Divergence in species and regulatory role of β-myosin heavy chain proximal promoter muscle-CAT elements

2002 ◽  
Vol 283 (6) ◽  
pp. C1761-C1775 ◽  
Author(s):  
Richard W. Tsika ◽  
John McCarthy ◽  
Natalia Karasseva ◽  
Yangsi Ou ◽  
Gretchen L. Tsika
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transgene Expression ◽  
Weight Bearing ◽  
Nuclear Extract ◽  
Proximal Promoter ◽  
Wild Type ◽  
Mobility Shift

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.

Control of beta-myosin heavy chain expression in systemic hypertension and caloric restriction in the rat heart

1995 ◽  
Vol 269 (4) ◽  
pp. C1025-C1033 ◽  
Author(s):  
S. J. Swoap ◽  
F. Haddad ◽  
P. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Caloric Restriction ◽  
Heavy Chain ◽  
Pressure Overload ◽  
Nuclear Extract ◽  
Systemic Hypertension ◽  
Sprague Dawley ◽  
Mobility Shift ◽  
Concomitant Decrease

In the rat left ventricle, both pressure overload induced by abdominal aortic constriction (Abcon) and caloric restriction (CR) induce an increase in the steady-state level of the beta-myosin heavy chain (MHC) protein and mRNA. Both models also induce a concomitant decrease in the alpha-MHC protein and mRNA. The goals of this study were to 1) determine if the changes in MHC expression in the models are due to altered transcription and 2) identify the relative levels of some key factors interacting with the regulatory regions of these genes. Female Sprague-Dawley rats were randomly assigned to the following groups: 1) normal control (NC), 2) Abcon, and 3) CR. After 5 wk of experimental manipulations, myocardial nuclei were isolated. These nuclei were used for 1) nuclear run-on assays or 2) nuclear extract, which was prepared and used for gel mobility shift assays (GMSAs). Nuclear run-on assays demonstrated that the increase in beta-MHC mRNA and protein expression in both Abcon and CR can be at least partially attributed to increased transcription. The concomitant decrease in alpha-MHC content can similarly be attributed to a decrease in transcription of this gene. Furthermore, GMSAs demonstrate that nuclear extract from each group interact differently with certain elements known to be important for expression in vitro. CR nuclear extracts have a 25.6 +/- 7.2% decrease (P < 0.05 vs. NC) in interaction with a thyroid-responsive element, a potential repressor of beta-MHC transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

scholarly journals Role of cytosol in the stimulation of RNA transport in vitro during cardiac hypertrophy in rats

1990 ◽  
Vol 267 (1) ◽  
pp. 133-140 ◽  
Author(s):  
A Subramaniam ◽  
M Thirunavukkarasu ◽  
C Rajamanickam
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Deae Cellulose ◽  
Gene Probe ◽  
Rna Transport ◽  
Transport Activity ◽  
Isolated Nuclei ◽  
Stimulation Of

The 100,000 g supernatant isolated from hypertrophic hearts on fractionation by (NH4)2SO4 and DEAE-cellulose chromatography showed an enhanced RNA-transport activity when incubated with isolated nuclei from sham-operated hearts in vitro. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are enriched in the DEAE-cellulose fractions exhibiting maximal transport activity, and they are phosphorylatable. Pretreatment of the cytosol with antibodies to the Mr-68,000 and -32,000 proteins decreases the transport activity of the cytosol from 14% to 4.25%. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are translocated from the cytosol to the nuclear envelope under conditions of RNA transport in vitro. Our results here suggest that at least two of these proteins, those of Mr 68,000 and 32,000, play an indispensible role in the nucleocytoplasmic RNA transport in vitro. By making use of a specific myosin heavy-chain B-gene probe and hybridization, we have also shown the effect of cytosol on the transport of myosin heavy-chain mRNA from nucleus to cytosol.

scholarly journals CREB Is a Positive Transcriptional Regulator of Gamma Interferon in Latent but Not Active Tuberculosis Infections

2010 ◽  
Vol 17 (9) ◽  
pp. 1377-1380 ◽  
Author(s):  
Yang Liu ◽  
Yan-Ling Guo ◽  
Shi-Jie Zhou ◽  
Fei Liu ◽  
Feng-Jiao Du ◽  
...  
Keyword(s):  
Western Blotting ◽  
Cell Function ◽  
Gamma Interferon ◽  
Regulatory Role ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Basic Leucine Zipper ◽  
Ifn Γ

ABSTRACT Gamma interferon (IFN-γ) is a crucial cytokine for protection against Mycobacterium tuberculosis, but the mechanism of IFN-γ transcription is still unclear. The cyclic AMP (cAMP) responsive element binding (CREB) proteins belong to the bZip (basic leucine zipper) family of transcription factors and are essential for T-cell function and cytokine production. This study focused on the capacity of CREB proteins to regulate IFN-γ transcription in CD3+ T cells obtained from tuberculosis (TB) patients and persons with latent tuberculosis infection (LTBI) in China. The electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and Western blotting were used to demonstrate the regulatory role of CREB. EMSA (in vitro) and ChIP (in vivo) experiments suggested CREB could bind to the IFN-γ proximal promoter in persons with LTBI, whereas no binding was detected in TB patients. Western blotting confirmed the expression of CREB proteins, especially serine-133-phosphorylated CREB, was markedly reduced in TB patients compared with persons with LTBI. These results suggested that CREB could promote the transcription and production of IFN-γ through binding with the IFN-γ proximal promoter, but the regulatory role of CREB was decreased in tuberculosis patients owing to diminished expression of CREB proteins, which in turn reduced the IFN-γ production.

scholarly journals Purα and Purβ Collaborate with Sp3 To Negatively Regulate β-Myosin Heavy Chain Gene Expression duringSkeletal Muscle Inactivity

2006 ◽  
Vol 27 (4) ◽  
pp. 1531-1543 ◽  
Author(s):  
Juan Ji ◽  
Gretchen L. Tsika ◽  
Hansjörg Rindt ◽  
Kathy L. Schreiber ◽  
John J. McCarthy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myosin Heavy Chain ◽  
Reporter Gene ◽  
Heavy Chain ◽  
Weight Bearing ◽  
Ectopic Expression ◽  
Reporter Gene Expression ◽  
Proximal Promoter ◽  
C2c12 Myotubes ◽  
Adult Skeletal Muscle

ABSTRACT Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased β-myosin heavy chain (βMyHC) gene expression. Our previous work showed that Sp3 contributes to decreased βMyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Purα, and Purβ proteins mediate repression of βMyHC expression under NWB conditions. Binding of Purα or Purβ to the single-stranded βMyHC distal negative regulatory element-sense strand (dβNRE-S) element is markedly increased under NWB conditions. Ectopic expression of Purα and Purβ decreasedβ MyHC reporter gene expression, while mutation of the dβNRE-S element increased expression in C2C12 myotubes. The dβNRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Purα and Purβ decreased endogenous Sp3 and Pur protein levels and increased βMyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Purα, Purβ, and Sp3, while chromatin immunoprecipitation assays demonstrated Purα, Purβ, and Sp3 binding to the βMyHC proximal promoter region harboring the dβNRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

scholarly journals Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

1994 ◽  
Vol 127 (6) ◽  
pp. 1933-1944 ◽  
Author(s):  
P Chen ◽  
B D Ostrow ◽  
S R Tafuri ◽  
R L Chisholm
Keyword(s):  
Myosin Heavy Chain ◽  
Conformational Changes ◽  
Heavy Chain ◽  
Biochemical Study ◽  
Neck Region ◽  
Light Chains ◽  
Dictyostelium Cell ◽  
Wild Type

Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment et al., 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987; Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild-type myosin, but the filaments then exhibit abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 873-881 ◽  
Author(s):  
Claire Lewis ◽  
Henrieta Skovierova ◽  
Gary Rowley ◽  
Bronislava Rezuchova ◽  
Dagmar Homerova ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protease Activity ◽  
Pdz Domain ◽  
Proximal Promoter ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Serovar Typhimurium

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ E (RpoE). S. Typhimurium htrA also possesses a σ E-dependent promoter; however, we found that the absence of σ E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ 70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

scholarly journals Follistatin Attenuates Myocardial Fibrosis in Diabetic Cardiomyopathy via the TGF-β–Smad3 Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Yinhui Wang ◽  
Kun Yu ◽  
Chengcheng Zhao ◽  
Ling Zhou ◽  
Jia Cheng ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Diabetic Cardiomyopathy ◽  
Heavy Chain ◽  
Transforming Growth Factor Beta ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Endogenous Protein ◽  
Matrix Metallopeptidase

Follistatin (FST) is an endogenous protein that irreversibly inhibits TGF-β superfamily members and plays an anti-fibrotic role in other diseases. However, the role of FST in diabetic cardiomyopathy remains unclear. In this study, we investigated the effects of FST on diabetic cardiomyopathy. The expression of FST was downregulated in the hearts of db/db mice. Remarkably, overexpressing FST efficiently protected against cardiac dysfunction. In addition, overexpression of FST promoted cardiac hypertrophy with an unchanged expression of atrial natriuretic peptide (ANP) and the ratio of myosin heavy chain-β/myosin heavy chain-α (MYH7/MYH6). Furthermore, FST reduced cardiac fibrosis and the production of reactive oxygen species (ROS), and enhanced matrix metallopeptidase 9 (MMP9) activities in db/db mouse hearts. We also observed that overexpressing FST decreased the level of transforming growth factor beta (TGF-β) superfamily members and the phosphorylation of Smad3; consistently, in vitro experiments also verified the above results. Our findings revealed the cardioprotective role of FST in attenuating diabetic cardiomyopathy through its anti-fibrotic effects through the TGF-β–Smad3 pathway and provided a promising therapeutic strategy for diabetic cardiomyopathy.

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