An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. Here we determined that GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activated expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the α-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy was suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND was associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacted with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. Taken together with previous observations, these results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Transcription factor GATA-4 plays critical roles in controlling heart development and cardiac hypertrophy. To understand how GATA-4 functions under diverse conditions, we sought to identify its coactivators. We tested p300 as a coactivator in GATA-4-dependent transient transcription assays in NIH3T3 cells and found that p300 synergistically activated GATA-4-dependent transcription on both synthetic and natural promoters. Direct physical interactions between the Nand C-zinc finger domains of GATA-4 and the cysteine/ histidine-rich region 3 (C/H3) of p300 were identified in immunoprecipitation and glutathione S-transferase pull-down experiments. Deletion of the C/H3 region of p300 abolished its coactivator activity indicating that the physical interaction was required for functional synergy. Through the use of a series of GATA-4 zinc ringer mutants, the amino acids WRR in the C finger were identified as critical to the interaction. The adenoviral E1A protein or a peptide encoding the C/H3 region of p300 could inhibit GATA-4-dependent transcription, presumably by competing for p300 binding. Furthermore, deletion of the region of p300 encoding the histone acetyltransferase activity abolished its effect on GATA-4-dependent transcriptional activity. These results establish that p300 acts as a GATA-4 coactivator and that the p300 histone acetyltransferase activity is necessary for the functional interaction.

The zinc finger-containing transcription factors GATA4 and GATA6 are important regulators of basal and inducible gene expression in cardiac and smooth muscle cell types. Here we demonstrate a direct functional role for GATA4 and GATA6 as regulators of cardiomyocyte hypertrophic growth and gene expression. To model the increase in endogenous GATA4 and GATA6 transcriptional activity that occurs in response to hypertrophic stimulation, each factor was overexpressed in cardiomyocytes using recombinant adenovirus. Overexpression of either GATA4 or GATA6 was sufficient to induce cardiomyocyte hypertrophy characterized by enhanced sarcomeric organization, a greater than 2-fold increase in cell surface area, and a significant increase in total protein accumulation. In vivo, transgenic mice with 2.5-fold overexpression of GATA4 within the adult heart demonstrated a slowly progressing increase in heart to body weight ratio, histological features of cardiomyopathy, and activation of hypertrophy-associated genes, suggesting that GATA factors are sufficient regulators of cardiomyocyte hypertrophy in vitro and in vivo. To evaluate the requirement of GATA factors as downstream transcriptional mediators of hypertrophy, a dominant negative GATA4-engrailed repressor fusion-encoding adenovirus was generated. Expression of GATA4-engrailed blocked GATA4- and GATA6-directed transcriptional responses and agonist-induced cardiomyocyte hypertrophy, demonstrating that cardiac-expressed GATA factors are necessary mediators of this process.

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, we tested 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.

Background Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals.Methods and Results The left ventricular expression of a directly injected reporter gene driven by 3542 bp of rat beta-myosin heavy chain (beta-MHC) promoter was increased 3.0-fold by aortic constriction (P<.005), an increment similar to the 3.2-fold increase in the level of the endogenous beta-MHC mRNA in the same left ventricles. Subsequent analysis identified a 107-bp beta-MHC promoter sequence (-303/-197) sufficient to convert a heterologous neutral promoter to one that is activated by aortic constriction. These sequences contain two M-CAT elements, which have previously been demonstrated to mediate inducible expression during alpha(1)-adrenergic-stimulated hypertrophy in cultured neonatal cardiac myocytes, and a GATA element. Although simultaneous mutation of both M-CAT elements markedly decreased the basal transcriptional activity of an injected 333-bp beta-MHC promoter, it had no effect on aortic constriction-stimulated transcription (3.5-fold increase, P<.005 for both wild type and mutant). In contrast, mutation of the GATA motif markedly attenuated aortic constriction-stimulated transcription (1.6-fold, P=NS) without affecting the basal transcriptional activity. This GATA site can interact with in vitro translated GATA-4 and compete with an established GATA site for GATA-4 binding activity in nuclear extracts from aortic constricted hearts.Conclusions Basal and aortic constriction-stimulated transcription of the beta-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within beta-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the alpha-skeletal actin promoter 1.7-fold whereas the alpha-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1. 6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle-specific protein involved in active force generation. The mechanism responsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory events involved in the induction and maintenance of the cardiac cell lineage. In this report we dissect the alpha-MHC regulatory region to identify the components necessary for directing high levels of cardiac muscle-restricted expression. Deletion, site-specific mutant and heterologous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-muscle cells. These studies indicated that sequences from -344 to -156 directed high levels of cardiac-muscle specific expression from a heterologous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box, alpha-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response factor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Together, these results identify two distinct regions with different regulatory function that are critical for the tissue restricted expression of the alpha-MHC gene.

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.