchie
Keyword(s):  
Transcription Factor ◽  
Creatine Kinase ◽  
Transcriptional Activation ◽  
Culture Medium ◽  
Nuclear Protein ◽  
C2c12 Cells ◽  
Nuclear Factor ◽  
Muscle Creatine Kinase ◽  
Responsive Element ◽  
Brain Creatine Kinase

Changes in gene expression occurring during skeletal muscle differentiation are exemplified by downregulation of brain creatine kinase (BCK) and induction of muscle creatine kinase (MCK). Although both are transcriptionally regulated, there appears to be no transcription factor-element overlap, suggesting that their coordinate expression results from culture medium-related influences. Basic fibroblast growth factor (bFGF) prevents myogenesis and represses MCK expression by inhibiting transcriptional activation. It was hypothesized that bFGF similarly influenced BCK by inducing its expression. Accordingly, BCK promoter constructs were transiently transfected into C2C12 cells and, after a switch to differentiation medium, were treated with bFGF, bFGF plus herbimycin, adenosine 3',5'-cyclic monophosphate (cAMP), or phorbol 12-myristate 13-acetate (PMA). Analyses demonstrated that bFGF responsiveness was contained within a 33-base pair element. Electromobility shift assays showed that bFGF induction increased the abundance of the nuclear factor binding the element. Both effects were prevented by herbimycin. Neither cAMP nor PMA specifically induced the construct containing the bFGF-responsive element. The induced factor required phosphorylation to bind, implying that bFGF-mediated increases in binding may be due to transcription factor phosphorylation.

Involvement of a NF-kappa B-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines

1990 ◽  
Vol 10 (2) ◽  
pp. 561-568
Author(s):  
H Shimizu ◽  
K Mitomo ◽  
T Watanabe ◽  
S Okamoto ◽  
K Yamamoto
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Interleukin 6 ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Nuclear Factor ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Mediators Of Inflammation

Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-alpha despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-kappa B transcription factor-binding motifs and binds an IL-1-TNF-alpha-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular localizations of this factor are indistinguishable from those of NF-kappa B. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-alpha in vivo. These results indicate that a nuclear factor indistinguishable from NF-kappa B is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-alpha.

scholarly journals The Paired-Domain Transcription Factor Pax8 Binds to the Upstream Enhancer of the Rat Sodium/Iodide Symporter Gene and Participates in Both Thyroid-Specific and Cyclic-AMP-Dependent Transcription

1999 ◽  
Vol 19 (3) ◽  
pp. 2051-2060 ◽  
Author(s):  
Makoto Ohno ◽  
Mariastella Zannini ◽  
Orlie Levy ◽  
Nancy Carrasco ◽  
Roberto di Lauro
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Thyroid Stimulating Hormone ◽  
Dependent Manner ◽  
Sodium Iodide Symporter ◽  
Paired Domain ◽  
Gene Encoding ◽  
Responsive Element

ABSTRACT The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides −2264 and −2495 in the 5′-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640 ◽  
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals Modulation of muscle creatine kinase promoter activity by the inducible orphan nuclear receptor TIS1

1997 ◽  
Vol 321 (2) ◽  
pp. 281-287 ◽  
Author(s):  
Wan-Lin YANG ◽  
Robert W. LIM
Keyword(s):  
Creatine Kinase ◽  
Nuclear Receptor ◽  
Reporter Gene ◽  
Direct Interaction ◽  
C2c12 Cells ◽  
Orphan Nuclear Receptor ◽  
Muscle Creatine Kinase ◽  
Muscle Creatine ◽  
Promoter Dna

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells but that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5ƀ flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.

scholarly journals Involvement of a NF-kappa B-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines.

1990 ◽  
Vol 10 (2) ◽  
pp. 561-568 ◽  
Author(s):  
H Shimizu ◽  
K Mitomo ◽  
T Watanabe ◽  
S Okamoto ◽  
K Yamamoto
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Interleukin 6 ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Nuclear Factor ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Mediators Of Inflammation

Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-alpha despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-kappa B transcription factor-binding motifs and binds an IL-1-TNF-alpha-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular localizations of this factor are indistinguishable from those of NF-kappa B. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-alpha in vivo. These results indicate that a nuclear factor indistinguishable from NF-kappa B is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-alpha.

Nuclear Factor of Activated T Cells (NFAT) Is Involved in the Depolarization-Induced Activation of Growth Hormone-Releasing Hormone Gene Transcription in Vitro

2004 ◽  
Vol 18 (12) ◽  
pp. 3011-3019 ◽  
Author(s):  
Masato Asai ◽  
Yasumasa Iwasaki ◽  
Masanori Yoshida ◽  
Noriko Mutsuga-Nakayama ◽  
Hiroshi Arima ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Gene Transcription ◽  
Transcriptional Activation ◽  
Site Directed Mutagenesis ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
High Potassium ◽  
Direct Binding

Abstract GHRH plays a pivotal role in the regulation of both synthesis and secretion of GH in the anterior pituitary. In this study, we examined the molecular mechanism of depolarization-induced GHRH gene transcription using the hypothalamus cell line, Gsh+/+, revealing the involvement of the transcription factor called nuclear factor of activated T cells (NFAT). GHRH, NFAT1, NFAT4, and related genes were endogenously expressed in Gsh+/+ cells and the rat arcuate nucleus, where NFAT1 and GHRH were colocalized. Cellular excitation with high potassium potently stimulated endogenous GHRH gene 5′-promoter activity as well as the NFAT-mediated gene transcription, the former being further enhanced by coexpression of NFAT. On the other hand, cyclosporin A (a calcineurin-NFAT inhibitor) or EGTA (a calcium chelator) significantly blocked the depolarization-induced GHRH gene transcription. EMSA and site-directed mutagenesis experiments showed the direct binding of NFAT at five sites of the GHRH promoter, among which the relative importance of three distal sites (−417/−403, −402/−387, −317/−301) was suggested. Finally, elimination of all five sites completely abolished the NFAT-induced GHRH gene up-regulation. Altogether, our results suggest that the transcription factor NFAT is involved in the depolarization-induced transcriptional activation of GHRH gene in the neuronal cells.

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