scholarly journals Structure/Function Analysis of Mouse Purβ, a Single-stranded DNA-binding Repressor of Vascular Smooth Muscle α-Actin Gene Transcription

2003 ◽  
Vol 278 (40) ◽  
pp. 38749-38757 ◽  
Author(s):  
Robert J. Kelm ◽  
Shu-Xia Wang ◽  
John A. Polikandriotis ◽  
Arthur R. Strauch
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Vascular Smooth Muscle ◽  
Structure Function ◽  
Gene Transcription ◽  
Function Analysis ◽  
Actin Gene ◽  
Single Stranded Dna

scholarly journals Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.

1995 ◽  
Vol 15 (5) ◽  
pp. 2429-2436 ◽  
Author(s):  
S Sun ◽  
E S Stoflet ◽  
J G Cogan ◽  
A R Strauch ◽  
M J Getz
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Vascular Smooth Muscle ◽  
Binding Site ◽  
Binding Activity ◽  
Actin Gene ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Alpha Actin

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

scholarly journals Structure-Function Analysis of Mouse Purβ II

2007 ◽  
Vol 282 (49) ◽  
pp. 35899-35909 ◽  
Author(s):  
Anna M. Knapp ◽  
Jon E. Ramsey ◽  
Shu-Xia Wang ◽  
Arthur R. Strauch ◽  
Robert J. Kelm
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Function Analysis ◽  
Dominant Factor ◽  
Actin Gene ◽  
Single Stranded Dna ◽  
Arginine Residues ◽  
Repressor Activity

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Purα and Purβ, in transcriptional repression of the smooth muscle α-actin gene in vascular cell types. Although Purα and Purβ share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Purβ is the dominant factor in gene regulation. To dissect the molecular basis of Purβ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Purβ with the smooth muscle α-actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Purβ repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Purβ and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison to the wild-type Purβ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased α-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Purβ protein capable of physical interactions necessary for smooth muscle α-actin gene repression.

scholarly journals Vascular Smooth Muscle α-Actin Gene Transcription during Myofibroblast Differentiation Requires Sp1/3 Protein Binding Proximal to the MCAT Enhancer

2002 ◽  
Vol 277 (39) ◽  
pp. 36433-36442 ◽  
Author(s):  
John G. Cogan ◽  
Sukanya V. Subramanian ◽  
John A. Polikandriotis ◽  
Robert J. Kelm ◽  
Arthur R. Strauch
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Protein Binding ◽  
Gene Transcription ◽  
Actin Gene ◽  
Myofibroblast Differentiation

Differential signaling pathways ofHO-1 gene expression in pulmonary and systemic vascular cells

1999 ◽  
Vol 277 (6) ◽  
pp. L1133-L1141 ◽  
Author(s):  
Cynthia L. Hartsfield ◽  
Jawed Alam ◽  
Augustine M. K. Choi
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Dna Binding ◽  
Vascular Smooth Muscle ◽  
Gene Transcription ◽  
Binding Activity ◽  
Activator Protein 1 ◽  
Distal Enhancer ◽  
Dna Binding Activity ◽  
Pulmonary Arterial

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-induced HO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B.-H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375–5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither induced HO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction of HO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5′-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or the HO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-induced HO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation of HO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-induced HO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.

scholarly journals Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.

1988 ◽  
Vol 107 (6) ◽  
pp. 2575-2586 ◽  
Author(s):  
D L Ruzicka ◽  
R J Schwartz
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Cardiac Cells ◽  
Actin Gene ◽  
Heart Tube ◽  
Specific Expression ◽  
Actin Genes ◽  
Beta Actin ◽  
Smooth Muscle Alpha Actin ◽  
Alpha Actin

The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger-Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha-actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells.

Sign in / Sign up

Export Citation Format

Share Document