Transcriptional regulation of the type I myosin heavy chain promoter in inactive rat soleus

2002 ◽  
Vol 282 (3) ◽  
pp. C528-C537 ◽  
Author(s):  
K. A. Huey ◽  
R. R. Roy ◽  
F. Haddad ◽  
V. R. Edgerton ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Gene Promoter ◽  
Type I ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Transcription Enhancer ◽  
Enhancer Factor

Chronic muscle inactivity with spinal cord isolation (SI) decreases expression of slow type I myosin heavy chain (MHC) while increasing expression of the faster MHC isoforms, primarily IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated transcriptionally and to identify cis-acting elements or regions of the rat type I MHC gene promoter involved in this response. One week of SI significantly decreased in vivo activity of the −3500-, −408-, −299-, −215-, and −171-bp type I MHC promoters. The activity of all tested deletions of the type I MHC promoter, relative to the human skeletal α-actin promoter, were significantly reduced in the SI soleus, except activity of the −171-bp promoter, which increased. Mutation of the βe3 element (−214/−190 bp) in the −215- and −408-bp promoters and deletion of this element (−171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to the βe3 element with SI, despite an increase in total binding to this region. These results demonstrate that type I MHC downregulation with SI is transcriptionally regulated and suggest that interactions between transcription enhancer factor-1 and the βe3 element are likely involved in this response.

scholarly journals Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression.

1997 ◽  
Vol 17 (7) ◽  
pp. 3924-3936 ◽  
Author(s):  
M P Gupta ◽  
C S Amin ◽  
M Gupta ◽  
N Hay ◽  
R Zak
Keyword(s):  
Myosin Heavy Chain ◽  
Reporter Gene ◽  
Heavy Chain ◽  
Troponin T ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Box ◽  
Two Factors ◽  
Transcription Enhancer ◽  
Enhancer Factor

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.

In vivo regulation of the β-myosin heavy chain gene in hypertensive rodent heart

2001 ◽  
Vol 280 (5) ◽  
pp. C1262-C1276 ◽  
Author(s):  
Carola E. Wright ◽  
P. W. Bodell ◽  
F. Haddad ◽  
A. X. Qin ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Promoter Activity ◽  
Proximal Promoter ◽  
Future Research ◽  
Chain Gene ◽  
Direct Gene Transfer ◽  
Basal Region ◽  
Mobility Shift

The main goal of this study was to examine the transcriptional activity of different-length β-myosin heavy chain (β-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by ∼45% relative to control. Results show that β-MHC promoter activity of all tested wild-type constructs, i.e., −3500, −408, −299, −215, −171, and −71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the −71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting βe1, βe2, GATA, and βe3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the β-MHC gene. Collectively, these results indicate that three separate regions on the β-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between −408 and −3500 bp, 2) a proximal region between −299 and −215 bp, and 3) a basal region within −71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate β-MHC transcriptional regulation in response to pressure overload.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals In vivo analysis of the murine beta-myosin heavy chain gene promoter.

1993 ◽  
Vol 268 (7) ◽  
pp. 5332-5338
Author(s):  
H. Rindt ◽  
J. Gulick ◽  
S. Knotts ◽  
J. Neumann ◽  
J. Robbins
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Gene Promoter ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Myosin Heavy Chain Gene ◽  
In Vivo Analysis

Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate

2007 ◽  
Vol 293 (1) ◽  
pp. C64-C74 ◽  
Author(s):  
Pawel R. Kiela ◽  
Nesrin Kuscuoglu ◽  
Anna J. Midura ◽  
Monica T. Midura-Kiela ◽  
Claire B. Larmonier ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Sodium Butyrate ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Upstream Site ◽  
Potent Inducer ◽  
Maximal Induction ◽  
Gel Mobility Shift

Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na+/H+ exchange. NaB induces Na+/H+ exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within −320/−34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position −58/−55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at −196/−175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a −196/−175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.

Transcriptional regulation of the type I myosin heavy chain gene in denervated rat soleus

2003 ◽  
Vol 284 (3) ◽  
pp. C738-C748 ◽  
Author(s):  
K. A. Huey ◽  
F. Haddad ◽  
A. X. Qin ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Chain Gene ◽  
Type I ◽  
Slow Type ◽  
Actin Promoter ◽  
Mrna Analysis

Denervation (DEN) of rat soleus is associated with a decreased expression of slow type I myosin heavy chain (MHC) and an increased expression of the faster MHC isoforms. The molecular mechanisms behind these shifts remain unclear. We first investigated endogenous transcriptional activity of the type I MHC gene in normal and denervated soleus muscles via pre-mRNA analysis. Our results suggest that the type I MHC gene is regulated via transcriptional processes in the denervated soleus. Deletion and mutational analysis of the rat type I MHC promoter was then used to identify cis elements or regions of the promoter involved in this response. DEN significantly decreased in vivo activity of the −3,500, −2,500, −914, −408, −299, and −215 bp type I MHC promoters, relative to the α-skeletal actin promoter. In contrast, normalized −171 promoter activity was unchanged. Mutation of the βe3 element (−214/−190) in the −215 promoter and deletion of this element (−171 promoter) blunted type I downregulation with DEN. In contrast, βe3 mutation in the −408 promoters was not effective in attenuating the DEN response, suggesting the existence of additional DEN-responsive sites between −408 and −215. Western blotting and gel mobility supershift assays demonstrated decreased expression and DNA binding of transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this decrease may contribute to type I MHC downregulation in denervated muscle.

Effect of unloading on type I myosin heavy chain gene regulation in rat soleus muscle

2005 ◽  
Vol 98 (4) ◽  
pp. 1185-1194 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Ming Zeng ◽  
Kenneth M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
Regulatory Elements ◽  
Hindlimb Suspension ◽  
Type I ◽  
Transcriptional Enhancer ◽  
Western Blots ◽  
E Box ◽  
Enhancer Factor

Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type I myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast-type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the downregulation of MHC1 in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC1 promoter was injected into soleus muscles of control rats and those subjected to 7 days of hindlimb suspension. Mutation analyses of six putative regulatory elements within the −408-bp region demonstrated that three elements, an A/T-rich, the proximal muscle-type CAT (βe3), and an E-box (−63 bp), play an important role in the basal level of MHC1 gene activity in the control soleus and function as unloading-responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the βe3 and E-box probes with nuclear extract from hindlimb suspension soleus compared with control soleus. Supershift assays indicated that transcriptional enhancer factor 1 and myogenin factors bind the βe3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the transcriptional enhancer factor 1 and myogenin factors were significantly attenuated in the unloaded soleus compared with the control muscle. We conclude that the downregulation of MHC1 in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements.

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression

1992 ◽  
Vol 12 (5) ◽  
pp. 2230-2240
Author(s):  
D M Stover ◽  
Z E Zehner
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Gene Activity ◽  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Silencer Element ◽  
Vimentin Gene

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

scholarly journals Transcription Enhancer Factor 1 Binds Multiple Muscle MEF2 and A/T-Rich Elements during Fast-to-Slow Skeletal Muscle Fiber Type Transitions

2003 ◽  
Vol 23 (15) ◽  
pp. 5143-5164 ◽  
Author(s):  
Natalia Karasseva ◽  
Gretchen Tsika ◽  
Juan Ji ◽  
Aijing Zhang ◽  
Xiaoqing Mao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Networks ◽  
Fiber Type ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Transcription Enhancer ◽  
Enhancer Factor

ABSTRACT In adult mouse skeletal muscle, β-myosin heavy chain (βMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload [MOV]). Our previous βMyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (βA/T-rich −269/−258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp βMyHC promoter (β293wt). Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms. By using the βA/T-rich element as bait in a yeast one-hybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific βA/T-rich binding factor. Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the βA/T-rich element is reacted with MOV-plantaris nuclear extract. Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes. In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp βMyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem βA/T-rich elements. These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.

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