Comparison on radiation effective dose and image quality of right coronary artery on prospective ECG-gated method between 320 row CT and 2nd generation (128-slice) dual source CT.

This retrospective study was to compare the image quality of right coronary artery (RCA) and effective radiation dose on prospective ECG-gated method between 320 row computed tomography (CT) and 2nd generation (128-slice) dual source CT. A total of 215 candidates underwent CT coronary angiography using prospective ECG-gated method, 120 patients enrolled in 320 row CT group, and 95 patients in dual source CT group. We divided RCA image quality scores as 1/2/3/4, which means excellent/good/adequate/not assessable and heart rates were considered, as well as the radiation dose. There is no statistically significant difference of RCA image quality of Score 1/2 between 320 row CT and 2nd generation dual source CT, but lower heart rate (<70/min) improved RCA image quality. Meanwhile, the 2nd generation dual source CT scan have significant lower radiation dose. For patients with high level heart rate variation, both prospective ECG-gated method of 320 row CT scan (Toshiba) and 2nd generation dual source CT scan (Siemens) basically provided good image quality on RCA. There is an advantage of effective radiation dose reduction in prospective ECG-gated method using the 2nd generation dual source CT scan. After the iodine contrast agent was injected into elbow vein, the threshold triggering method was used to carry out prospective gated scanning, and the acquired fault image was reconstructed by the standard post-processing software of each manufacturer. The radiation dose value is obtained through the dose report automatically generated after each scan.

Reduced Compensatory ß-Cell Proliferation in Nfatc3-Deficient Mice Fed on High-Fat Diet.

BACKGROUND: High-fat-diet induces pancreatic ß-cell compensatory proliferation, and impairments in pancreatic ß-cell proliferation and function can lead to defects in insulin secretion and diabetes. NFATc3 is important for HFD-induced adipose tissue inflammation. But it is unknown whether NFATc3 is required for ß cell compensatory growth in mice fed with HFD. METHODS: NFATc3 mRNA and protein expression levels were quantified by RT-qPCR and Western blotting, respectively, in pancreatic islets of WT mice fed on HFD for 12-20 weeks. Adenoviral-mediated overexpression of NFATc3 were conducted in Min6 cells and cultured primary mouse islets. NFATc3-/- mice and WT control mice were fed with HFD and metabolic and functional parameters were measured. RESULTS: We observed that the NFATc3 expression level was reduced in the islets of high-fat-diet (HFD)-fed mice. Adenovirus-mediated overexpression of NFATc3 enhanced glucose-stimulated insulin secretion and ß-cell gene expression in cultured primary mouse islets. Nfatc3-/- mice initially developed similar glucose tolerance at 2-4 weeks after HFD feeding than HFD-fed WT mice, but Nfatc3-/- mice developed improved glucose tolerance and insulin sensitivity after 8 weeks of HFD feeding compared to Nfatc3+/+fed with HFD. Furthermore, Nfatc3-/- mice on HFD exhibited decreased ß-cell mass and reduced expression of genes important for ß-cell proliferation and function compared to Nfatc3+/+mice on HFD. CONCLUSIONS: The findings suggested that NFATc3 plays a role in maintaining the pancreatic ß-cell compensatory growth and gene expression in response to obesity.

SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic ß cells.

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of ß-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic ß cells.

Mobilized bone marrow progenitor cells serve as donors of cytoprotective genes for cardiac repair.

We proposed here that mobilized progenitor cells (MPCs) from the bone marrow are special cell types which carry cytoprotective proteins for cardiac repair following ischemia. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery (LAD) in mice. Progenitor cells in peripheral blood were analyzed by fluorescence-activated cell sorting (FACS). The expression of cytoprotective genes was assayed by ELISA, RT-PCR, and/or real-time PCR. G-CSF was markedly up-regulated in the ischemic myocardium. A good correlation was observed between serum G-CSF and progenitor cells in circulation following LAD ligation. MPCs overexpressed cardiac transcription factor, GATA-4, and anti-apoptotic factor, Bcl-2, besides expression of the surface markers of bone marrow stem cells (BMSCs). Transplantation of cultured MPCs into the ischemic border area significantly improved cardiac function by reducing infarction size. More importantly, MPCs significantly protected cardiomyocytes against apoptosis when co-cultured with cardiomyocytes. The cardiac protection by MPCs was blocked by Bcl-2 neutralizing antibody and GATA-4 siRNA. In contrast, transfection of BMSCs with GATA-4 provided increased protection of myocytes against apoptosis. It is concluded that MPCs are highly cytoprotective and carry protective genes responsible for cardiac repair.

The DnaJ-related factor Mrj interacts with nuclear factor of activated T cells c3 and mediates transcriptional repression through class II histone deacetylase recruitment.

The calcium-regulated protein phosphatase calcineurin (PP2B) functions as a regulator of gene expression in diverse tissues through the dephosphorylation and activation of a family of transcription factors known as nuclear factor of activated T cells (NFAT). Here we show that NFATc3, in addition to being calcium responsive, is regulated through an indirect recruitment of class II histone deacetylases (HDACs). Specifically, yeast two-hybrid screening with the rel homology domain of NFATc3 identified the chaperone mammalian relative of DnaJ (Mrj) as a specific interacting factor. Mrj and NFATc3 were shown to directly associate with one another in mammalian cells and in vitro. Mrj served as a potent inhibitor of NFAT transcriptional activity within the nucleus through a mechanism involving histone deacetylase recruitment in conjunction with heat shock stimulation. Indeed, Mrj was determined to interact with class II histone deacetylases, each of which translocated to the nucleus following heat shock stimulation. Mrj also decreased NFATc3 occupancy of the tumor necrosis factor-alpha promoter in cardiomyocytes in an HDAC-dependent manner, and Mrj blocked calcineurin-induced cardiomyocyte hypertrophic growth. Conversely, small-interfering-RNA-mediated reduction of Mrj augmented NFAT transcriptional activity and spontaneously induced cardiac myocyte growth. Collectively, our results define a novel response pathway whereby NFATc3 is negatively regulated by class II histone deacetylases through the DnaJ (heat shock protein-40) superfamily member Mrj.

Regulation of calcineurin through transcriptional induction of the calcineurin A beta promoter in vitro and in vivo.

The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin A(beta) (CnA(beta)) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnA(beta) upregulation is not clear. Here, we isolated the mouse CnA(beta) promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnA(beta) promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated beta-galactosidase (LacZ) containing transgenic mice under the control of the CnA(beta) 2.3-kb promoter. CnA(beta)-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnA(beta)-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnA(beta) 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnA(beta) expression as an additional means of regulating calcineurin activity in the heart.

Direct and indirect interactions between calcineurin-NFAT and MEK1-extracellular signal-regulated kinase 1/2 signaling pathways regulate cardiac gene expression and cellular growth.

MEK1, a member of the mitogen-activated protein kinase (MAPK) cascade that directly activates extracellular signal-regulated kinase (ERK), induces cardiac hypertrophy in transgenic mice. Calcineurin is a calcium-regulated protein phosphatase that also functions as a positive regulator of cardiac hypertrophic growth through a direct mechanism involving activation of nuclear factor of activated T-cell (NFAT) transcription factors. Here we determined that calcineurin-NFAT and MEK1-ERK1/2 signaling pathways are interdependent in cardiomyocytes, where they directly coregulate the hypertrophic growth response. For example, genetic deletion of the calcineurin Abeta gene reduced the hypertrophic response elicited by an activated MEK1 transgene in the heart, while inhibition of calcineurin or NFAT in cultured neonatal cardiomyocytes also blunted the hypertrophic response driven by activated MEK1. Conversely, targeted inhibition of MEK1-ERK1/2 signaling in cultured cardiomyocytes attenuated the hypertrophic growth response directed by activated calcineurin. However, targeted inhibition of MEK1-ERK1/2 signaling did not directly affect calcineurin-NFAT activation, nor was MEK1-ERK1/2 activation altered by targeted inhibition of calcineurin-NFAT. Mechanistically, we show that MEK1-ERK1/2 signaling augments NFAT transcriptional activity independent of calcineurin, independent of changes in NFAT nuclear localization, and independent of alterations in NFAT transactivation potential. In contrast, MEK1-ERK1/2 signaling enhances NFAT-dependent gene expression through an indirect mechanism involving induction of cardiac AP-1 activity, which functions as a necessary NFAT-interacting partner. As a second mechanism, MEK1-ERK1/2 and calcineurin-NFAT proteins form a complex in cardiac myocytes, resulting in direct phosphorylation of NFATc3 within its C terminus. MEK1-ERK1/2-mediated phosphorylation of NFATc3 directly augmented its DNA binding activity, while inhibition of MEK1-ERK1/2 signaling reduced NFATc3 DNA binding activity. Collectively, these results indicate that calcineurin-NFAT and MEK1-ERK1/2 pathways constitute a codependent signaling module in cardiomyocytes that coordinately regulates the growth response through two distinct mechanisms.

NFATc3 deficiency reduces the classical activation of adipose tissue macrophages

Nuclear factors of activated T cells (NFAT) c3 have a prominent role in the regulation of proinflammatory factors in immune cells. The classically activated M1 macrophages are key players in the initiation and maintenance of adipose tissue (AT) inflammation. The role of NFATc3 in obesity and AT inflammation is unknown. We set out to determine how deficiency of NFATc3 effected macrophage polarization, inflammation and insulin resistance in visceral AT of high-fat diet (HFD)-fed mice. Nfatc3−/− and WT mice were fed a HFD for 8­17 weeks. Epididymal white AT (eWAT) F4/80(+) cells were characterized by fluorescence-activated cell sorting and quantitative RT-PCR. Results showed that Nfatc3−/− mice developed HFD-induced obesity similar to WT mice, but insulin sensitivity and glucose tolerance were improved, and liver fat accumulation was reduced in Nfatc3−/− mice compared to WT control mice. Moreover, M1 macrophage content and proinflammatory factors were reduced, whereas the alternatively activated M2 macrophage content was increased in eWAT of HFD-fed Nfatc3−/− mice compared to that of WT mice. In addition, eWAT insulin signaling was improved in HFD-fed Nfatc3−/− mice. Importantly, after bone-marrow-derived macrophages had been isolated from Nfatc3−/− mice and cultured in vitro, treatment of these cells with interferon-γ and lipopolysaccharide resulted in reduction of M1 inflammatory markers, suggesting that NFATc3 promoted M1 polarization by a cell-autonomous mechanism. The results demonstrated that NFATc3 played an important role in M1 macrophage polarization, AT inflammation and insulin resistance in response to obesity through transcriptional activation of proinflammatory genes.

Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling.

The MAPKs are important transducers of growth and stress stimuli in virtually all eukaryotic cell types. In the mammalian heart, MAPK signaling pathways have been hypothesized to regulate myocyte growth in response to developmental signals or physiologic and pathologic stimuli. Here we generated cardiac-specific transgenic mice expressing dominant-negative mutants of p38alpha, MKK3, or MKK6. Remarkably, attenuation of cardiac p38 activity produced a progressive growth response and myopathy in the heart that correlated with the degree of enzymatic inhibition. Moreover, dominant-negative p38alpha, MKK3, and MKK6 transgenic mice each showed enhanced cardiac hypertrophy following aortic banding, Ang II infusion, isoproterenol infusion, or phenylephrine infusion for 14 days. A mechanism underlying this enhanced-growth profile was suggested by the observation that dominant-negative p38alpha directly augmented nuclear factor of activated T cells (NFAT) transcriptional activity and its nuclear translocation. In vivo, NFAT-dependent luciferase reporter transgenic mice showed enhanced activation in the presence of the dominant-negative p38alpha transgene before and after the onset of cardiac hypertrophy. More significantly, genetic disruption of the calcineurin Abeta gene rescued hypertrophic cardiomyopathy and depressed functional capacity observed in p38-inhibited mice. Collectively, these observations indicate that reduced p38 signaling in the heart promotes myocyte growth through a mechanism involving enhanced calcineurin-NFAT signaling.

Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy.

Calcineurin (PP2B) is a calcium/calmodulin-activated, serine-threonine phosphatase that transmits signals to the nucleus through the dephosphorylation and translocation of nuclear factor of activated T cell (NFAT) transcription factors. Whereas calcineurin-NFAT signaling has been implicated in regulating the hypertrophic growth of the myocardium, considerable controversy persists as to its role in maintaining versus initiating hypertrophy, its role in pathological versus physiological hypertrophy, and its role in heart failure. To address these issues, NFAT-luciferase reporter transgenic mice were generated and characterized. These mice showed robust and calcineurin-specific activation in the heart that was inhibited with cyclosporin A. In the adult heart, NFAT-luciferase activity was upregulated in a delayed, but sustained manner throughout eight weeks of pathological cardiac hypertrophy induced by pressure-overload, or more dramatically following myocardial infarction-induced heart failure. In contrast, physiological hypertrophy as produced in two separate models of exercise training failed to show significant calcineurin-NFAT coupling in the heart at multiple time points, despite measurable increases in heart to body weight ratios. Moreover, stimulation of hypertrophy with growth hormone-insulin-like growth factor-1 (GH-IGF-1) failed to activate calcineurin-NFAT signaling in the heart or in culture, despite hypertrophy, activation of Akt, and activation of p70 S6K. Calcineurin Abeta gene-targeted mice also showed a normal hypertrophic response after GH-IGF-1 infusion. Lastly, exercise- or GH-IGF-1-induced cardiac growth failed to show induction of hypertrophic marker gene expression compared with pressure-overloaded animals. Although a direct cause-and-effect relationship between NFAT-luciferase activity and pathological hypertrophy was not proven here, our results support the hypothesis that separable signaling pathways regulate pathological versus physiological hypertrophic growth of the myocardium, with calcineurin-NFAT potentially serving a regulatory role that is more specialized for maladaptive hypertrophy and heart failure.

Calcineurin Abeta gene targeting predisposes the myocardium to acute ischemia-induced apoptosis and dysfunction.

Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Abeta gene-targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Abeta-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Abeta-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.

Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes.

BACKGROUND: Bone marrow stromal cells (BMSCs) have the potential to differentiate into various cells and can transdifferentiate into myocytes if an appropriate cellular environment is provided. However, the molecular signals that underlie this process are not fully understood. In this study, we show that BMSC differentiation is dependent on communication with cells in their microenvironment. METHODS AND RESULTS: BMSCs were isolated from green fluorescent protein (GFP)-transgenic mice and cocultured with myocytes in a ratio of 1:40. Myocytes were obtained from neonatal rat ventricles. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction. Before coculturing, the BMSCs were negative for alpha-actinin and exhibited a nucleus with many nucleoli. After 7-day coculture with myocytes, some BMSCs became alpha-actinin-positive and formed gap junctions with native myocytes. However, BMSCs separated from myocytes by a semipermeable membrane were still negative for alpha-actinin. Transdifferentiated myocytes from BMSCs were microdissected from cocultures by laser captured microdissection to determine the changes in gene expression. BMSCs cocultured with myocytes expressed mouse cardiac transcription factor GATA-4. CONCLUSIONS: When cocultured with myocytes, BMSCs can transdifferentiate into cells with a cardiac phenotype. Differentiated myocytes express cardiac transcription factors GATA-4 and myocyte enhancer factor-2. The transdifferentiation processes rely on intercellular communication of BMSCs with myocytes.

Transcription factor Ets1 regulates expression of thioredoxin-interacting protein and inhibits insulin secretion in pancreatic ß-cells.

Long-term activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucotoxicity and inhibit insulin gene expression in ß-cells. Transcription factor Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38 residue. We hypothesize that Ets1 plays an important role in mediating ERK1/2 induced glucotoxicity in ß-cells. We determined the role of Ets1 in Min6 cells and isolated mouse islets using overexpression and siRNA mediated knockdown of Ets1. The results show that Ets1 was localized in insulin-staining positive cells but not in glucagon-staining positive cells. Overexpression of Ets1 reduced glucose-stimulated insulin secretion in primary mouse islets. Overexpression of Ets1 in Min6 ß-cells and mouse islets increased expression of thioredoxin-interacting protein (TXNIP). Conversely, knockdown of Ets1 by siRNA reduced expression of TXNIP in Min6 cells. Ets1 was associated with the txnip promoter in min6 cells and transfection of 293 cells with Ets1 and p300 synergistically increased txnip promoter reporter activity. Moreover, overexpression of Ets1 inhibited Min6 cell proliferation. Our results suggest that Ets1, by promoting TXNIP expression, negatively regulates ß-cell function. Thus, over-activation of Ets1 may contribute to diet-induced ß-cell dysfunction.

Cdk2ap1 is required for epigenetic silencing of Oct4 during murine embryonic stem cell differentiation.

Oct4 is a known master regulator of stem cell renewal and differentiation. Expression of Oct4 during differentiation is regulated by promoter methylation by the nucleosome remodeling and histone deacetylation (NuRD) complex. Here, we show that Cdk2ap1, a negative regulator of Cdk2 function and cell cycle, promotes Oct4 promoter methylation during murine embryonic stem cell differentiation to down-regulate Oct4 expression. We further show that this repressor function of Cdk2ap1 is dependent on its physical interaction with the methyl DNA-binding protein, Mbd3. Our data support a potential molecular link between the known differentiation promoters, including bone morphogenetic proteins and transforming growth factor signaling, and embryonic stem cell differentiation.

The basic helix-loop-helix factor Hand 2 regulates autonomic nervous system development.

Mammalian autonomic nervous system (ANS) development requires the combinatorial action of a number of transcription factors, which include Mash 1, Phox 2b, and GATA 3. Here we show that the bHLH transcription factor, Hand 2 (dHAND), is expressed concurrently with Mash 1 during sympathetic nervous system (SNS) development and that the expression of Hand 2 is not dependent on Mash 1. This suggests that these two bHLH factors work in parallel during SNS development. We also show that ectopic expression of Hand 2 activates the neuronal program and promotes the acquisition of a phenotype corresponding to peripheral neurons including neurons of the SNS lineage in P19 embryonic carcinoma cells. We propose that Hand 2 works in parallel with other members of the transcriptional network to regulate ANS developmental but can ectopically activate the program by a cross-regulatory mechanism that includes the activation of Mash 1. We show that this function is dependent on its interaction with the histone acetyltransferase p300/CBP, indicating that Hand 2 functions to promote ANS development as part of a larger transcriptional complex.

The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer.

HAND2 (dHAND) is a basic helix-loop-helix (bHLH) transcription factor expressed in numerous tissues during development including the heart, limbs, and a subset of neural crest derivatives. Functional analysis has shown that HAND2 is involved in development of the branchial arches, heart, limb, vasculature, and nervous system. Although it is essential for development of numerous tissues, little is known about its mode of action. To this end, we have characterized HAND2 transcriptional regulatory mechanisms. Using mammalian one-hybrid analysis we show that HAND2 contains a strong transcriptional activation domain in the amino-terminal third of the protein. Like most tissue-restricted bHLH factors, HAND2 heterodimerizes with the broadly expressed bHLH factors, the E-proteins. We determined the consensus DNA binding site of HAND2 and show that HAND2 binds a subset of E-boxes as a heterodimer with E12. Yeast two-hybrid screening of a neuroblastoma cDNA library for HAND2-interacting proteins selected HAND2 and numerous additional members of the E-protein family. Although HAND2 homodimer formation was confirmed by in vitro analysis, HAND2 fails to homodimerize in a mammalian two-hybrid assay but demonstrates robust HAND2/E12 interaction. We conclude that HAND2 functions as a transcription activator by binding a subset of E-boxes as a heterodimer with E-proteins.

The transcription factors GATA4 and dHAND physically interact to synergistically activate cardiac gene expression through a p300-dependent mechanism.

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 alpha-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.

JAB1 enhances HAND2 transcriptional activity by regulating HAND2 DNA binding.

HAND2 (also known as dHAND) is a basic helix-loop-helix (bHLH) transcription factor essential for development of the heart, limbs, and neural crest-derived lineages. HAND2 expression is observed in a number of tissues derived from the neural crest, including components of the peripheral nervous system, where it has been shown to regulate sympathetic nervous system development. Here we show that HAND2 is expressed in both the sympathetic and the parasympathetic divisions of the autonomic nervous system (ANS). How HAND2 functions during development of these neuronal lineages is uncertain. An important mechanism involved in HAND2's function is its interactions with other proteins. To understand better the molecular interactions regulating HAND2 during ANS development, we employed a yeast two-hybrid screen to identify HAND2-interacting proteins. One protein identified in this screen, Jun activation domain-binding protein (JAB1), is involved in numerous cell processes, including regulation of transcription and protein turnover. We show that JAB1 binds directly to the HLH domain of HAND2 and increases HAND2 transcription-stimulating activity. However, JAB1 does not contain a transcriptional activation domain, nor does it recruit an activation domain to HAND2. Our data indicate that JAB1 augments HAND2 transcriptional activity by enhancing HAND2 DNA binding. We further show that enhanced HAND2 DNA binding is mediated through the HLH domain and not through the DNA binding domain. These results show that JAB1 regulates the transcriptional activity of HAND2 in a unique manner that may account, in part, for the apparent ability of this bHLH factor to regulate gene expression through numerous mechanisms.