Georges Nemer

A Murine Model of Holt-Oram Syndrome Defines Roles of the T-Box Transcription Factor Tbx5 in Cardiogenesis and Disease

Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.

Cooperative Interaction between GATA-4 and GATA-6 Regulates Myocardial Gene Expression

ABSTRACT Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the α- and β-myosin heavy-chain (α- and β-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes.

Transcriptional activation of BMP-4 and regulation of mammalian organogenesis by GATA-4 and -6

Transcription factors GATA-4, -5, and -6 constitute an evolutionary conserved subfamily of vertebrate zinc finger regulators highly expressed in the developing heart and gut. Genetic evidence suggests that each protein is essential for embryonic development, but their exact functions are not fully elucidated. Moreover, because all three proteins share similar transcriptional properties in vitro, and because transcripts for two or more GATA genes are present in similar tissues, the molecular basis underlying in vivo specificity of GATA factors remains undefined. Knowledge of the exact cell types expressing each protein and identification of downstream targets would greatly help define their function. We have used high-resolution immunohistochemistry to precisely determine the cellular distribution of the GATA-4, -5, and -6 proteins in murine embryogenesis. The results reveal novel sites of expression in mesodermal and ectodermal cells. In particular, GATA-4 and -6 expression was closely associated with yolk sac vasculogenesis and early endoderm-mesoderm signaling. Additionally, GATA-6 was strongly expressed in the embryonic ectoderm, neural tube, and neural crest-derived cells. This pattern of expression closely paralled that of BMP-4, and the BMP-4 gene was identified as a direct downstream target for GATA-4 and -6. These findings offer new insight into the function of GATA-4 and -6 during early stages of embryogenesis and reveal the existence of a positive cross-regulatory loop between BMP-4 and GATA-4. They also raise the possibility that part of the early defects in GATA-4 and/or GATA-6 null embryos may be due to impaired BMP-4 signaling.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

Background Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations. Methods The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation. Results We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased. Conclusions We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.

Regulation of heart development and function through combinatorial interactions of transcription factors.

Understanding the molecular mechanisms controlling cardiac-specific gene transcription requires the dissection of the cis-elements that govern the complex spatio-temporal expression of these genes. The four-chambered vertebrate heart is formed during the late phases of fetal development following a series of complex morphogenetic events that require the functional presence of different proteins. The gradient-like expression of some genes, as well as the chamber-specific expression of others, is tightly regulated by combinatorial interactions of several transcription factors and their cofactors. Chamber- and stage-specific cardiac myocyte cultures have been invaluable for identifying transcription factor binding sites involved in basal, chamber-specific, and inducible expression of many cardiac promoters: these studies, which were largely confirmed in vivo in transgenic mouse models, led to the isolation of key regulators of heart development. In addition, the use of pluripotent embryonic stem cells helped elucidate the early molecular events controlling cardiomyocyte differentiation. Together, these studies point to a major role for GATA transcription factors and their interacting partners in transcriptional control of heart development. In addition, members of the T-box family of transcription factors and homeodomain containing proteins, together with chamber-restricted transcriptional repressors and co-repressors play critical roles in heart septation and chamber specification. These fine-tuned cooperative interactions between different classes of proteins are at the basis of normal cardiac function, and alteration in their expression level or function leads to cardiac pathologies.

The Kruppel-like transcription factor KLF13 is a novel regulator of heart development.

In humans, congenital heart defects occur in 1–2% of live birth, but the molecular mechanisms and causative genes remain unidentified in the majority of cases. We have uncovered a novel transcription pathway important for heart morphogenesis. We report that KLF13, a member of the Krüppel‐like family of zinc‐finger proteins, is expressed predominantly in the heart, binds evolutionarily conserved regulatory elements on cardiac promoters and activates cardiac transcription. KLF13 is conserved across species and knockdown of KLF13 in Xenopus embryos leads to atrial septal defects and hypotrabeculation similar to those observed in humans or mice with hypomorphic GATA‐4 alleles. Physical and functional interaction with GATA‐4, a dosage‐sensitive cardiac regulator, provides a mechanistic explanation for KLF13 action in the heart. The data demonstrate that KLF13 is an important component of the transcription network required for heart development and suggest that KLF13 is a GATA‐4 modifier; by analogy to other GATA‐4 collaborators, mutations in KLF13 may be causative for congenital human heart disease.

Familial Hypercholesterolemia: The Lipids or the Genes?

Familial Hypercholesterolemia (FH) is a common cause of premature cardiovascular disease and is often undiagnosed in young people. Although the disease is diagnosed clinically by high LDL cholesterol levels and family history, to date there are no single internationally accepted criteria for the diagnosis of FH. Several genes have been shown to be involved in FH; yet determining the implications of the different mutations on the phenotype remains a hard task. The polygenetic nature of FH is being enhanced by the discovery of new genes that serve as modifiers. Nevertheless, the picture is still unclear and many unknown genes contributing to the phenotype are most likely involved. Because of this evolving polygenetic nature, the diagnosis of FH by genetic testing is hampered by its cost and effectiveness.In this review, we reconsider the clinical versus genetic nomenclature of FH in the literature. After we describe each of the genetic causes of FH, we summarize the known correlation with phenotypic measures so far for each genetic defect. We then discuss studies from different populations on the genetic and clinical diagnoses of FH to draw helpful conclusions on cost-effectiveness and suggestions for diagnosis.

GATA4 loss-of-function mutations underlie familial tetralogy of fallot.

Tetralogy of Fallot (TOF) represents the most common form of cyanotic congenital heart disease and accounts for significant morbidity and mortality in humans. Emerging evidence has implicated genetic defects in the pathogenesis of TOF. However, TOF is genetically heterogeneous and the genetic basis for TOF in most patients remains unclear. In this study, the GATA4 gene were sequenced in 52 probands with familial TOF, and three novel heterozygous mutations, including A9P and L51V both located in the putative first transactivational domain and N285S in the C‐terminal zinc finger, were identified in three probands, respectively. Genetic analysis of the pedigrees demonstrated that in each family the mutation cosegregated with TOF with complete penetrance. The missense mutations were absent in 800 control chromosomes and the altered amino acids were highly conserved evolutionarily. Functional analysis showed that the GATA4 mutants were consistently associated with diminished DNA‐binding affinity and decreased transcriptional activity. Furthermore, the N285S mutation completely disrupted the physical interaction between GATA4 and TBX5. To our knowledge, this report associates GATA4 loss‐of‐function mutations with familial TOF for the first time, providing novel insight into the molecular mechanism involved in TOF and suggesting potential implications for the early prophylaxis and allele‐specific therapy of TOF.

Modulation of COX-2 expression by statins in human monocytic cells

Macrophage cyclooxygenase‐2 (COX‐2) plays an important role in prostaglandin E2 and throm‐boxane A2 production. Statins are inhibitors of HMG CoA (3‐Hydroxy‐3‐methylgrutaryl coenzyme A) reduc‐tases and cholesterol synthesis, which block the expression of several inflammatory proteins independent of their capacity to lower endogenous cholesterol. In the present study, we investigated the effect of simvastatin and mevastatin on COX‐2 induction in human mono‐cytic cell line U937 and analyzed the underlying mechanisms. Pretreatment of U937 cells with simvastatin or mevastatin for 24 h resulted in a significant reduction in the lipopolysaccharide (LPS)‐dependent induction of prostaglandin E2, thromboxane A2 synthesis, and COX‐2 expression. Mevalonate, the direct metabolite of HMG CoA reductase, and farnesyl pyrophosphate and geranylgeranyl‐pyrophosphate, intermediates of the mevalonate pathway, significantly reversed the inhibitory effect of statins on COX‐2. An inhibitor of geranylgeranyl transferases, GGTI‐286 mimicked the effect of statins on COX‐2 expression. Cytonecrotic factor‐1 increased LPS‐dependent expression of COX‐2. Treatment of cells with NSC 23766, an inhibitor of Rac, which we demonstrated to block Rac 2 activation, resulted in an inhibition of the LPS‐depen‐dent expression of COX‐2. Whereas no effect was obtained with RhoA/C blocker, C3 exoenzyme. Gel retardation experiments and NF‐κB‐p65 transcription factor assay showed that simvastatin and NSC 23766 decrease significantly NF‐κB complex formation. In macrophages, the antiinflammatory effects of statins are mediated in part through the inhibition of COX‐2 and prostanoids. Rac GTPase protein is identified as one of the targets of statins in this regulation.—Habib A., Shamseddeen, I., Nasrallah, M. S., Antoun, T. A., Nemer, G., Bertoglio J., Badreddine R., Badr K. F. Modulation of COX‐2 expression by statins in human monocytic cells. FASEB J. 21, 1665–1674 (2007)

Homozygous familial hypercholesterolemia in Lebanon: A genotype/phenotype correlation

Familial hypercholesterolemia (FH) is an inherited disease characterized by the deposition of LDL in tissues causing premature atherosclerosis. Many genes are implicated in FH resulting in a large variability in the phenotype. DNA sequencing of the LDLR gene was done for forty patients clinically diagnosed with homozygous FH and forty family members variably affected. Patients underwent noninvasive heart and vascular studies. Statistical and pedigree analyses were used to correlate the different genotypes with the phenotypes. The prevalence of homozygosity at the Lebanese allele (2043C>A) is 45%. However, 27.5% of the patients have no mutations at all in the LDLR gene, and 27.5% are either heterozygous for the 2043C>A mutation, heterozygous for a mutation in another exon of the LDLR gene, or combined heterozygous for two different mutations. We confirm previous reports on the higher prevalence of FH in Lebanon. Our results do, however contradict previous reports on an assumed higher prevalence among the Christian Lebanese. Mutations in the LDLR especially combined heterozygosity can cause a severe phenotype similar to the homozygous mutation in the Lebanese allele. This information is particularly important in targeting the more prevalent heterozygotes in the general population with early diagnosis and intervention.