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GATA-4 and heart morphogenesis (part 2/2)

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. Evidence is provided 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 (Morin, 2000).

Vitamin A is essential for normal embryonic cardiogenesis. The vitamin A-deficient phenotype in the avian embryo includes an abnormal heart tube closed at the sinus venosus and the absence of large vessels that normally connect the embryonic heart to the developing circulatory system. In vitamin A-deficient embryos the expression of cardiomyocyte differentiation genes, including atrial-specific myosin heavy chain, ventricular-specific myosin, and sarcomeric myosins as well as the putative cardiomyocyte specification gene Nkx-2.5, is not altered. However, the expression of transcription factor GATA-4 is severely decreased in the heart-forming regions of vitamin A-deficient stage 7-10 embryos. Significantly, GATA-4 transcripts are completely lacking in the lateral mesoderm, posterior to the heart, in the area of the developing cardiac inflow tract that later displays prominent morphological defects, including a closed nonseptated heart lacking a sinus venosus. The administration of retinol to the vitamin A-deficient embryo restores GATA-4 expression and completely rescues the vitamin A-deficient phenotype. These results indicate that GATA-4 is a component of the retinoid-mediated cardiogenic pathway unlinked to cardiomyocyte differentiation, but involved in the morphogenesis of the posterior heart tube and the development of the cardiac inflow tract (Kostetskii, 1999).

The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac alpha-actin (alphaCA) promoter in fibroblasts. Nkx-2.5 is shown to also cooperate with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the alphaCA promoter. A similar cooperation also takes place on 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 stimulates robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitates Nkx-2.5 DNA binding activity. The following simple model is proposed: 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, alphaCa 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 (Sepulveda, 1998).

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, a test was performed to see whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways (Lee, 1998).

In vertebrates, heart development is a multistep process that starts with formation and patterning of the primitive heart tube and is followed by complex morphological events to give rise to the mature four-chambered heart. These various stages are characterized by distinct patterns of gene expression. Although chamber specificity and developmental regulation can be demonstrated in transgenic mice using short promoter fragments, the mechanism underlying spatial and temporal specificity within the heart remains largely unclear. Combinatorial interaction between a limited number of cardiac-specific and ubiquitous transcription factors may account for the diverse genetic inputs required to generate the complex transcriptional patterns that characterize the developing myocardium. The cardiac atrial natriuretic peptide (ANP) promoter was used to test this hypothesis. The ANP gene is transcribed in a spatial- and temporal-specific manner in the heart, and a 500 bp promoter fragment is sufficient to recapitulate both chamber and developmental specificity. This promoter is composed of three modules: a "basal" cardiac promoter that is essential for transcription in embryonic and postnatal atrial and ventricular myocytes, and two other independent modules that behave as chamber-specific enhancers. The basal cardiac promoter is the target of two cardiac-specific transcription factors, the zinc finger GATA-4 protein and the Nkx2-5 homeodomain, which bind to contiguous elements within this region. At low concentrations--a situation that likely occurs during the very first stages of cardiac cell fate determination--the two proteins synergistically activate transcription from the ANP promoter. This functional synergy requires physical interaction between the GATA-4 protein and an extended C-terminal homeodomain on Nkx2-5. This interaction, which unmasks an activation domain present just N-terminal of the homeodomain, is specific for GATA-4 and-5, but is not observed with the other cardiac GATA factor, GATA-6. Optimal synergy requires binding of both proteins to their cognate sites, although modest synergy also could be observed on heterologous promoters containing only multimerized Nkx binding sites, suggesting that Nkx2-5 is able to recruit GATA-4 into a transcriptionally active complex. The GATA/Nkx interaction, which appears to have been evolutionarily conserved in nematode, fly, and mammals, provides a paradigm for analyzing transcription factor interaction during organogenesis (Durocher, 1998).

In many vertebrates, removal of early embryonic heart precursors can be repaired, leaving the heart and embryo without visible deficit. One possibility is that this 'regulation' involves a cell fate switch whereby cells, perhaps in regions surrounding normal progenitors, are redirected to the heart cell fate. However, the lineage and spatial relationships between cells that are normal heart progenitors and those that can assume that role after injury are not known, nor are their molecular distinctions. A laser-activated technique was adapted to label single or small patches of cells in the lateral plate mesoderm of the zebrafish and to track their subsequent lineage. The heart precursor cells are found clustered in a region adjacent to the prechordal plate, just anterior to the notochord tip. Complete unilateral ablation of all heart precursors with a laser does not disrupt heart development, if performed before the 18-somite stage. By combining extirpation of the heart precursors with cell labeling, it is found that cells anterior to the normal cardiogenic compartments constitute the source of regulatory cells that compensate for the loss of the progenitors. One of the earliest embryonic markers of the premyocardial cells is the divergent homeodomain gene, Nkx2.5. Interestingly, normal cardiogenic progenitors derive from only the anterior half of the Nkx2.5-expressing region in the lateral plate mesoderm. The posterior half, adjacent to the notochord, does not include cardiac progenitors and the posterior Nkx2.5-expressing cells do not contribute to the heart, even after ablation of the normal cardiogenic region. The cells that can acquire a cardiac cell fate after injury to the normal progenitors also reside near the prechordal plate, but anterior to the Nkx2.5-expressing domain. These cells express Nkx2.7. The overlap between GATA4 and Nkx2.7 expressing cells may explain why Nkx2.5 mutation in mice does not prevent assembly of a heart tube. Normally these anterior cells give rise to head mesenchyme. In common with cardiac progenitors, they share early expression of GATA 4. The location of the different elements of the cardiac field, and their response to injury, suggests that the prechordal plate supports and/or the notochord suppresses the cardiac fate (Serbedzija, 1998).

The slow myosin heavy chain (MyHC) 3 gene was used to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter (a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif) control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity are found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increases in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form (Wang, 1998).

nkx-2.5 is one of the first genes expressed in the developing heart of early stage vertebrate embryos. Cardiac expression of nkx-2.5 is maintained throughout development and the gene is also expressed in the developing pharyngeal arches, spleen, thyroid and tongue. Genomic sequences flanking the mouse nkx-2.5 gene were analyzed for early developmental regulatory activity in transgenic mice. Approximately 3 kb of 5' flanking sequence is sufficient to activate gene expression in the cardiac crescent as early as E7.25 and in limited regions of the developing heart at later stages. Expression also is detected in the developing spleen anlage at least 24 hours before the earliest reported spleen marker and in the pharyngeal pouches and their derivatives including the thyroid. The observed expression pattern from the -3 kb construct represents a subset of the endogenous nkx-2.5 expression pattern, which is evidence for compartment-specific nkx-2.5 regulatory modules. A 505 bp regulatory element has been identified that contains multiple GATA, NKE, bHLH, HMG and HOX consensus binding sites. This element is sufficient for gene activation in the cardiac crescent and in the heart outflow tract, pharynx and spleen when linked directly to lacZ or when positioned adjacent to the hsp68 promoter. Mutation of paired GATA sites within this element eliminates gene activation in the heart, pharynx and spleen primordia of transgenic embryos. The dependence of this nkx-2.5 regulatory element on GATA sites for gene activity is evidence for a GATA-dependent regulatory mechanism controlling nkx-2.5 gene expression. The presence of consensus binding sites for other developmentally important regulatory factors within the 505 bp distal element suggests that combinatorial interactions between multiple regulatory factors are responsible for the initial activation of nkx-2.5 in the cardiac, thyroid and spleen primordia (Searcy, 1998).

The homeobox gene Nkx2-5 is the earliest known marker of the cardiac lineage in vertebrate embryos. Nkx2-5 expression is first detected in mesodermal cells specified to form heart at embryonic day 7.5 in the mouse and expression is maintained throughout the developing and adult heart. In addition to the heart, Nkx2-5 is transiently expressed in the developing pharynx, thyroid and stomach. To investigate the mechanisms that initiate cardiac transcription during embryogenesis, the Nkx2-5 upstream region was analyzed for regulatory elements sufficient to direct expression of a lacZ transgene in the developing heart of transgenic mice. A cardiac enhancer is described. Located about 9 kilobases upstream of the Nkx2-5 gene, the enhancer fully recapitulates the expression pattern of the endogenous gene in cardiogenic precursor cells from the onset of cardiac lineage specification and throughout the linear and looping heart tube. Thereafter, as the atrial and ventricular chambers become demarcated, enhancer activity becomes restricted to the developing right ventricle. Transcription of Nkx2-5 in pharynx, thyroid and stomach is controlled by regulatory elements separable from the cardiac enhancer. This distal cardiac enhancer contains a high-affinity binding site for the cardiac-restricted zinc finger transcription factor GATA4. GATA4 is essential for transcriptional activity. These results reveal a novel GATA-dependent mechanism for activation of Nkx2-5 transcription in the developing heart and indicate that regulation of Nkx2-5 is controlled in a modular manner, with multiple regulatory regions responding to distinct transcriptional networks in different compartments of the developing heart. At least two other regulatory regions that direct Nkx2-5 expression in the developing heart have also been identified and negative regulatory elements have been identified as well (Lien, 1999).

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', the T-box-containing transcription factor Tbx5 was isolated; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells shows that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicate that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 are necessary for their interaction. Tbx5 and Nkx2-5 directly binds to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors show synergistic activation. Deletion analysis shows that both the N-terminal domain and T-box of Tbx5 are important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, does not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activates the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 start to beat earlier and express cardiac-specific genes more abundantly than do parental P19CL6 cells, whereas cell lines expressing the G80R mutant do not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development (Hiroi, 2001).

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Isolated cardiac septal defects in a large pedigree were shown to be linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, a frame-shift mutation of GATA4 (E359del) was identified that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5 (Garg, 2003).

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. Tbx20 expression has been analyzed during murine cardiac development, and DNA-binding and transcriptional properties of Tbx20 isoforms have been assessed. Tbx20 is expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carries strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bind specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacts with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, leads to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behavior in an in vivo context. It is proposed that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development (Stennard, 2003).

BMP signals act in concert with FGF8, WNT11 and WNT antagonists to induce the formation of cardiac tissue in the vertebrate embryo. In an effort to understand how these signaling pathways control the expression of key cardiac regulators, the cis-regulatory elements of the chick tinman homolog chick Nkx2.5 have been characterized. At least three distinct cardiac activating regions (CARs) of chick Nkx2.5 cooperate to regulate early expression in the cardiac crescent and later segmental expression in the developing heart. In this report, attention was focused on a 3' BMP-responsive enhancer, termed CAR3, which directs robust cardiac transgene expression. By systematic mutagenesis and gel shift analysis of this enhancer, it has been demonstrated that GATA4/5/6, YY1 and SMAD1/4 are all necessary for BMP-mediated induction and heart-specific expression of CAR3. Adjacent YY1 and SMAD-binding sites within CAR3 constitute a minimal BMP response element, and interaction of SMAD1/4 with the N terminus of YY1 is required for BMP-mediated induction of CAR3. These data suggest that BMP-mediated activation of this regulatory region reflects both the induction of GATA genes by BMP signals, as well as modulation of the transcriptional activity of YY1 by direct interaction of this transcription factor with BMP-activated SMADs (Lee, 2004).

How might the interaction of SMADs with YY1 modulate the activity of this transcription factor when bound to CAR3? Because YY1 can function as either a transcriptional activator or repressor, SMAD association with YY1 may serve to recruit co-activators that modulate the activity of this transcription factor to become an efficient transcriptional activator. Indeed, recruitment of co-activators such as p300 by TGFß activated SMADs is a well-characterized mechanism for SMAD target gene activation. Similarly, known interacting partners of YY1 also include several members of the histone deacetylase family as well as a histone H4 methylase, which have been implicated in either transcriptional repression or activation of YY1 regulated target genes, respectively. It will be interesting to determine if SMAD association with YY1 alters the interaction of this transcription factor with either of these families of histone modifying enzymes, and to what extent chromatin modification is responsible for appropriate regulation of Nkx2.5 (Lee, 2004).

SMAD-mediated modulation of YY1 activity adds an interesting new facet to the repertoire of functions of YY1 during heart development, which also includes direct recruitment of transcriptional co-activators to promote the expression of cardiac B-type natriuretic peptide, inhibition of the expression of the cardiac {alpha}-actin gene, and both activation and inhibition of the expression of the cardiac-specific Mlc2 gene. Clearly, the context within which YY1 functions is of great importance, and it is likely that transcription factors such as GATA and SMAD proteins, when bound to neighboring cognate binding sites, modulate either the association of co-factors with adjacently bound YY1 or the activity of such co-factors. In addition to the GATA, YY1- and SMAD-binding sites, linker scanning mutational analysis of the chick Nkx2.5 CAR3 BMPRE has revealed other sites yet to be characterized that also have a significant impact on the BMP response of this regulatory element. A complete understanding of complex enhancers such as Nkx2.5 CAR3 will require not only the identification of the transcription factors that regulate their expression but also elucidation of the transcriptional co-factors that are recruited to such regulatory elements in a combinatorial fashion (Lee, 2004).

The vertebrate heart forms initially as a linear tube derived from a primary heart field in the lateral mesoderm. Recent studies in mouse and chick have demonstrated that the outflow tract and right ventricle originate from a separate source of mesoderm that is anterior to the primary heart field. The discovery of this anterior, or secondary, heart field has led to a greater understanding of the morphogenetic events involved in heart formation; however, many of the underlying molecular events controlling these processes remain to be determined. The MADS domain transcription factor MEF2C is required for proper formation of the cardiac outflow tract and right ventricle, suggesting a key role in anterior heart field development. Therefore, as a first step toward identifying the transcriptional pathways upstream of MEF2C, a lacZ reporter gene was introduced into a bacterial artificial chromosome (BAC) encompassing the murine Mef2c locus and this recombinant was used to generate transgenic mice. This BAC transgene was sufficient to recapitulate endogenous Mef2c expression, and comparative sequence analyses revealed multiple regions of significant conservation in the noncoding regions of the BAC. One of these conserved noncoding regions represents a transcriptional enhancer that is sufficient to direct expression of lacZ exclusively to the anterior heart field throughout embryonic development. This conserved enhancer contains two consensus GATA binding sites that are efficiently bound by the zinc finger transcription factor GATA4 and are completely required for enhancer function in vivo. This enhancer also contains two perfect consensus sites for the LIM-homeodomain protein ISL1. These elements are specifically bound by ISL1 and are essential for enhancer function in transgenic embryos. Thus, these findings establish Mef2c as the first direct transcriptional target of ISL1 in the anterior heart field and support a model in which GATA factors and ISL1 serve as the earliest transcriptional regulators controlling outflow tract and right ventricle development (Dodou, 2004).

Normal heart development is orchestrated by a set of highly conserved transcription factors that includes GATA4, Nkx2-5, and Tbx5. Heterozygous mutation of each of these genes causes congenital heart disease in humans. In mouse models, haploinsufficiency for Nkx2-5 or Tbx5 results in an increased incidence of structural heart disease, confirming that normal heart development is sensitive to small changes in expression levels of Nkx2-5 and Tbx5. However, mice haploinsufficient for GATA4 have not been reported to have cardiac abnormalities. Two new GATA4 alleles, GATA4H and GATA4flox, were generated. GATA4flox/flox embryos express 50% less GATA4 protein in the heart and survive normally. In contrast, GATA4H/H embryos expres 70% less GATA4 protein in the heart and die between days 13.5 and 16.5 of gestation. These embryos have common atrioventricular canal (CAVC), double outlet right ventricle (DORV), hypoplastic ventricular myocardium, and normal coronary vasculature. Myocardial hypoplasia is associated with diminished cardiomyocyte proliferation. Hemodynamic measurements demonstrate that these embryos have normal systolic function, severe diastolic dysfunction, and atrioventricular regurgitation. Surprisingly, expression levels of the putative GATA4 target genes ANF, BNP, MEF2C, Nkx2-5, cyclin D2, and BMP4 were unchanged in mutant hearts, suggesting that GATA4 is not a dose-limiting regulator of the expression of these genes during later stages of embryonic cardiac development. These data demonstrate that multiple aspects of embryonic cardiac morphogenesis and function are exquisitely sensitive to small changes in GATA4 expression levels (Pu, 2004).

The cardiac conduction system comprises a specialized tract of electrically coupled cardiomyocytes responsible for impulse propagation through the heart. Abnormalities in cardiac conduction are responsible for numerous forms of cardiac arrhythmias, but relatively little is known about the gene regulatory mechanisms that control the formation of the conduction system. This study demonstrates that a distal enhancer for the connexin 30.2 (Cx30.2, also known as Gjd3) gene, which encodes a gap junction protein required for normal atrioventricular (AV) delay in mice, is necessary and sufficient to direct expression to the developing AV conduction system (AVCS). Moreover, this enhancer requires Tbx5 and Gata4 for proper expression in the conduction system, and Gata4+/- mice have short PR intervals indicative of accelerated AV conduction. These results implicate Gata4 in conduction system function and provide a clearer understanding of the transcriptional pathways that impact normal AV delay (Munshi, 2009).

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. This study shows that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), referred to as 'cardiac TFs,' have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, chromatin occupancy by these TFs and p300 were defined genome wide and unbiased support for this hypothesis is provided. This principle was used to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. These data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers (He, 2011).

The cardiac transcription network modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. A systems biology study is presented integrating mRNA profiles with DNA-binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi-mediated knockdown. Finally, conclusions primarily obtained in cardiomyocyte cell culture were confirmed in a time-course of cardiac maturation in mouse around birth. Insights are provided into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, it was found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, it was found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, an Srf-centered transcription network is presented providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, this study shows the combinatorial contribution of four DNA-binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders (Schlesinger, 2011; full text of article).

Epicardial GATA factors regulate early coronary vascular plexus formation

During early development, GATA factors have been shown to be important for key events of coronary vasculogenesis, including formation of the epicardium. Myocardial GATA factors are required for coronary vascular (CV) formation; however, the role of epicardial localized GATAs in this process has not been addressed. The current study was conducted to investigate the molecular mechanisms by which the epicardium controls coronary vasculogenesis, focusing on the role of epicardial GATAs in establishing the endothelial plexus during early coronary vasculogenesis. To address the role of epicardial GATAs, GATA4 and GATA6 transcription factors were specifically from the mouse epicardium, and the number of endothelial cells in the sub-epicardium was found to be drastically reduced, and concomitant coronary vascular plexus formation was significantly compromised. This study has presented evidence for a novel role for epicardial GATA factors in controlling plexus formation by recruiting endothelial cells to the sub-epicardium (Kolander, 2014).

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serpent: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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