Da-Zhi Wang

The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation

Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of ∼22 nucleotides that post-transcriptionally regulate gene expression. Increasing evidence points to the potential role of miRNAs in various biological processes. Here we show that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). Our results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, our studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development.

Activation of Cardiac Gene Expression by Myocardin, a Transcriptional Cofactor for Serum Response Factor

Serum response factor (SRF) regulates transcription of numerous muscle and growth factor-inducible genes. Because SRF is not muscle specific, it has been postulated to activate muscle genes by recruiting myogenic accessory factors. Using a bioinformatics-based screen for unknown cardiac-specific genes, we identified a novel and highly potent transcription factor, named myocardin, that is expressed in cardiac and smooth muscle cells. Myocardin belongs to the SAP domain family of nuclear proteins and activates cardiac muscle promoters by associating with SRF. Expression of a dominant negative mutant of myocardin in Xenopus embryos interferes with myocardial cell differentiation. Myocardin is the founding member of a class of muscle transcription factors and provides a mechanism whereby SRF can convey myogenic activity to cardiac muscle genes.

MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure

Cardiovascular disease is the leading cause of human morbidity and mortality. Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy associated with heart failure. Here, we report that cardiac-specific knockout of Dicer, a gene encoding a RNase III endonuclease essential for microRNA (miRNA) processing, leads to rapidly progressive DCM, heart failure, and postnatal lethality. Dicer mutant mice show misexpression of cardiac contractile proteins and profound sarcomere disarray. Functional analyses indicate significantly reduced heart rates and decreased fractional shortening of Dicer mutant hearts. Consistent with the role of Dicer in animal hearts, Dicer expression was decreased in end-stage human DCM and failing hearts and, most importantly, a significant increase of Dicer expression was observed in those hearts after left ventricle assist devices were inserted to improve cardiac function. Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions.

Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression

Smooth muscle cells switch between differentiated and proliferative phenotypes in response to extracellular cues, but the transcriptional mechanisms that confer such phenotypic plasticity remain unclear. Serum response factor (SRF) activates genes involved in smooth muscle differentiation and proliferation by recruiting muscle-restricted cofactors, such as the transcriptional coactivator myocardin, and ternary complex factors (TCFs) of the ETS-domain family, respectively. Here we show that growth signals repress smooth muscle genes by triggering the displacement of myocardin from SRF by Elk-1, a TCF that acts as a myogenic repressor. The opposing influences of myocardin and Elk-1 on smooth muscle gene expression are mediated by structurally related SRF-binding motifs that compete for a common docking site on SRF. A mutant smooth muscle promoter, retaining responsiveness to myocardin and SRF but defective in TCF binding, directs ectopic transcription in the embryonic heart, demonstrating a role for TCFs in suppression of smooth muscle gene expression in vivo. We conclude that growth and developmental signals modulate smooth muscle gene expression by regulating the association of SRF with antagonistic cofactors.

Myocardin is a master regulator of smooth muscle gene expression

Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF–myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.

Potentiation of serum response factor activity by a family of myocardin-related transcription factors.

Myocardin is a SAP (SAF-A/B, Acinus, PIAS) domain transcription factor that associates with serum response factor (SRF) to potently enhance SRF-dependent transcription. Here we describe two myocardin-related transcription factors (MRTFs), A and B, that also interact with SRF and stimulate its transcriptional activity. Whereas myocardin is expressed specifically in cardiac and smooth muscle cells, MRTF-A and -B are expressed in numerous embryonic and adult tissues. In SRF-deficient embryonic stem cells, myocardin and MRTFs are unable to activate SRF-dependent reporter genes, confirming their dependence on SRF. Myocardin and MRTFs comprise a previously uncharacterized family of SRF cofactors with the potential to modulate SRF target genes in a wide range of tissues.

Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex

Calcineurin, which binds to the Z-disc in cardiomyocytes via alpha-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and alpha-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCF(atrogin-1) complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCF(atrogin-1) ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.

Myocardin Is a Key Regulator of CArG-Dependent Transcription of Multiple Smooth Muscle Marker Genes

Abstract— The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM &agr;-actin, SM-myosin heavy chain, and SM22&agr; by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.

The serum response factor coactivator myocardin is required for vascular smooth muscle development

Formation of the vascular system requires differentiation and patterning of endothelial and smooth muscle cells (SMCs). Although much attention has focused on development of the vascular endothelial network, the mechanisms that control vascular SMC development are largely unknown. Myocardin is a smooth and cardiac muscle-specific transcriptional coactivator of serum response factor, a ubiquitous transcription factor implicated in smooth muscle gene expression. When expressed ectopically in nonmuscle cells, myocardin can induce smooth muscle differentiation by its association with serum response factor. Here we report that mouse embryos homozygous for a myocardin loss-of-function mutation die by embryonic day 10.5 and show no evidence of vascular SMC differentiation. Myocardin is the only transcription factor known to be necessary and sufficient for vascular SMC differentiation.