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Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo  [PDF]
Chaitali Misra,Nita Sachan,Caryn Rothrock McNally,Sara N. Koenig,Haley A. Nichols,Anuradha Guggilam,Pamela A. Lucchesi,William T. Pu,Deepak Srivastava,Vidu Garg
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002690
Abstract: Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.
NKX2-5 Regulates the Expression of β-Catenin and GATA4 in Ventricular Myocytes  [PDF]
Ali M. Riazi, Jun K. Takeuchi, Lisa K. Hornberger, Syed Hassan Zaidi, Fariba Amini, John Coles, Benoit G. Bruneau, Glen S. Van Arsdell
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005698
Abstract: Background The molecular pathway that controls cardiogenesis is temporally and spatially regulated by master transcriptional regulators such as NKX2-5, Isl1, MEF2C, GATA4, and β-catenin. The interplay between these factors and their downstream targets are not completely understood. Here, we studied regulation of β-catenin and GATA4 by NKX2-5 in human fetal cardiac myocytes. Methodology/Principal Findings Using antisense inhibition we disrupted the expression of NKX2-5 and studied changes in expression of cardiac-associated genes. Down-regulation of NKX2-5 resulted in increased β-catenin while GATA4 was decreased. We demonstrated that this regulation was conferred by binding of NKX2-5 to specific elements (NKEs) in the promoter region of the β-catenin and GATA4 genes. Using promoter-luciferase reporter assay combined with mutational analysis of the NKEs we demonstrated that the identified NKX2-5 binding sites were essential for the suppression of β-catenin, and upregulation of GATA4 by NKX2-5. Conclusions This study suggests that NKX2-5 modulates the β-catenin and GATA4 transcriptional activities in developing human cardiac myocytes.
DNA Methylation Restricts Lineage-specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation  [PDF]
Masaaki Oda equal contributor,Yuichi Kumaki equal contributor,Masaki Shigeta equal contributor,Lars Martin Jakt,Chisa Matsuoka,Akiko Yamagiwa,Hitoshi Niwa,Masaki Okano
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003574
Abstract: DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.
Conserved Usage of Alternative 5′ Untranslated Exons of the GATA4 Gene  [PDF]
Séverine Mazaud Guittot,Marie France Bouchard,Jean-Philippe Robert-Grenon,Claude Robert,Cynthia G. Goodyer,David W. Silversides,Robert S. Viger
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008454
Abstract: GATA4 is an essential transcription factor required for the development and function of multiple organs. Despite this important role, our knowledge of how the GATA4 gene is regulated remains limited. To better understand this regulation, we characterized the 5′ region of the mouse, rat, and human GATA4 genes.
PIAS1 Is a GATA4 SUMO Ligase That Regulates GATA4-Dependent Intestinal Promoters Independent of SUMO Ligase Activity and GATA4 Sumoylation  [PDF]
Narasimhaswamy S. Belaguli, Mao Zhang, Andres-Hernandez Garcia, David H. Berger
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035717
Abstract: GATA4 confers cell type-specific gene expression on genes expressed in cardiovascular, gastro-intestinal, endocrine and neuronal tissues by interacting with various ubiquitous and cell-type-restricted transcriptional regulators. By using yeast two-hybrid screening approach, we have identified PIAS1 as an intestine-expressed GATA4 interacting protein. The physical interaction between GATA4 and PIAS1 was confirmed in mammalian cells by coimmunoprecipitation and two-hybrid analysis. The interacting domains were mapped to the second zinc finger and the adjacent C-terminal basic region of GATA4 and the RING finger and the adjoining C-terminal 60 amino acids of PIAS1. PIAS1 and GATA4 synergistically activated IFABP and SI promoters but not LPH promoters suggesting that PIAS1 differentially activates GATA4 targeted promoters. In primary murine enterocytes PIAS1 was recruited to the GATA4-regulated IFABP promoter. PIAS1 promoted SUMO-1 modification of GATA4 on lysine 366. However, sumoylation was not required for the nuclear localization and stability of GATA4. Further, neither GATA4 sumoylation nor the SUMO ligase activity of PIAS1 was required for coactivation of IFABP promoter by GATA4 and PIAS1. Together, our results demonstrate that PIAS1 is a SUMO ligase for GATA4 that differentially regulates GATA4 transcriptional activity independent of SUMO ligase activity and GATA4 sumoylation.
An Ebox Element in the Proximal Gata4 Promoter Is Required for Gata4 Expression In Vivo  [PDF]
Alain Boulende Sab, Marie-France Bouchard, Mélanie Béland, Bruno Prud'homme, Ouliana Souchkova, Robert S. Viger, Nicolas Pilon
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029038
Abstract: GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5′ origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5′ flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult) revealed that the Ebox mutation leads to a robust and specific decrease (up to 89%) of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.
Identification of Functional Mutations in GATA4 in Patients with Congenital Heart Disease  [PDF]
Erli Wang, Shuna Sun, Bin Qiao, Wenyuan Duan, Guoying Huang, Yu An, Shuhua Xu, Yufang Zheng, Zhixi Su, Xun Gu, Li Jin, Hongyan Wang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062138
Abstract: Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.
The db/db Mouse: A Useful Model for the Study of Diabetic Retinal Neurodegeneration  [PDF]
Patricia Bogdanov, Lidia Corraliza, Josep A. Villena, Andrea R. Carvalho, José Garcia-Arumí, David Ramos, Jesús Ruberte, Rafael Simó, Cristina Hernández
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097302
Abstract: Background To characterize the sequential events that are taking place in retinal neurodegeneration in a murine model of spontaneous type 2 diabetes (db/db mouse). Methods C57BLKsJ-db/db mice were used as spontaneous type 2 diabetic animal model, and C57BLKsJ-db/+ mice served as the control group. To assess the chronological sequence of the abnormalities the analysis was performed at different ages (8, 16 and 24 weeks). The retinas were evaluated in terms of morphological and functional abnormalities [electroretinography (ERG)]. Histological markers of neurodegeneration (glial activation and apoptosis) were evaluated by immunohistochemistry. In addition glutamate levels and glutamate/aspartate transporter (GLAST) expression were assessed. Furthermore, to define gene expression changes associated with early diabetic retinopathy a transcriptome analyses was performed at 8 week. Furthermore, an additional interventional study to lower blood glucose levels was performed. Results Glial activation was higher in diabetic than in non diabetic mice in all the stages (p<0.01). In addition, a progressive loss of ganglion cells and a significant reduction of neuroretinal thickness were also observed in diabetic mice. All these histological hallmarks of neurodegeneration were less pronounced at week 8 than at week 16 and 24. Significant ERG abnormalities were present in diabetic mice at weeks 16 and 24 but not at week 8. Moreover, we observed a progressive accumulation of glutamate in diabetic mice associated with an early downregulation of GLAST. Morphological and ERG abnormalities were abrogated by lowering blood glucose levels. Finally, a dysregulation of several genes related to neurotransmission and oxidative stress such as UCP2 were found at week 8. Conclusions Our results suggest that db/db mouse reproduce the features of the neurodegenerative process that occurs in the human diabetic eye. Therefore, it seems an appropriate model for investigating the underlying mechanisms of diabetes-induced retinal neurodegeneration and for testing neuroprotective drugs.
GATA4 and DcR1 methylation in glioblastomas
Paulina Vaitkiene, Daina Skiriute, Kestutis Skauminas, Arimantas Tamasauskas
Diagnostic Pathology , 2013, DOI: 10.1186/1746-1596-8-7
Abstract: Methylation status of GATA4 and DcR1 promoters was investigated by methylation specific PCR in 99 glioblastoma patients. Statistical analyses were conducted to investigate the association between clinical variables and overall survival time.GATA4 and DcR1 were aberrantly methylated in 23.2% and 27.6% of glioblastoma tumors, but not in normal brain. GATA4 promoter hypermethylation showed significant association with patients age (p = 0.027). Relationship between genes promoter methylation and glioblastoma patient survival was not determined.The present work demonstrated that GATA4 and DcR1 promoter hypermethylation is tumor specific event in glioblastoma but they promoter methylation cannot be considered as a prognostic marker of glioblastoma survival. Virtual slides http://www.diagnosticpathology.diagnomx.eu/vs/1381170351801852
Molecular Model of the Contractile Ring  [PDF]
D. Biron,E. Alvarez-Lacalle,Tsvi Tlusty,Elisha Moses
Quantitative Biology , 2010, DOI: 10.1103/PhysRevLett.95.098102
Abstract: We present a model for the actin contractile ring of adherent animal cells. The model suggests that the actin concentration within the ring and consequently the power that the ring exerts both increase during contraction. We demonstrate the crucial role of actin polymerization and depolymerization throughout cytokinesis, and the dominance of viscous dissipation in the dynamics. The physical origin of two phases in cytokinesis dynamics ("biphasic cytokinesis") follows from a limitation on the actin density. The model is consistent with a wide range of measurements of the midzone of dividing animal cells.
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