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Background: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated. Methods: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms. Results: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes‚ whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes. Conclusions: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Rationale: Fetal growth and survival depend critically on proper development and integrity of the vascular system. Fli1 (Friend leukemia integration 1), a member of the Ets family of transcription factors, plays critical roles in vascular morphogenesis and homeostasis at mid-gestation, the developmental stage at which expression of its upstream regulator, Etv2, ceases. However, molecular mechanisms of Fli1 action in vascular morphogenesis remain incompletely understood. Objective: To dissect molecular mechanisms of vascular morphogenesis governed by Fli1. Methods and Results: Utilizing Fli1 promoter-driven lineage-specific LacZ expression, Fli1 loss-of-function strategies, and a series of molecular techniques, we demonstrate that Fli1 expression in multipotent myogenic progenitor cells (MPCs) occurs independent of Etv2, and loss of Fli1 expression results in a significant increase in LacZ + cells in mesoderm within somites and limb buds, leading to reciprocal regulation of the expression of several key endothelial and myogenic genes and vascular abnormalities. Conversely, embryos harboring conditional Fli1 gain-of-function in MPCs manifested aberrant vasculogenesis with lack of myogenesis. Mechanistically, elevated Fli1 activity in myoblasts and in adult MPCs (also called satellite cells) of X-linked muscular dystrophic mdx mice markedly induced endothelial, but attenuated myogenic, gene expression and differentiation. Importantly, ectopic expression of Myf5 or MyoD, two key myogenic regulators, in Fli1-expressing myoblasts restored their differentiation potential, indicating that levels of Fli1 and myogenic regulators in MPCs inversely regulate their endothelial versus myogenic potential. Conclusions: Fli1 governs vascular morphogenesis by regulating endothelial potential by inversely regulating endothelial versus myogenic programs in MPCs. Our data uncover an important and previously unrecognized mechanism of vascular morphogenesis governed by Fli1 and highlight the physiological significance of the fine tuning of Fli1 activity in multipotent progenitors for proper vascular and muscle morphogenesis during development and disease.

Supplemental Digital Content is available in the text., Background: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. Methods: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. Results: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload–induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. Conclusions: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes., Heart failure with preserved ejection fraction (HFpEF) is a global, major health issue for which no effective therapies are available. Here, the authors discover that the interplay between two transcription factors, Xbp1s and FoxO1, is critical for metabolic adaptation and lipid handling in HFpEF-stressed cardiomyocytes.

Rationale: Reperfusion injury accounts for up to half of myocardial infarct size, and meaningful clinical therapies targeting it do not exist. We have reported previously that autophagy is reduced during reperfusion and that HDAC (histone deacetylase) inhibition enhances cardiomyocyte autophagy and blunts ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, whether inducing autophagy per se, as opposed to other effects triggered by HDAC inhibition, is sufficient to protect against reperfusion injury is not clear. Objective: We set out to test whether augmentation of autophagy using a specific autophagy-inducing peptide, TB (Tat-Beclin), protects the myocardium through reduction of reactive oxygen species (ROS) during reperfusion injury. Methods and Results: Eight- to 12-week-old, WT (wild type) C57BL6 mice and tamoxifen-inducible cardiomyocyte-specific ATG7 KO (ATG7 knockout) mice (to test the dependency on autophagy) were randomized into 2 groups: exposed to a control TS (Tat-scrambled) peptide or a TB peptide. Each group was subjected to I/R surgery (45-minute coronary ligation, 24-hour reperfusion). Infarct size, systolic function, autophagic flux, and ROS were assayed. Cultured neonatal rat ventricular myocytes were exposed to TB during simulated I/R injury. ATG7 knockdown by small interfering RNA in neonatal rat ventricular myocytes was used to evaluate the role of autophagy. TB treatment at reperfusion reduced infarct size by 20% (absolute reduction; 50% relative reduction) and improved contractile function. Improvement correlated with increased autophagic flux in the border zone with less oxidative stress. ATG7 KO mice did not manifest TB-promoted cardioprotection during I/R. In neonatal rat ventricular myocytes subjected to I/R, TB reduced cell death by 41% and reduced I/R-induced ROS generation. Conversely, ATG7 knockdown in neonatal rat ventricular myocytes abolished these beneficial effects of TB on cell death and ROS reduction. Conclusions: Induction of autophagy at the time of reperfusion is sufficient to mitigate myocardial reperfusion injury by reducing ROS and cell death. Maintenance of appropriate autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in acute myocardial infarction.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure (HF). Limited insight into underlying mechanisms has culminated in the longstanding absence of evidence-based therapies capable of mitigating the substantial morbidity and mortality associated with the syndrome. Existing clinical and epidemiological evidence suggests that excessive body fat and lipid mishandling contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are unknown. We recently developed a novel, clinically relevant, murine model of HFpEF, uncovering suppression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) signaling pathway as a critical driver of HFpEF pathogenesis. Objectives: To define and manipulate mechanisms downstream of Xbp1s in HFpEF and decipher its cardioprotective actions. Methods and Results: In the myocardium of experimental HFpEF, we detected cardiomyocyte steatosis coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 depletion, as well as Xbp1s over-expression, in cardiomyocytes each ameliorated the HFpEF phenotype and reduced myocardial lipid accumulation. Strikingly, forced expression of Xbp1s in cardiomyocytes triggered proteasomal degradation of FoxO1. Furthermore, we discovered that FoxO1 is ubiquitinated upon Xbp1s over-expression, and Xbp1s-induced proteasomal degradation of FoxO1 occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1), a protein we identified as a novel and direct transcriptional target of Xbp1s. Conclusions: Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1–Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1–Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1–Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis., Disease stress-induced cardiac hypertrophy is a major mechanism of pathological cardiac remodeling. Here, the authors unveil a previously unrecognized role of a FoxO1–Dio2 signaling axis in maladaptive, afterload-induced cardiac hypertrophy and intracellular thyroid hormone homeostasis.

Background: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. Methods: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. Results: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. Conclusions: Our results reveal a critical role of the XBP1s–FKBP11–mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Background: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Methods: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. Results: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Conclusions: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1–SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Background: Reperfusion injury accounts for a significant portion of myocardial damage in acute coronary syndromes. Autophagy, a process of cell catabolism, plays a vital role in the heart’s response to stress. We have reported that re-induction of ischemia/reperfusion (I/R)-suppressed cardiomyocyte autophagy with histone deacetylase (HDAC) inhibitors affords significant cardioprotection. However, as HDACs govern many processes and may have off-target effects, we set out to modulate autophagy in a manner independent of HDAC activity. Here, we hypothesized that induction of autophagy with a novel agent, Tat-Beclin, at the time of reperfusion, will reduce I/R injury and rescue cardiac function. Methods: Wild type and ATG7 (protein required for autophagic flux) knockout mice were randomized among 3 treatment groups prior to surgical I/R injury [45 min LAD artery ligation; 24h reperfusion]: vehicle control (VC), Tat-Scrambled (TS), or Tat-Beclin (TB). Each agent was delivered at coronary reperfusion. To define molecular mechanisms, cultured adult and neonatal rat ventricular cardiomyocytes (ARVMs/NRVMs) were subjected to simulated I/R. Results: Induction of cardiomyocyte autophagy at reperfusion reduced infarct size 20.1% (±6.3%, n=23, p Conclusion: Direct induction of cardiomyocyte autophagy reduces infarct size and declines in contractile function. Autophagy rescues I/R injury in part through reduction of oxidative stress. Critically, this cardioprotection was observed when intervention occurred at the time of reperfusion, the clinically relevant context.

SummaryThe hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Establishment of the maternal-fetal circulation during embryonic development is a fundamental process required for effective exchange of nutrients, waste products and signaling factors critical to all subsequent stages of fetal growth and development. Recent work has uncovered a previously unrecognized role of the transcription factor FoxO1 in the orchestration of molecular events underlying establishment of maternal-fetal circulatory interaction. These new data contribute to a larger body of literature implicating this protein in the governance of a wide array of processes during development and beyond.

Forkhead box O1 (FoxO1), a member of the Forkhead box-containing O family of transcription factors, is a key regulator of numerous genes that govern a wide array of cellular functions, including differentiation, homeostasis, and survival. However, the role of FoxO1 in development remains elusive. Here, we describe an essential and previously undefined role for FoxO1 in placental development. We demonstrate that FoxO1 -null embryos up to embryonic day 9.0 (E9.0) are indistinguishable, including their morphology, cardiovascular structure, and vascular gene expression, from wild-type (WT) littermates. However, FoxO1 -nulls manifested a profoundly swollen/hydropic allantois, which failed to fuse with the chorion, a phenotype that leads to subsequent cardiovascular malformation, progressive apoptotic cell death, and embryonic lethality at E10.5. Quantitative RT-PCR analysis of genes involved in placental development revealed significant attenuation of VCAM1 expression in FoxO1 -null embryos. Using immunohistochemical, transcriptional, and chromatin immunoprecipitation assays, we further discovered that FoxO1 is an essential upstream regulator of the VCAM1 gene. Collectively, our findings provide critical molecular insight into a unique FoxO1 – VCAM1 axis that governs placental morphogenesis, a process that is essential for subsequent normal cardiovascular development and fetal life.

Background— Recent studies suggest that the hematopoietic and cardiac lineages have close ontogenic origins, and that an early mesodermal cell population has the potential to differentiate into both lineages. Studies also suggest that specification of these lineages is inversely regulated. However, the transcriptional networks that govern the cell fate specification of these progenitors are incompletely defined. Methods and Results— Here, we show that Nkx2-5 regulates the hematopoietic/erythroid fate of the mesoderm precursors early during cardiac morphogenesis. Using transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1 , in the Nkx2-5 –null embryos. We further observed that overexpression of Nkx2-5 with an Nkx2-5–inducible embryonic stem cell system significantly repressed Gata1 gene expression and suppressed the hematopoietic/erythroid potential, but not the endothelial potential, of the embryonic stem cells. This suppression was cell-autonomous, and was partially rescued by overexpressing Gata1. In addition, we demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene. Conclusions— Our results demonstrate that the hematopoietic/erythroid cell fate is suppressed via Nkx2-5 during mesodermal fate determination, and that the Gata1 gene is one of the targets that are suppressed by Nkx2-5.

In response to changes in workload, the heart grows or shrinks. Indeed, the myocardium is capable of robust and rapid structural remodeling. In the setting of normal, physiological demand, the heart responds with hypertrophic growth of individual cardiac myocytes, a process that serves to maintain cardiac output and minimize wall stress. However, disease-related stresses, such as hypertension or myocardial infarction, provoke a series of changes that culminate in heart failure and/or sudden death. At the other end of the spectrum, cardiac unloading, such as occurs with prolonged bed rest or weightlessness, causes the heart to shrink. In recent years, considerable strides have been made in deciphering the molecular and cellular events governing pro- and anti-growth events in the heart. Prominent among these mechanisms are those mediated by FoxO (Forkhead box-containing protein, O subfamily) transcription factors. In many cell types, these proteins are critical regulators of cell size, viability, and metabolism, and their importance in the heart is just emerging. Also in recent years, evidence has emerged for a pivotal role for autophagy, an evolutionarily conserved pathway of lysosomal degradation of damaged proteins and organelles, in cardiac growth and remodeling. Indeed, evidence for activated autophagy has been detected in virtually every form of myocardial disease. Now, it is clear that FoxO is an upstream regulator of both autophagy and the ubiquitin-proteasome system. Here, we discuss recent advances in our understanding of cardiomyocyte autophagy, its governance by FoxO, and the roles each of these plays in cardiac remodeling.

Recent studies support the existence of a common progenitor for the cardiac and endothelial cell lineages, but the underlying transcriptional networks responsible for specification of these cell fates remain unclear. Here we demonstrated that Ets-related protein 71 (Etsrp71), a newly discovered ETS family transcription factor, was a novel downstream target of the homeodomain protein, Nkx2–5. Using genetic mouse models and molecular biological techniques, we demonstrated that Nkx2–5 binds to an evolutionarily conserved Nkx2–5 response element in the Etsrp71 promoter and induces the Etsrp71 gene expression in vitro and in vivo. Etsrp71 was transiently expressed in the endocardium/endothelium of the developing embryo (E7.75-E9.5) and was extinguished during the latter stages of development. Using a gene disruption strategy, we found that Etsrp71 mutant embryos lacked endocardial/endothelial lineages and were nonviable. Moreover, using transgenic technologies and transcriptional and chromatin immunoprecipitation (ChIP) assays, we further established that Tie2 is a direct downstream target of Etsrp71. Collectively, our results uncover a novel functional role for Nkx2–5 and define a transcriptional network that specifies an endocardial/endothelial fate in the developing heart and embryo.

Destabilization of activator-DNA complexes by the proteasomal ATPases can inhibit transcription by limiting activator interaction with DNA. Modification of the activator by monoubiquitylation protects the activator from this destabilization activity. In this study, we probe the mechanism of this protective effect of monoubiquitylation. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and Rpt1. This interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. A model is proposed in which activator monoubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.

Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic as well as oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. We observed that the cardiac SP cells increase in number more than 3-fold within 3 days of injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals increased expression of cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide and is associated with increased levels of alpha-glutathione reductase protein expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene, we demonstrated that hypoxia-inducible factor (HIF)-2alpha binds an evolutionary conserved HIF-2alpha response element in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of Abcg2 expression by HIF-2alpha. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2alpha which functions with other factors to initiate a cytoprotective program for this progenitor SP cell population that resides in the adult heart.

We recently reported that the 19S regulatory subunit of the yeast 26S proteasome stimulates transcription elongation by RNA polymerase II. However, because of basic differences between yeast and mammals in the components and cellular location of the proteasome, it is crucial to assess whether this is a general phenomenon. Here we address this question and demonstrate that (1) the nonproteolytic activity of the 19S (PA700) complex of the proteasome is required for efficient activated transcription in the mammalian in vitro system, (2) this requirement applies to both natural and artificial activators, and (3) highly purified PA700 can provide this activity. In vitro transcription assays using HeLa cell nuclear extracts reveal that antibodies against human Trip1p/Rpt6 (mammalian Sug1p), one of the six ATPases in the PA700, significantly inhibit activated transcription. Similarly, immunodepletion of the PA700 from the extract also significantly reduces activated, but not basal, transcription and add-back of the highly purified mammalian PA700 restores the activity. Finally, inhibitors of the proteasome's peptidase activities do not affect transcription although the peptidase activity is almost completely inhibited. These findings indicate that the requirement for a nonproteolytic activity of the 19S complex in transcription is general in eukaryotes.

Background: Mesp1 and Etv2 are essential transcription factors in the regulation of mesodermal lineage development, but their relationship is unclear. Results: Mesp1 interacts physically with Creb1 and transcriptionally regulates Etv2 gene expression. Conclusion: Etv2 is a direct downstream target gene of Mesp1. Significance: This is the first report to identify Creb1 as a coactivator of Mesp1 to regulate gene expression. Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP+ cells are derived from Mesp1-Cre+ cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.

Background: The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in glucose metabolism and multiple diseases, regulation of the HBP remains largely undefined. Methods & Results: Here, we show that spliced Xbp1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers activation of the HBP by means of Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions known to promote O-GlcNAc modification. We go on to demonstrate that Xbp1s, acutely stimulated by ischemia/reperfusion (I/R) in heart, confers robust cardioprotection against I/R injury. We also show that HBP induction is required for this cardioprotective response. Mechanistically, HBP may mediate the adaptive branch of the UPR by activating autophagy and ER-associated degradation. Conclusion: These studies reveal that Xbp1s couples the UPR to the HBP, promoting robust cardioprotection during I/R.

Rationale: Cardiovascular health depends on proper development and integrity of blood vessels. Ets variant 2 (Etv2), a member of the E26 transforming–specific family of transcription factors, is essential to initiate a transcriptional program leading to vascular morphogenesis in early mouse embryos. However, endothelial expression of the Etv2 gene ceases at midgestation; therefore, vascular development past this stage must continue independent of Etv2. Objective: To identify molecular mechanisms underlying transcriptional regulation of vascular morphogenesis and homeostasis in the absence of Etv2. Methods and Results: Using loss- and gain-of-function strategies and a series of molecular techniques, we identify Friend leukemia integration 1 ( Fli1 ), another E26 transforming–specific family transcription factor, as a downstream target of Etv2. We demonstrate that Etv2 binds to conserved Ets-binding sites within the promoter region of the Fli1 gene and governs Fli1 expression. Importantly, in the absence of Etv2 at midgestation, binding of Etv2 at Ets-binding sites in the Fli1 promoter is replaced by Fli1 protein itself, sustaining expression of Fli1 as well as selective Etv2-regulated endothelial genes to promote endothelial cell survival and vascular integrity. Consistent with this, we report that Fli1 binds to the conserved Ets-binding sites within promoter and enhancer regions of other Etv2-regulated endothelial genes, including Tie2 , to control their expression at and beyond midgestation. Conclusions: We have identified a novel positive feed-forward regulatory loop in which Etv2 activates expression of genes involved in vasculogenesis, including Fli1 . Once the program is activated in early embryos, Fli1 then takes over to sustain the process in the absence of Etv2.

The ability to isolate highly proliferative hepatocytes is important for gaining insight into the molecular mechanisms of liver regeneration as well as for the development of a bio-hybrid artificial liver, because it is difficult to proliferate hepatocytes in vitro. The aim of this study was to isolate highly proliferative hepatocytes in the adult liver. We tried to separate hepatocytes expressing low levels of asialoglycoprotein receptor (ASGP-R) based on differences of adhesion of hepatocytes for poly[N-p-vinylbenzyl-O-β-d-galactopyranosyl-(1→-4)-d-gluconamide] (PVLA). PVLA is a β-galactose-carrying styrene polymer. This polymer is regarded as an artificial ligand for ASGP-R. We were able to get hepatocytes that could not adhere to PVLA when we weakened the interaction between PVLA and hepatocytes by decreasing calcium ion in the incubation buffer. Those hepatocytes that could not adhere to PVLA and had low ASGP-R expression were approximately 5% to 15% of the total number of hepatocytes. It is of interest that the hepatocytes that could not adhere to PVLA, which had lower ASGP-R expression levels, had higher (more than two times) DNA synthesizing activity (i.e., were more proliferative) than the hepatocytes that could adhere to PVLA, which had higher ASGR-R expression levels. These results suggest that the hepatocytes with lower adhesion to PVLA due to their low ASGP-R expression may be potential candidates for progenitor-like hepatocytes due to their highly proliferative capacity. These findings indicate that isolation of highly proliferative hepatocytes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.

It is generally thought that the primary or even sole activity of the 19S regulatory particle of the 26S proteasome is to facilitate the degradation of polyubiquitinated proteins by the 20S-core subunit. However, we present evidence that the 19S complex is required for efficient elongation of RNA polymerase II (RNAP II) in vitro and in vivo. First, yeast strains carrying alleles of SUG1 and SUG2, encoding 19S components, exhibit phenotypes indicative of elongation defects. Second, in vitro transcription is inhibited by antibodies raised against Sug1, or by heat-inactivating temperature-sensitive Sug1 mutants with restoration of elongation by addition of immunopurified 19S complex. Finally, Cdc68, a known elongation factor, coimmunoprecipitates with the 19S complex, indicating a physical interaction. Inhibition of the 20S proteolytic core of the proteasome has no effect on elongation. This work defines a nonproteolytic role for the 19S complex in RNAP II transcription.

We previously reported a novel strategy to stabilize purine motif triplex DNA within a mammalian gene promoter at physiologically relevant pH, temperature, and potassium (K(+)) concentrations by a comb-type poly(L-lysine)-graft-dextran copolymer [Ferdous et al., (1998) Nucleic Acids Res. 26, 3949-3954]. Here we describe the major contribution(s) of the copolymer to stabilize the purine motif triplex DNA at physiological K(+) concentrations. Self-aggregation through guanine-quartet formation of guanine-rich (G-rich) triplex-forming oligonucleotides (TFOs) has long been proposed for K(+)-mediated inhibition of the purine motif triplex formation. However, this was not the case for the severe inhibitory effect of K(+) observed under our reaction conditions. Rather significant decrease in rate of triplex formation involving a G-rich TFO was a major factor to confer K(+) inhibition. Interestingly, in the presence of the copolymer the rate of triplex formation was tremendously increased and K(+)-induced dissociation of preformed triplexes was not observed. Moreover, the triplex-promoting/stabilizing efficiency of the copolymer was amazingly higher than that of physiological concentrations of spermine. An absolute increase in binding constant of the TFO to the target duplex could therefore be the predominant mechanistic source for the copolymer-mediated triplex stabilization under physiological conditions in vitro.

We have previously demonstrated that the polycation comb-type copolymer having abundant grafts of hydrophilic polymer chains significantly stabilizes DNA duplexes and triplexes [Maruyama et al., Bioconjugate Chem., 8 (1997) 3, Ferdous et al., Nucleic Acids Res., 26 (1998) 39]. This study was designed to estimate the mechanisms involved in the copolymer-mediated stabilization of DNA duplexes and triplexes. The melting temperatures, T m , of DNA duplex and triplex increased with increasing salt concentration, as well documented by the Poisson–Boltzmann and counterion condensation theories that were originally proposed by Manning [J. Chem. Phys., 51 (1969) 924] and further elaborated by Manning [Biopolymers 11 (1972) 937, Biopolymers. 15 (1976) 2385] and Record [Biopolymers, 14 (1975) 2137–2158, Biopolymers, 15 (1976) 893]. In the presence of the copolymer, however, the T m values of DNA duplexes and triplexes did not show significant change with salt concentration. It was concluded that the copolymer is capable of reducing the counterion condensation effects to stabilize DNA duplexes and triplexes. Strong but exchangeable interaction between the copolymer and DNA is seemingly involved in the stabilization behavior.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (l-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Three-dimensional (3D) and two-dimensional (2D) cultures of hepatocytes in various concentrations (0.3–0.7%) of agarose gel revealed that the hepatocytes under 3D cultures in 0.3% agarose gel possess long-term (>3 weeks) viability, significant self-assembly to form tissue like aggregates, low lactate dehydrogenase release and high albumin synthesis. These were in contrast to 2D culture of hepatocytes. Our results suggest that the 3D culture of hepatocytes in agarose gel favors aggregate formation of functionally active cells and would be useful for liver transplantation as well as to analyze hepatocytes biology.

By employing a reductive amination reaction between the epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of the hydrophilic dextran (Dex) side chain, we have prepared different comb-type copolymers which varied in the degree of grafting and the length of the hydrophilic Dex chains. The resulting copolymers, poly(L-lysine)-graft-dextran (PLL-g-Dex), were tested for their ability to stabilize triplex DNA in vitro under physiologically relevant conditions. Thermal denaturation (UV-Tm) and circular dichroism experiments revealed that the graft copolymer with the higher degree of grafting of long Dex chains significantly increased the thermal stability of triplex structure of poly(dA). 2poly(dT) by more than 50 degreesC without affecting the transition between triplex and single-stranded DNA or the native structure of DNA. Of importance is that when triplex formation involving a 30-mer target duplex from rat alpha1 (I) collagen promoter was analyzed by an in vitro electrophoretic mobility shift assay, the graft copolymer also remarkably diminished potassium inhibition of the purine motif triplex formation up to 200 mM as well as pH-dependence of the pyrimidine motif triplex formation. Moreover the triplex-stabilizing efficiency of the copolymer was significantly higher than that of other oligocations like spermine and spermidine. We suggest that a molecular design of comb-type copolymers consisting of various types of polycation backbones (e.g., PLL) grafted with different hydrophilic side chains (e.g., Dex) is a novel strategy to create efficient triplex stabilizers that will certainly shed light on possible in vivo application of the antigene strategy.

Triplex DNA formation involving unmodified triplex-forming oligonucleotides (TFOs) is very unstable under physiological conditions. Here, we report a novel strategy to stabilize both purine and pyrimidine motif triplex DNA within the rat alpha1 (I) collagen gene promoter under physiologically relevant conditions by a poly(L-lysine)- graft -dextran copolymer. Using an in vitro electrophoretic mobility shift assay, we show that the copolymer almost completely abrogates the inhibitory effects of physiological concentrations of monovalent cations, particularly potassium ion (K+), on purine motif triplex formation involving very low concentrations of an unmodified guanine-rich TFO. Of importance, pH dependency in pyrimidine motif triplex formation involving an unmodified cytosine-rich TFO is also significantly overcome by the copolymer. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of other oligocations, like spermine and spermidine. We suggest that the ability of the graft copolymer to stabilize triplex DNA under physiologically relevant pH and salt concentrations will be a cue for further progress in the antigene strategy.

We have developed a simple method to purify sequence-specific DNA-binding proteins directly from crude cell extracts by using DNA affinity latex beads. The method enabled us to purify not only DNA-binding proteins, but also their associated proteins. Using beads bearing the ATF/E4TF3 site from the adenovirus E4 gene promoter, a protein kinase activity was copurified with the ATF/E4TF3 family. We found that the kinase interacted with ATF1 in vitro efficiently. The kinase did not bind directly to DNA. The kinase mainly phosphorylated ATF1 on serine 36, which was one of target amino acids for casein kinase (CK) II. Biological features of the kinase were the same as those of CKII and an anti-CKII serum reacted with the kinase, indicating that the kinase was CKII. Moreover, it was clearly shown that one of CKII subunits, the CKII alpha protein bound to glutathione-S-transferase (GST) fusion ATF1 but not GST in vitro. It has been reported that a specific CKII inhibitor, 5,6-dichloro-1-beta-D-ribo-furanosylbenzimidazole (DRB) inhibits transcription by RNA polymerase II [Zandomeni et al., (1986) J. Biol. Chem. 261, 3414-3419]. Taken together, these results suggest that ATF/E4TF3 may recruit the CKII activity to a transcription initiation machinery and stimulate transcription.

Congenital Heart Disease (CHD) is the most frequent and deadly birth defect. Patients with CHD that survive the neonatal period often progress to develop advanced heart failure requiring specialized treatment including cardiac transplantation. A full understanding of the transcriptional networks that direct cardiac progenitors during heart development will enhance our understanding of both normal cardiac function and pathological states. These findings will also have important applications for emerging therapies and the treatment of congenital heart disease. Furthermore, a number of shared transcriptional pathways or networks have been proposed to regulate the development and regeneration of tissues such as the heart. We have utilized transgenic technology to isolate and characterize cardiac progenitor cells from the developing mouse heart and have begun to define specific transcriptional networks of cardiovascular development. Initial studies identified Tdgf1 as a potential target of Nkx2- 5. To mechanistically dissect the regulation of this molecular program, we utilized an array of molecular biological techniques to confirm that Nkx2-5 is an upstream regulator of the Tdgf1 gene in early cardiac development. These studies further define Nkx2-5 mediated transcriptional networks and enhance our understanding of cardiac morphogenesis.

Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide, even despite recent scientific and technological advances and comprehensive preventive strategies. The cardiac myocyte is a voracious consumer of energy, and alterations in metabolic substrate availability and consumption are hallmark features of these disorders. Autophagy, an evolutionarily ancient response to metabolic insufficiency, has been implicated in the pathogenesis of a wide range of heart pathologies. However, the precise role of autophagy in these contexts remains obscure owing to its multifarious actions. Here, we review recently derived insights regarding the role of autophagy in cardiac hypertrophy and heart failure, highlighting its effects on metabolism.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP+ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

C-kit-ligand, also known as stem cell factor, is expressed broadly and has a functional role during hematopoesis, gametogenesis, melanogenesis, mast cell growth and differentiation. Although the receptor for c-kit-ligand, c-kit, has been utilized as a marker to identify cardiac stem cell and progenitor cell populations, the transcriptional regulation and biological function of c-kit-ligand during cardiogenesis has not been defined. Here we demonstrate that c-kit-ligand is a novel downstream target of Nkx2–5. The homeodomain transcription factor, Nkx2–5, is one of the earliest markers of the cardiac lineage and mice lacking this transcription factor are nonviable. To identify potential Nkx2–5 downstream target genes, we utilized ES/EBs that were engineered to overexpress Nkx2–5 and undertook transcriptome analysis of embyroid bodies with and without Nkx2–5 induction. We observed a significant increase in c-kit-ligand expression following Nkx2–5 induction suggesting a role for Nkx2–5 in the activation of c-kit-ligand. Furthermore, analysis of the c-kit-ligand promoter revealed three evolutionarily conserved Nkx2–5 response elements, supporting the notion that Nkx2–5 is a transcriptional regulator of gene expression. We undertook transcriptional assays and transfected the c-kit-ligand promoter-luciferase reporter in the absence and presence of increasing amounts of Nkx2–5. We observed that Nkx2–5, in a dose dependent fashion, was a potent transcriptional activator of c-kit-ligand. These studies enhance our understanding of Nkx2–5 mediated transcriptional networks and further emphasize that Nkx2–5 is an important transcriptional regulator of cardiac progenitor cell populations.

Recent analysis of a Gal4 mutant (Gap71) carrying three point mutations (S22D, K23Q and K25F) in its DNA-binding domain (DBD), has demonstrated that it cannot occupy GAL promoters efficiently in cells and that it is not mono-ubiquitylated, suggesting a functional link between this modification and stable DNA binding in cells. The mechanistic underpinning of this phenotype is that this protein is hypersensitive to a newly discovered activity of the proteasomal ATPases--their ability to actively dissociate transcription factor-DNA complexes after direct interaction with the activation domain. In this paper, we examine the roles of each of the three point mutations contained in Gap71 individually. These experiments have revealed that serine 22 is a site of phosphorylation in the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event that is critical for the activity of this paradigmatic transcription factor.

Recent studies have shown that the intersection between transcription and proteins involved in the ubiquitin–proteasome pathway encompasses both proteolytic and nonproteolytic functions. Examples of the latter type include evidence that monoubiquitylation of some transcriptional activators stimulates their activity. In addition, the proteasomal ATPases are recruited to many active promoters through binding to activators and play an important, nonproteolytic role in promoter escape and elongation. In this study, we report the discovery of a new nonproteolytic activity of the proteasome (specifically the proteasomal ATPases): the active destabilization of activator–promoter complexes. This reaction depends on the presence of an activation domain and ATP. Destabilization is inhibited in vitro and in vivo if the protein is monoubiquitylated or if ubiquitin is genetically fused to the activator. The fact that monoubiquitylated activator is resistant to the “stripping” activity of the proteasomal ATPases may explain, in part, why some activators require this modification in order to function efficiently.

Synthetic activators that mimic the ability of native transcription factors to recruit the RNA polymerase holoenzyme to specific promoters could, in principle, be constructed by joining a sequence-specific DNA binding moiety with a molecule able to bind the holoenzyme. We report here that a peptide nucleic acid (PNA)-peptide chimera is capable of activating transcription in vitro in a HeLa nuclear extract. Specifically, a promoter-targeted PNA alone acts as a strong inhibitor of basal transcription in a HeLa nuclear extract, presumably due to structural modification of the promoter. However, the fusion of a Gal80-binding peptide to the PNA, but not control peptides, reactivates transcription. The Gal80-binding peptide was selected solely on the basis of its ability to bind the yeast repressor.

Developmental and regenerative mechanisms are directed by stem cell populations. Skeletal muscle is a dynamic tissue that is capable of adapting to stress and severe injury due to a resident somatic stem cell population. In response to a severe injury that destroys upward of 90% of the tissue, skeletal muscle efficiently and reproducibly regenerates damaged tissue and restores the cellular architecture within a 2-wk period. Recent studies have localized and examined the molecular regulation of skeletal muscle stem cell populations using emerging molecular biological technologies. These studies enhance the understanding of the regulatory mechanisms that direct the somatic stem cell populations and the role they play in development and regeneration. Furthermore, these basic science studies will serve as a platform for future therapies directed toward patients with myopathic diseases.

The polyionic interaction between DNA and polycations grafted with hydrophilic dextran side chains was evaluated. The comb-type copolymers, poly(L-lysine)-graft-dextran, were successfully prepared by employing a reductive amination reaction between epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of dextran (Dex). A coupling efficacy on the order of 70% was obtained regardless of intrinsic philicities of the solvents used, either aqueous buffer or DMSO. The resulting graft copolymers, which varied in the degree of grafting and the length of hydrophilic side chains, formed a soluble complex with DNA. They also affected the melting behavior of double-stranded DNA (dsDNA) in different ways. Copolymers having a high degree of grafting thermally stabilized dsDNA without affecting its reversible transition between single-stranded and double-stranded forms. However, copolymers with a low degree of grafting or with a high degree of grafting of short dextran chains impeded the reversibility of this transition. Furthermore, highly grafted copolymers also accelerated the hybridization of DNA strands in a low-ionic strength medium. It is of particular note that these copolymers scarcely altered circular dichroismic signals of dsDNA even when the copolymers were added in excess. This suggested that the copolymer interacted with dsDNA without affecting its native structure or physicochemical properties. Finally, the copolymer even formed a stable complex with a short oligonucleotide (20 bases). We, therefore, concluded that, by regulating the degree of grafting and the molecular weight of grafted side chains, it would be possible to design novel different graft copolymers capable of acting as carriers of functional genes to target cells or tissue.

We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

The binding constant of a homopyrimidine oligonucleotide with its target duplex for the pyrimidine-motif triplex formation at neutral pH was analyzed and found to be remarkably higher in the presence of the poly (L-lysine)-graft-dextran copolymer than that without any triplex stabilizer or in the presence of spermine. The promoting effect mainly results from the considerable increase in the association rate constant.

Various comb-type copolymer containing a polycation as a main chain was design to construct delivery systems of DNAs. The comb-type copolymers having cell-specific polysaccharides were proved to be useful to deliver DNA to the target cells in vivo. Of interest, the copolymers with abundant side chains of hydrophilic polymers are capable of stabilizing DNA triplex. Further, injectable nanoparticles for controlled releases of DNAs were fabricated from the copolymer and a biodegradable polymer.

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL-g-HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL-g-HA complexed to a beta-galactosidase expression plasmid (pSV beta-Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier-DNA complex was distributed exclusively in SECs. A large number of SECs expressing beta-galactosidase was detected along the sinusoidal lining after transfection with PLL-g-HA/pSV beta-Gal. Moreover, PLL-g-HA effectively stabilized DNA triplex formation. In conclusion, the new PLL-g-HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Triplex-stabilizing effect of a graft copolymer under physiologically relevant conditions has been evaluated and compared with other polyamines. Here we show that the graft copolymer significantly stabilizes triplex DNAs with amazingly higher efficacy than that of physiological concentrations of spermine and spermidine.

Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. Electrophoretic mobility shift assays using a 30-mer homopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat alpha 1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein-DNA interaction. When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a plasmid DNA, copolymer-mediated triplex stabilization also remarkably competes endonuclease activity of BamH1. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of spermine and benzo[e]pyridoindole. Our results indicate that the copolymer, regardless of the length of the target duplex, stabilizes triplexes for significant inhibition of protein-DNA interaction and endonuclease activity. Since stable triplex formation within a short region out of a long native duplex is a prerequisite to confer the therapeutic potential of antigene strategy, triplex stabilization on a long target duplex and inhibition of nuclear protein-DNA interaction may open the possible in vivo applicability of the copolymer.