Mechanisms Responsible for the Promoter-specific Effects of Myocardin

Zhou, Jiliang; Herring, B. Paul

doi:10.1074/jbc.m411586200

Cited by 46 publications

(61 citation statements)

References 26 publications

(34 reference statements)

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“…Previous studies have demonstrated that SRF binds to this CArG box in the telokin promoter, in intact chromatin, and in smooth muscle cells and that myocardin can strongly activate the promoter through its interaction with SRF bound to this site (36). Myocardin has been shown to be a powerful coactivator of SRF on smooth muscle-specific genes and to be required for vascular smooth muscle development (4,7,24,32,35).…”

Section: Discussionmentioning

confidence: 99%

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Touw¹,

Hoggatt²,

Simon³

et al. 2007

American Journal of Physiology-Cell Physiology

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Section: Discussionmentioning

confidence: 99%

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Touw¹,

Hoggatt²,

Simon³

et al. 2007

American Journal of Physiology-Cell Physiology

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“…Western Blotting-Western blot analysis was carried out essentially as described previously (22)(23)(24). 30 g of protein was fractionated on 5 or 15% SDS-polyacrylamide gels, electrophoretically transferred to a nitrocellulose membrane.…”

Section: Quantitative Real Time Rt-pcr (Qrt-pcr) Analysis-totalmentioning

confidence: 99%

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Wang

Saunders

et al. 2010

Journal of Biological Chemistry

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Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

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“…The telokin promoter, which drives expression of a gene encoding the carboxyl-terminal end of SM myosin light chain kinase, contains a single CArG element that is responsive to both SRF and MYOCD (14,15). The SM myosin light chain kinase gene itself was shown to harbor conserved CArG elements also reactive to SRF-MYOCD (16,17).…”

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confidence: 99%

The Smooth Muscle Cell-restricted KCNMB1 Ion Channel Subunit Is a Direct Transcriptional Target of Serum Response Factor and Myocardin

Long

Tharp

Georger

et al. 2009

Journal of Biological Chemistry

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Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise ␣ and ␤ subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype. Smooth muscle cells (SMCs)2 are of crucial importance in maintaining normal structural and contractile integrity of various organs and tissues throughout the vertebrate body. Unlike skeletal and cardiac muscle cells, which largely undergo irreversible terminal differentiation, SMCs have the inherent ability to alter their differentiated state in response to diverse stimulatory cues. This process of SMC phenotypic modulation is a hallmark of several human pathological conditions, including vascular occlusive disease, asthma, intestinal and bladder obstruction, and Alzheimer disease. SMC phenotypic modulation is often defined in terms of unique molecular signatures of gene expression involved with contraction and cytoskeletal architecture as well as extracellular matrix remodeling (1, 2). For example, vascular SMCs display reduced levels of contractile filaments following injury to the vessel wall, adopting the so-called synthetic phenotype characterized by an overabundance of rough endoplasmic reticulum (3). On the other hand, recent studies have shown an exaggerated SMC contractile phenotype thought to contribute to disease progression (4, 5).The majority of SMC-restricted genes contain one or more conserved CArG elements that bind the widely express...

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Mechanisms Responsible for the Promoter-specific Effects of Myocardin

Cited by 46 publications

References 26 publications

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

The Smooth Muscle Cell-restricted KCNMB1 Ion Channel Subunit Is a Direct Transcriptional Target of Serum Response Factor and Myocardin

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