B. Paul Herring

A novel role of Brg1 in the regulation of SRF/MRTFA-dependent smooth muscle-specific gene expression.

Serum response factor (SRF) is a key regulator of smooth muscle differentiation, proliferation, and migration. Myocardin-related transcription factor A (MRTFA) is a co-activator of SRF that can induce expression of SRF-dependent, smooth muscle-specific genes and actin/Rho-dependent genes, but not MAPK-regulated growth response genes. How MRTFA and SRF discriminate between these sets of target genes is still unclear. We hypothesized that SWI/SNF ATP-dependent chromatin remodeling complexes, containing Brahma-related gene 1 (Brg1) or Brahma (Brm), may play a role in this process. Results from Western blotting and qRT-PCR analysis demonstrated that dominant negative Brg1 blocked the ability of MRTFA to induce expression of smooth muscle-specific genes, but not actin/Rho-dependent early response genes, in fibroblasts. In addition, dominant negative Brg1 attenuated expression of smooth muscle-specific genes in primary cultures of smooth muscle cells. MRTFA overexpression did not induce expression of smooth muscle-specific genes in SW13 cells, which lack endogenous Brg1 or Brm. Reintroduction of Brg1 or Brm into SW13 cells restored their responsiveness to MRTFA. Immunoprecipitation assays revealed that Brg1, SRF, and MRTFA form a complex in vivo, and Brg1 directly binds MRTFA, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated the ability of MRTFA to increase SRF binding to the promoters of smooth muscle-specific genes, but not early response genes. Together these data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in regulating expression of SRF/MRTFA-dependent smooth muscle-specific genes but not SRF/MRTFA-dependent early response genes.

The SWI/SNF Chromatin Remodeling Complex Regulates Myocardin-Induced Smooth Muscle–Specific Gene Expression

Objective—Regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle–specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. Methods and Results—We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle–specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle–specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, which lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle–specific genes. Whereas, reconstitution of wild-type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle–specific genes. Brg1 and Brm directly bind to the N terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. Conclusion—These data demonstrate that the ability of myocardin to induce smooth muscle–specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes.

Smooth Muscle-Specific Genes Are Differentially Sensitive to Inhibition by Elk-1

ABSTRACT Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22α, and smooth muscle α-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22α, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22α without affecting expression of smooth muscle α-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.

Identification of Barx2B, a Serum Response Factor-associated Homeodomain Protein

CC(A/T)6GG or serum response elements represent a common regulatory motif important for regulating the expression of many smooth muscle-specific genes. They are multifunctional elements that bind serum response factor (SRF) and are important for serum induction of genes, expression of muscle-specific genes, and differentiation of vascular smooth muscle cells. In the current study, a yeast two-hybrid screen was used to identify proteins from mouse intestine that interact with SRF. A novel homeodomain-containing transcription factor, called Barx2b, was identified that specifically interacts with SRF and promotes the DNA binding activity of SRF. Northern blotting, RNase protection analysis, and Western blotting revealed that Barx2b mRNA and protein are expressed in several smooth muscle-containing tissues, as well as in skeletal muscle and brain. In vitro binding studies using bacterial fusion proteins revealed that the DNA-binding domain of SRF interacts with a region of Barx2b located amino-terminal of the homeobox domain. The results of these studies support the hypothesis that interaction of SRF with different homeodomain-containing proteins may play a critical role in determining the cell-specific functions of SRF.

Mechanisms Responsible for the Promoter-specific Effects of Myocardin

Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases such as atherosclerosis, restenosis, and asthma. Recent studies have demonstrated that the interaction of SRF with the co-activator myocardin is a critical determinant of smooth muscle development. It has been proposed that the specific transcriptional activation of smooth muscle-restricted genes (as opposed to other SRF-dependent genes) by myocardin results from the presence of multiple CArG boxes in smooth muscle genes that facilitate myocardin homodimer formation. This proposal was further tested in the current study. Our results show that the SMC-specific telokin promoter, which contains only a single CArG box, is strongly activated by myocardin. Furthermore, myocardin and a dimerization defective mutant myocardin induce expression of endogenous telokin but not c-fos in 10T1/2 fibroblast cells. Knocking down myocardin by small interfering RNA decreased telokin promoter activity and expression in A10 SMCs. A series of telokin and c-fos promoter chimeric and mutant reporter genes was generated to determine the mechanisms responsible for the promoter-specific effects of myocardin. Data from these experiments demonstrated that the ets binding site in the c-fos promoter partially blocks the activation of this promoter by myocardin. However, the binding of ets factors alone was not sufficient to explain the promoter-specific effects of myocardin. Elements 3′ of the CArG box in the c-fos promoter act in concert with the ets binding site to block the ability of myocardin to activate the promoter. Conversely, elements 5′ and 3′ of the CArG box in the telokin promoter act in concert with the CArG box to facilitate myocardin stimulation of the promoter. Together these data suggest that the promoter specificity of myocardin is dependent on complex combinatorial interactions of multiple cis elements and their trans binding factors.

Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes

Results show that smooth muscle-specific promoters represent novel downstream targets of the winged helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1). HFH-1 strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells. HFH-1 was also found to repress transcription of several other smooth muscle-specific promoters, including the SM22α promoter. HFH-1 inhibits telokin promoter activity, by binding to a forkhead consensus site located within an AT-rich region of the telokin promoter. The DNA-binding domain alone was sufficient to mediate inhibition, suggesting that binding of HFH-1 blocks the binding of other positive-acting factors. HFH-1 does not disrupt serum response factor binding to an adjacent CArG box within the telokin promoter, implying that HFH-1 must compete with other unidentified trans-activators to mediate repression. The localization of HFH-1 mRNA to the epithelial cell layer of mouse bladder and stomach implicates HFH-1 in repressing telokin expression in epithelial cells. This suggests that cell-specific expression of telokin is likely mediated by both positive-acting factors in smooth muscle cells and negative-acting factors in nonmuscle cell types. We propose a model in which the smooth muscle specificity of the telokin promoter is regulated by interactions between positive- and negative-acting members of the hepatocyte nuclear factor-3/forkhead family of transcription factors.

Impaired Collateral Artery Development in Spontaneously Hypertensive Rats

Objective: To determine whether collateral artery development is impaired in spontaneously hypertensive (SHR) relative to normotensive (WKY) rats.

SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development.

ABSTRACT SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1flox/flox mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.

Cell-Specific Regulatory Modules Control Expression of Genes in Vascular and Visceral Smooth Muscle Tissues

Abstract— A novel approach with chimeric SM22&agr;/telokin promoters was used to identify gene regulatory modules that are required for regulating the expression of genes in distinct smooth muscle tissues. Conventional deletion or mutation analysis of promoters does not readily distinguish regulatory elements that are required for basal gene expression from those required for expression in specific smooth muscle tissues. In the present study, the mouse telokin gene was isolated, and a 370-bp (−190 to 180) minimal promoter was identified that directs visceral smooth muscle–specific expression in vivo in transgenic mice. The visceral smooth muscle–specific expression of the telokin promoter transgene is in marked contrast to the reported arterial smooth muscle–specific expression of a 536-bp minimal SM22&agr; (−475 to 61) promoter transgene. To begin to identify regulatory elements that are responsible for the distinct tissue-specific expression of these promoters, a chimeric promoter in which a 172-bp SM22&agr; gene fragment (−288 to −116) was fused to the minimal telokin promoter was generated and characterized. The −288 to −116 SM22&agr; gene fragment significantly increased telokin promoter activity in vascular smooth muscle cells in vitro and in vivo. Conversely, a fragment of the telokin promoter (−94 to −49) increased the activity of the SM22&agr; promoter in visceral smooth muscle cells of the bladder. Together, these data demonstrate that both vascular- and visceral smooth muscle–specific regulatory modules direct gene expression in subsets of smooth muscle tissues.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

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.