Lynda Robitaille

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.

GATA-dependent recruitment of MEF2 proteins to target promoters

The myocyte enhancer factor‐2 (MEF2) proteins are MADS‐box transcription factors that are essential for differentiation of all muscle lineages but their mechanisms of action remain largely undefined. In mammals, the earliest site of MEF2 expression is the heart where the MEF2C isoform is detectable as early as embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers including atrial natriuretic factor (ANF). However, most of these promoters contain no or low affinity MEF2 binding sites and they are not significantly activated by any MEF2 proteins in heterologous cells suggesting a dependence on a cardiac‐enriched cofactor for MEF2 action. We provide evidence that MEF2 proteins are recruited to target promoters by the cell‐specific GATA transcription factors, and that MEF2 potentiates the transcriptional activity of this family of tissue‐restricted zinc finger proteins. Functional MEF2/GATA‐4 synergy involves physical interaction between the MEF2 DNA‐binding domain and the carboxy zinc finger of GATA‐4 and requires the activation domains of both proteins. However, neither MEF2 binding sites nor MEF2 DNA binding capacity are required for transcriptional synergy. The results unravel a novel pathway for transcriptional regulation by MEF2 and provide a molecular paradigm for elucidating the mechanisms of action of MEF2 in muscle and non‐muscle cells.

A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription.

In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A- and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP- and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.

Inhibition of transcription factor GATA-4 expression blocks in vitro cardiac muscle differentiation.

Commitment of mesodermal cells to the cardiac lineage is a very early event that occurs during gastrulation, and differentiation of cardiac muscle cells begins in the presomite stage prior to formation of the beating heart tube. However, the molecular events, including gene products that are required for differentiation of cardiac muscle cells, remain essentially unknown. GATA-4 is a recently characterized cardiac muscle-restricted transcription factor whose properties suggest an important regulatory role in heart development. We tested the role of GATA-4 in cardiac differentiation, using the pluripotent P19 embryonal carcinoma cells, which can be differentiated into beating cardiac muscle cells. In this system, GATA-4 transcripts and protein are restricted to cells committed to the cardiac lineage, and induction of GATA-4 precedes expression of cardiac marker genes and appearance of beating cells. Inhibition of GATA-4 expression by antisense transcripts blocks development of beating cardiac muscle cells and interferes with expression of cardiac muscle markers. These data indicate that GATA-4 is necessary for development of cardiac muscle cells and identify for the first time a tissue-specific transcription factor that may be crucial for early steps of mammalian cardiogenesis.

Developmental stage-specific regulation of atrial natriuretic factor gene transcription in cardiac cells.

Cardiac myocytes undergo a major genetic switch within the first week of postnatal development, when cell division ceases terminally and many cardiac genes are either activated or silenced. We have developed stage-specific cardiocyte cultures to analyze transcriptional control of the rat atrial natriuretic factor (ANF) gene to identify the mechanisms underlying tissue-specific and developmental regulation of this gene in the heart. The first 700 bp of ANF flanking sequences was sufficient for cardiac muscle- and stage-specific expression in both atrial and ventricular myocytes, and a cardiac muscle-specific enhancer was localized between -136 and -700 bp. Deletion of this enhancer markedly reduced promoter activity in cardiac myocytes and derepressed ANF promoter activity in nonexpressing cells. Two distinct domains of the enhancer appeared to contribute differentially to cardiac specificity depending on the differentiation stage of the myocytes. DNase I footprinting of the enhancer domain active in differentiated cells revealed four putative regulatory elements including an A+T-rich region and a CArG element. Deletion mutagenesis and promoter reconstitution assays revealed an important role for the CArG-containing element exclusively in cardiac cells, where its activity was switched on in differentiated myocytes. Transcriptional activity of the ANF-CArG box correlated with the presence of a cardiac- and stage-specific DNA-binding complex which was not recognized by the c-fos serum response element. Thus, the use of this in vitro model system representing stage-specific cardiac development unraveled the presence of different regulatory mechanisms for transcription of the ANF gene during cardiac differentiation and may be useful for studying the regulatory pathways of other genes that undergo switching during cardiac myogenesis.

Comparison of Risk Factors for Human Metapneumovirus and Respiratory Syncytial Virus Disease Severity in Young Children

Abstract Background. Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading pediatric pathogens. However, risk factors for severe hMPV disease remain unknown. We comparatively assessed environmental, host, and viral determinants for severe hMPV and RSV infections. Methods. We studied a prospective cohort of >1000 children aged <3 years hospitalized in or presenting to a pediatric clinic for acute respiratory infection. We collected clinical data at enrollment and 1-month follow-up and tested nasopharyngeal secretions for respiratory viruses. Disease severity was defined as hospitalization and was also assessed with a severity score (1 point/variable) calculated on the basis of fraction of inhaled O2 ≥ 30%, hospitalization >5 days, and pediatric intensive care unit admission. Results. hMPV was identified in 58 of 305 outpatient children (19.0%) and 69 of 734 hospitalized children (9.4%), second only to RSV (48.2% and 63.6%, respectively). In multivariate regression analysis of hMPV cases, age <6 months and household crowding were associated with hospitalization. Among hospitalized patients, risk factors for severe hMPV disease were female sex, prematurity, and genotype B infection. Age <6 months, comorbidities, and household crowding were risk factors for RSV hospitalization; breast-feeding and viral coinfection were protective. Age <6 months and prematurity were associated with severe RSV cases among hospitalized children. Conclusions. hMPV and RSV severity risk factors may differ slightly. These findings will inform hMPV prevention strategies.

Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.

fos/jun repression of cardiac-specific transcription in quiescent and growth-stimulated myocytes is targeted at a tissue-specific cis element.

Unlike that of skeletal muscle cells in which growth and differentiation appear mutually exclusive, growth stimulation of cardiac cells is characterized by transient expression of early response nuclear proto-oncogenes as well as induction of several cardiac-specific markers. This observation led to the speculation that these proto-oncogenes, particularly c-fos and c-jun, might act as positive regulators of cardiac transcription. We have examined the role of c-jun and c-fos in basal and growth-stimulated cardiac transcription, using the cardiac-specific atrial natriuretic factor (ANF) gene as a marker. The results indicate that c-jun and c-fos are negative regulators of ANF transcription. Inducers of jun and fos activity, such as mitogens and growth factors, inhibited endogenous ANF transcripts. In transient cotransfection assays, jun and fos were able to trans-repress the ANF promoter in both quiescent and alpha 1-adrenergic stimulated myocytes. This repression was specific to myocyte cultures and was not observed in nonmuscle cells. Deletion analysis indicated that repression does not require typical AP-1-binding sites (tetradecanoyl phorbol acetate response elements) or serum response elements but is targeted at a cardiac-specific element within the ANF promoter. Various Fos-related proteins, including Fra-1, Fos B, and v-Fos, were able to trans-repress ANF transcription. In addition, C-terminal c-fos mutants which no longer repress transcription of such early growth response genes as c-fos and EGR-1 retained the ability to repress ANF transcription. Repression by c-jun occurs via the N-terminal activation domain and does not require the DNA-binding domain, suggesting that proto-oncogene repression involves interaction with one or more limiting cardiac-specific coactivators.

The transcription factor Egr1 regulates the HIF-1α gene during hypoxia

Using oligonucleotide expression microarrays we have examined the modulation of gene expression in the DU145 prostate cancer cell line. Our findings confirm that the Egr1 transcription factor is rapidly and transiently upregulated by hypoxia. Furthermore, we have demonstrated that HIF‐1α mRNA is also transiently upregulated, as is its target gene VEGF. To elucidate the mechanism of the transcriptional upregulation of the HIF‐1α gene, we have shown that Egr1 is able to directly bind to the HIF‐1α promoter using chromatin immunoprecipitation. We also provide evidence that the binding of Egr1 is necessary for the trans‐activation of the HIF‐1α promoter. These studies highlight the importance for the Egr1 transcription factor in the hypoxic response in cultured prostate cancer cell lines, and indicate that the response of Egr1 is upstream of HIF‐1 in these cells. These studies are the first demonstration that the HIF‐1α transcription factor is targeted directly by Egr1 in hypoxia.

TRAF6 and IRF7 Control HIV Replication in Macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.