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 DNA Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

2006 ◽  
Vol 188 (12) ◽  
pp. 4577-4580 ◽  
Author(s):  
Rajesh Kasiviswanathan ◽  
Jae-Ho Shin ◽  
Zvi Kelman
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Minichromosome Maintenance ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Mcm Helicase ◽  
Methanothermobacter Thermautotrophicus

ABSTRACT The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

Single-stranded DNA binding activity of XPBI, but not XPBII, from Sulfolobus tokodaii causes double-stranded DNA melting

Extremophiles ◽  
2010 ◽  
Vol 15 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Xiaoqing Ma ◽  
Ye Hong ◽  
Wenyuan Han ◽  
Duohong Sheng ◽  
Jinfeng Ni ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Dna Melting ◽  
Single Stranded Dna ◽  
Sulfolobus Tokodaii ◽  
Dna Binding Activity ◽  
Double Stranded Dna

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.

Src-Dependent Erk1/2-Mediated Signaling Pathways and Associated Ang Ii-Stimulated Growth Responses Are Altered in Vascular Smooth Muscle Cells from Arteries of Hypertensive Patients.

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 721-721
Author(s):  
Rhian M Touyz ◽  
Gang He ◽  
Xiao-Hua Wu ◽  
Jeong Bae Park ◽  
Mohammed El Mabrouk ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Vascular Remodeling ◽  
Binding Activity ◽  
Ang Ii ◽  
Hypertensive Patients ◽  
Dna Binding Activity

P154 Molecular mechanisms underlying vascular remodeling in human hypertension are unclear. We investigated c-Src and ERK1/2 activity, proto-oncogene expression, activating protein 1 (AP-1) DNA-binding activity, and DNA and protein synthesis in Ang II-stimulated vascular smooth muscle cells (VSMC) derived from small peripheral arteries from normotensive subjects (NT) (n=5) and age-matched untreated hypertensive patients (HT) (n=10). The media and media:lumen ratio were greater (p<0.05) in arteries from HT than NT, indicating vascular remodeling. Ang II-dose-dependently increased synthesis of DNA and protein, with enhanced effects (p<0.01) in VSMCs from HT. PD98059, a selective inhibitor of the ERK1/2 pathway, attenuated (p<0.01) Ang II-stimulated growth. PD98059 effects were greater in HT than NT. In NT, Ang II transiently increased ERK1/2 phosphorylation. In HT, Ang II-stimulated ERK1/2 activation was significantly augmented (p<0.05 vs NT) and sustained. PP2, a selective Src inhibitor, reduced ERK1/2 activity and normalized responses in HT. Ang II-induced c-Src phosphorylation was 2-3 fold greater in HT than NT. In HT, kinase activation was followed by overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. PD98059 attenuated these responses. In HT cells transfected with c-fos antisense oligodeoxynucleotide (ODN), Ang II-stimulated DNA synthesis was reduced compared with sense ODN. These novel findings suggest that vascular remodeling is associated with augmented Ang II-stimulated VSMC growth, which is mediated via hyperactivation of c-Src-regulated, ERK1/2-dependent pathways leading to overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. Our data define a signal transduction pathway in VSMCs that could contribute to structural changes and vascular remodeling in essential hypertension.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

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