The Hippo signaling pathway regulates growth of the heart and other tissues. Hippo pathway kinases influence the activity of various targets, including the transcriptional coactivator Yap, but the specific role of Yap in heart growth has not been investigated. We show that Yap is necessary and sufficient for embryonic cardiac growth in mice. Deletion of Yap in the embryonic mouse heart impeded cardiomyocyte proliferation, causing myocardial hypoplasia and lethality at embryonic stage 10.5. Conversely, forced expression of a constitutively active form of Yap in the embryonic heart increased cardiomyocyte number and heart size. Yap activated the insulin-like growth factor (IGF) signaling pathway in cardiomyocytes, resulting in inactivation of glycogen synthase kinase 3β, which led to increased abundance of β-catenin, a positive regulator of cardiac growth. Our results point to Yap as a critical downstream effector of the Hippo pathway in the control of cardiomyocyte proliferation and a nexus for coupling the IGF, Wnt, and Hippo signaling pathways with the developmental program for heart growth.
Systemic loss-of-function studies have demonstrated that Pax3 transcription factor expression is essential for dorsal neural tube, early neural crest and muscle cell lineage morphogenesis. Cardiac neural crest cells participate in both remodeling of the pharyngeal arch arteries and outflow tract septation during heart development, but the lineage specific role of Pax3 in neural crest function has not yet been determined. To gain insight into the requirement of Pax3 within the neural crest, we conditionally deleted Pax3 in both the premigratory and migratory neural crest populations via Wnt1-Cre and Ap2α-Cre and via P0-Cre in only the migratory neural crest, and compared these phenotypes to the pulmonary atresia phenotype observed following the systemic loss of Pax3. Surprisingly, using Wnt1-Cre deletion there are no resultant heart defects despite the loss of Pax3 from the premigratory and migratory neural crest. In contrast, earlier premigratory and migratory Ap2α-Cre mediated deletion resulted in double outlet right ventricle alignment heart defects. In order to assess the tissue-specific contribution of neural crest to heart development, genetic ablation of neural crest lineage using a Wnt1-Cre-activated diphtheria toxin fragment-A cell-killing system was employed. Significantly, ablation of Wnt1-Cre-expressing neural crest cells resulted in fully penetrant persistent truncus arteriosus malformations. Combined, the data show that Pax3 is essential for early neural crest progenitor formation, but is not required for subsequent cardiac neural crest progeny morphogenesis involving their migration to the heart or septation of the outflow tract.
mouse embryo; Pax3; cardiac neural crest; congenital heart defects; lineage mapping; conditional knockout; genetic cell abaltion
Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.
Female mice lacking smooth muscle alpha-actin have a lactation defect that results from the inability of myoepithelial cells to generate sufficient contractile force in response to oxytocin to promote milk ejection.
cytoskeleton; lactation; mammary glands; milk ejection; myoepithelial cell; pregnancy; smooth muscle alpha-actin; transgenic/knockout model
Protein tyrosine phosphatase-like A (PTPLa) has been implicated in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. The molecular mechanisms of PTPLa in myogenesis are unknown. In this report, we demonstrate that PTPLa is required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes. The repressed MyoG expression was responsible for the impaired myoblast differentiation. Meanwhile, impeded cell growth, with an obvious S-phase arrest and compromised G2/M transition, was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes, along with a compromised G2/M transition due to the decreased CDK1 (cyclin-dependent kinase 1) activity and upregulated p21, contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of the PTPLa gene was explored. We identified PTPLa as a new target gene of the serum response factor (SRF). Skeletal- and cardiac-muscle-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of the PTPLa gene in mice.
Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth.
By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus.
In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.
Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.
The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. While emerging evidence shows that the fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF-signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2α, an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates critical aspects of FGF-dependent OFT development. Ablation of Frs2α in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2α mutants had defective endothelial-mesenchymal-transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. The results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.
receptor tyrosine kinase; cell signaling; heart development; second heart field; mouse model
Heart valves develop from precursor structures called cardiac cushions, an endothelial-lined cardiac jelly that resides in the inner side of the heart tube. The cushions are then invaded by cells from different sources, undergo a series of complicated and poorly understood remodeling processes, and give rise to valves. Disruption of the fibroblast growth factor (FGF) signaling axis impairs morphogenesis of the outflow tract (OFT). Yet, whether FGF signaling regulates OFT valve formation is unknown.
To study how OFT valve formation is regulated and how aberrant cell signaling causes valve defects.
Methods and results
By employing mouse genetic manipulation, cell lineage tracing, ex vivo heart culture, and molecular biology approaches, we demonstrated that FGF signaling in the OFT myocardium upregulated Bmp4 expression, which then enhanced smooth muscle differentiation of neural crest cells (NCCs) in the cushion. FGF signaling also promoted OFT myocardial cell invasion to the cushion. Disrupting FGF signaling interrupted cushion remodeling with reduced NCCs differentiation into smooth muscle and less cardiomyocyte invasion, and resulted in malformed OFT valves.
The results demonstrate a novel mechanism by which the FGF-BMP signaling axis regulates formation of OFT valve primordia by controlling smooth muscle differentiation of cushion NCCs.
FGF; BMP; heart development; NCC differentiation; cardiac valve defect
Serum response factor (SRF), a cardiac enriched transcription factor, is required for the appearance of beating sarcomeres in the heart. SRF may also direct the expression of microRNAs (miRs) that inhibit the expression of cardiac regulatory factors. The recent knockout of miR-1-2, an SRF gene target, showed defective heart development, caused in part by the induction of GATA6, Irx4/5, and Hand2, that may alter cardiac morphogenesis, channel activity and cell cycling. SRF and co-factors play an obligatory role in cardiogenesis, as major determinants of myocyte differentiation not only by regulating the biogenesis of muscle contractile proteins but also by driving the expression of silencer miRNA.
SUMO-specific protease 2 (SENP2) has a broad de-SUMOylation activity in vitro. However, the biological function of SENP2 is largely unknown. Here, we show that deletion of SENP2 gene in mouse causes defects in the embryonic heart and reduces the expression of Gata4 and Gata6, which are essential for cardiac development. SENP2 regulates transcription of Gata4 and Gata6 mainly through alteration of occupancy of Pc2/CBX4, a Polycomb Repressive Complex 1 (PRC1) subunit, on its promoters. We demonstrate that Pc2/CBX4 is a target of SENP2 in vivo and that SUMOylation is essential for Pc2/CBX4-mediated PRC1 recruitment to methylated histone 3 at K27 (H3K27me3). In SENP2 null embryo, SUMOylated Pc2/CBX4 accumulates and Pc2/CBX4 occupancy on the promoters of PcG target genes is markedly increased, leading to repression of Gata4 and Gata6 transcription. Our results reveal a critical role for de-SUMOylation in the regulation of PcG target gene expression through a novel mechanism.
Although the molecular pathways governing the development of the anterior pole of the heart have been the subject of intense investigation, little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart which generates the atria, pulmonary veins and portions of the atrio-ventricular canal. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm in a domain encompassing the dorsal mesocardium and dorsal mesenchymal protrusion which generates portions of the atria and atrio-ventricular cushions. Loss of Wnt2 results in defective development of the atrial myocardium, atrio-ventricular canal, and pulmonary veins resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The dorsal mesocardium and dorsal mesenchymal protrusion overlaps spatially with posterior second heart field progenitors and we show that the number and proliferation of these progenitors is reduced in Wnt2-/- mutants. Remarkably, these defects can be rescued in vivo in a temporally restricted manner through pharmacological inhibition of Gsk-3β, indicating that Wnt2 regulates canonical Wnt signaling in the posterior cardiac mesoderm. Molecular and genetic analysis shows that Wnt2 works in a feed-forward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal an important new molecular pathway regulating cardiac inflow tract development and demonstrates that such defects in the second heart field can be rescued pharmacologically in vivo.
Background & Aims
Expansion and patterning of the endoderm generate a highly ordered, multi-organ digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The NK homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development but its function in the mammalian gut is less clear.
We generated a Bapx1Cre knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development.
Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach, near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1Cre/Cre gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggest a hierarchy between these two factors; Bapx1 expression is lost in the absence of Barx1.
This study reveals the non-redundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.
Gastrointestinal development; stomach; mesoderm; endoderm; Nkx3-2; Bapx1; pyloric sphincter; Barx1; antrum
Highly proliferative, CD34+/CD45+ fibroblasts derived from monocytic, blood-borne precursor cells play a critical role in the development of fibrosis in a murine ischaemic/reperfusion cardiomyopathy (I/RC) model. The differentiation of human monocytes into fibroblasts in vitro occurs after transendothelial migration (TEM) induced by monocyte chemoattractant protein 1 (MCP-1). Because Rho-associated kinase-1 (ROCK-1) has been implicated in fibrosis and leukocyte TEM, we investigated its involvement in I/RC.
Methods and results
We subjected mice with genetic deletion of ROCK-1 to I/RC. We found that ROCK-1−/− mice did not develop the fibrosis and cardiac dysfunction characteristic for I/RC: compared with wild-type, ROCK-1−/− hearts showed markedly lower numbers of I/RC-induced α-smooth muscle actin+ fibroblasts and CD34+/CD45+ fibroblast precursors. Isolated cardiac fibroblasts from ROCK-1−/− mice undergoing I/RC were large and slowly proliferating, similar to fibroblasts isolated from sham-treated hearts. We also performed in vitro assays in which human peripheral blood mononuclear cells (PBMC) migrated through endothelial cells in response to MCP-1. Prior to migration, PBMC were incubated with ROCK-1-targeting small interfering RNA to silence ROCK-1 expression. We found that an 80% reduction of ROCK-1 protein did not inhibit TEM, but significantly reduced the amount of mononuclear cells that differentiated into fibroblasts by >20-fold.
Our data implicate an important role for ROCK-1 in the differentiation, but not in the TEM of monocytes that mature into cardiac fibroblasts. These cells mediate non-adaptive fibrosis.
Cardiac fibroblasts; Monocytes; Rho-associated kinase-1; Endothelial transmigration; Fibrosis
The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters α-myosin heavy chain (αMyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of αMyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin.
adipocytes; progenitor cells; Wnt signaling; desmosomes; heart failure
SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy
Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression, and thereby could be used to manipulate ES cell differentiation, has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133 promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1’s effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell fate decisions from pluripotent ES cells.
Expression of genes with tight and precise temporal and spatial control is desired in a wide variety of applications ranging from cultured cells and transgenic animals to gene therapy. While current inducible systems, such as RU486 and chemical inducers of dimerization (CID), have improved earlier inducible models (Gossen et al., 1995) (Wang et al., 1994), no single system is perfect at present. One potential drawback of these systems is leakage of transgene expression, causing limitations of each system. We have developed an inducible model containing both RU486 and CID systems, which in addition to inducing caspase activation, has potential applicability specifically to other genes encoding proteins that require a dimerization event for activation. This Double-Inducible Gene Activation System generates two barriers for the target gene expression and protein activation thereby minimizing leakage.
RU486; CID; keratin 14; inducible gene expression; double-inducible system
To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed.
Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1-3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis.
We have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage), heart tube looping (28-somite stage), and outflow track septation (38-somite stage). Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation.
The results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and motility as well as cellular redox status, which may contribute to cardiovascular abnormalities in mouse embryos lacking Folr1 gene activity.
Myocardin, a serum response factor (SRF)-dependent cofactor, is a potent activator of smooth muscle gene activity but a poor activator of cardiogenic genes in pluripotent 10T1/2 fibroblasts. Posttranslational modification of GATA4, another myocardin cofactor, by sumoylation strongly activated cardiogenic gene activity. Here, we found that myocardin's activity was strongly enhanced by SUMO-1 via modification of a lysine residue primarily located at position 445 and that the conversion of this residue to arginine (K445R) impaired myocardin transactivation. PIAS1 was involved in governing myocardin activity via its E3 ligase activity that stimulated myocardin sumoylation on an atypical sumoylation site(s) and by its physical association with myocardin. Myocardin initiated the expression of cardiac muscle-specified genes, such as those encoding cardiac α-actin and α-myosin heavy chain, in an SRF-dependent manner in 10T1/2 fibroblasts, but only in the presence of coexpressed SUMO-1/PIAS1. Thus, SUMO modification acted as a molecular switch to promote myocardin's role in cardiogenic gene expression.
Mutations in developmental regulatory genes have been found to be responsible for some cases of congenital heart defects. One such regulatory gene is Gata4, a zinc finger transcription factor. In order to circumvent the early embryonic lethality of Gata4-null embryos and to investigate the role of myocardial Gata4 expression in cardiac development, we used Cre/loxP technology to conditionally delete Gata4 in the myocardium of mice at an early and a late time point in cardiac morphogenesis. Early deletion of Gata4 by Nkx2-5Cre resulted in hearts with striking myocardial thinning, absence of mesenchymal cells within the endocardial cushions, and selective hypoplasia of the RV. RV hypoplasia was associated with downregulation of Hand2, a transcription factor previously shown to regulate formation of the RV. Cardiomyocyte proliferation was reduced, with a greater degree of reduction in the RV than in the LV. Late deletion of Gata4 by Cre recombinase driven by the α myosin heavy chain promoter did not selectively affect RV development or generation of endocardial cushion mesenchyme but did result in marked myocardial thinning with decreased cardiomyocyte proliferation, as well as double-outlet RV. Our results demonstrate a general role of myocardial Gata4 in regulating cardiomyocyte proliferation and a specific, stage-dependent role in regulating the morphogenesis of the RV and the atrioventricular canal.
Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal α-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.
Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFΔ5 to SRF. Increased levels of SRFΔ5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFΔ5 variant relative to wild-type SRF. SRFΔ5 forms DNA binding-competent homodimers and heterodimers. SRFΔ5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal α-actin, cardiac α-actin, smooth α-actin, SM22α, and SRF promoter-luciferase reporter activities. Expression of SRFΔ5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal α-actin and myogenin mRNAs. SRFΔ5 repressed the serum-induced activity of the c-fos serum response element. SRFΔ5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFΔ5, may play an important regulatory role in modulating SRF-dependent gene expression.
The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac α-actin (αCA) promoter in fibroblasts (C. Y. Chen and R. J. Schwartz, Mol. Cell. Biol. 16:6372–6384, 1996). We demonstrate here that Nkx-2.5 also cooperates with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the αCA promoter and a minimal promoter, containing only multimerized Nkx-2.5 DNA binding sites (NKEs), in heterologous CV-1 fibroblasts. Transcriptional activity requires the N-terminal activation domain of Nkx-2.5 and Nkx-2.5 binding activity through its homeodomain but does not require GATA-4’s activation domain. The minimal interactive regions were mapped to the homeodomain of Nkx-2.5 and the second zinc finger of GATA-4. Removal of Nkx-2.5’s C-terminal inhibitory domain stimulated robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitated Nkx-2.5 DNA binding activity. We postulate the following simple model: GATA-4 induces a conformational change in Nkx-2.5 that displaces the C-terminal inhibitory domain, thus eliciting transcriptional activation of promoters containing Nkx-2.5 DNA binding targets. Therefore, αCa promoter activity appears to be regulated through the combinatorial interactions of at least three cardiac tissue-enriched transcription factors, Nkx-2.5, GATA-4, and SRF.