Yu Chung Yang

The essential role of Cited2, a negative regulator for HIF-1α, in heart development and neurulation

Cited2 is a cAMP-responsive element-binding protein (CBP)/p300 interacting transcriptional modulator and a proposed negative regulator for hypoxia-inducible factor (HIF)-1α through its competitive binding with HIF-1α to CBP/p300. Disruption of the gene encoding Cited2 is embryonic lethal because of defects in the development of heart and neural tube. Morphological and Doppler echocardiographic analyses of Cited2−/− embryos reveal severe cardiovascular abnormalities, including pulmonic arterial stenosis and ventricular septal defects accompanied by high peak outflow velocities, features of the human congenital cardiac defect termed tetralogy of Fallot. The mRNA levels of several HIF-1α-responsive genes, such as vascular endothelial growth factor (VEGF), Glut1, and phosphoglycerate kinase 1, increased in the Cited2−/− hearts. The increase of VEGF levels is significant, because defects in the Cited2−/− embryos closely resemble the major defects observed in the VEGF transgenic embryos. Finally, compared with wild-type, cultured fibroblasts from Cited2−/− embryos demonstrate an enhanced expression of HIF-1α-responsive genes under hypoxic conditions. These observations suggest that functional loss of Cited2 is responsible for defects in heart and neural tube development, in part because of the modulation of HIF-1 transcriptional activities in the absence of Cited2. These findings demonstrate that Cited2 is an indispensable regulatory gene during prenatal development.

TIP60 is a co-repressor for STAT3

Tip60 (Tat-interactiveprotein, 60 kDa), a cellular protein with intrinsic histone acetyltransferase activity, is involved in DNA damage repair and apoptosis. Recent studies have suggested that Tip60 acts either as a co-activator or a co-repressor to modulate transcription. In this study, we demonstrate that Tip60 represses reporter gene expression when it is fused to the Gal4 DNA binding domain. We also show that Tip60 associates with histone deacetylase 7 (HDAC7) through its N-terminal zinc finger-containing region and that HDAC7 activity is required for the repressive effect of Tip60. Because endogenous Tip60 interacts with STAT3, we hypothesized that Tip60 might complex with STAT3 and HDAC7 and modulate STAT3-mediated trans-activation. Consistent with this hypothesis, the overexpression of Tip60 represses STAT3-driven reporter gene expression, which can be further potentiated by the co-transfection of HDAC7. Furthermore, interleukin-9-induced c-myc expression, which depends on STAT3 activity, is abrogated by exogenous expression of Tip60. This is the first demonstration of which Tip60 represses STAT3 activity in part through the recruitment of HDAC7.

Cited2, a coactivator of HNF4α, is essential for liver development

The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2−/− fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell‐cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver‐enriched transcription factor HNF4α. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4α‐responsive promoters and the reduced HNF4α binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4α.

HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency

Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.

Cited2 is required for fetal lung maturation

Lung maturation at the terminal sac stage of lung development is characterized by a coordinated increase in terminal sac formation and vascular development in conjunction with the differentiation of alveolar type I and type II epithelial cells. The Cited2-Tcfap2a/c complex has been shown to activate transcription of Erbb3 and Pitx2c during mouse development. In this study, we show that E17.5 to E18.5 Cited2-null lungs had significantly reduced terminal sac space due to an altered differentiation of type I and type II alveolar epithelial cells. In addition, E17 Cited2-null lungs exhibited a decrease in the number of apoptotic cells, contributing to the loss in airspace. Consistent with the phenotype, genes associated with alveolar cell differentiation and survival were differentially expressed in Cited2-null fetal lungs compared to those of wild-type littermates. Moreover, expression of Cebpa, a key regulator of airway epithelial maturation, was significantly decreased in Cited2-null fetal lungs. Cited2 and Tcfap2c were present on the Cebpa promoter in E18.5 lungs to activate Cebpa transcription. We propose that the Cited2-Tcfap2c complex controls lung maturation by regulating Cebpa expression. Understanding the function of this complex may provide novel therapeutic strategies for patients with respiratory distress syndromes.

Expression of Active Notch1 in Avian Coronary Development

Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear‐localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro‐epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD‐positive endothelial cells were detected within the nascent vessel plexus at the atrioventricular junction and within the compact myocardium (Hamburger and Hamilton stage [HH] 25–HH30). The endothelial cells expressing N1ICD were surrounded by N1ICD‐positive smooth muscle cells after coronary orifice formation (HH32–HH35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1‐VEGF‐Notch pathway may play a role in epicardial cell interactions that promote epicardial epithelial/mesenchymal transition and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions. Developmental Dynamics 238:162–170, 2009.

Conditional Deletion of Cited2 Results in Defective Corneal Epithelial Morphogenesis and Maintenance

Cited2 is an important transcriptional cofactor involved in multiple organ development. Gene profile analysis has identified Cited2 as one of the transcription factors expressed at high levels in adult mouse cornea. To address the function of Cited2 in corneal morphogenesis, we deleted Cited2 in surface ectoderm derived ocular structures including cornea by crossing Cited2-floxed mice with Le-Cre transgenic mice. Cited2(flox/flox);Le-Cre(+) eyes invariably displayed corneal opacity and developed spontaneous corneal neovascularization at older age. Fewer layers of corneal epithelial cells and the absence of cytokeratin 12 (K12) expression featured Cited2 deficient postnatal and adult eyes. Cited2 deficient cornea exhibited impaired healing in response to corneal epithelial debridement by manifesting abnormal histology, lack of K12 expression and corneal neovascularization. Moreover, mechanistic studies suggest that Cited2 may play a role in corneal morphogenesis in part through modulating the expression of Pax6 and Klf4. Collectively, these findings demonstrate a novel function of Cited2 in postnatal corneal morphogenesis and maintenance. Our study will help better understand the molecular mechanisms involved in corneal biology, and more importantly, it may provide a valuable animal model for testing therapeutics in the treatment of corneal disorders, especially blindness as a result of corneal epithelial cell deficiency.

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene

Background: Role of Cited2 in murine embryonic stem cell pluripotency and differentiation remains elusive. Results: Cited2 knock-out ESCs exhibit defective cardiomyocyte differentiation with delayed silence of Oct4 expression. Conclusion: Cited2 is essential for proper ESC differentiation toward cardiomyocytes. Significance: This study identifies Cited2 as an important mediator of ESC cell fate decision through direct regulation of Oct4 expression. Cited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail 2) is a transcriptional modulator critical for the development of multiple organs. Although many Cited2-mediated phenotypes and molecular events have been well characterized using in vivo genetic murine models, Cited2-directed cell fate decision in embryonic stem cells (ESCs) remains elusive. In this study, we examined the role of Cited2 in the maintenance of stemness and pluripotency of murine ESCs by a gene-targeting approach. Cited2 knock-out (Cited2Δ/−, KO) ESCs display defective differentiation. Loss of Cited2 in differentiating ESCs results in delayed silencing of the genes involved in the maintenance of pluripotency and self-renewal of stem cells (Oct4, Klf4, Sox2, and c-Myc) and the disturbance in cardiomyocyte, hematopoietic, and neuronal differentiation. In addition, Cited2 KO ESCs experience a delayed induction of cardiomyocyte differentiation-associated proteins, NFAT3 (along with the reduced expression of NFAT3 target genes, Nkx2.5 and β-MHC), N-cadherin, and smooth muscle actin. CITED2 is recruited to the Oct4 promoter to regulate its expression during early ESC differentiation. This is the first demonstration that Cited2 controls ESC pluripotency and differentiation via direct regulation of Oct4 gene expression.

Cited2 Is Required for the Maintenance of Glycolytic Metabolism in Adult Hematopoietic Stem Cells

Mammalian adult hematopoietic stem cells (HSCs) reside in the hypoxic bone marrow microenvironment and display a distinct metabolic phenotype compared with their progenitors. It has been proposed that HSCs generate energy mainly through anaerobic glycolysis in a pyruvate dehydrogenase kinase (Pdk)-dependent manner. Cited2 is an essential regulator for HSC quiescence, apoptosis, and function. Herein, we show that conditional deletion of Cited2 in murine HSCs results in elevated levels of reactive oxygen species, decreased cellular glutathione content, increased mitochondrial activity, and decreased glycolysis. At the molecular level, Cited2 deficiency significantly reduced the expression of genes involved in metabolism, such as Pdk2, Pdk4, and lactate dehydrogenases B and D (LDHB and LDHD). Cited2-deficient HSCs also exhibited increased Akt signaling, concomitant with elevated mTORC1 activity and phosphorylation of FoxOs. Further, inhibition of PI3/Akt, but not mTORC1, partially rescued the repression of Pdk4 caused by deletion of Cited2. Altogether, our results suggest that Cited2 is required for the maintenance of adult HSC glycolytic metabolism likely through regulating Pdk2, Pdk4, LDHB, LDHD, and Akt activity.

Cited2, a Transcriptional Modulator Protein, Regulates Metabolism in Murine Embryonic Stem Cells

Background: The function of HIF-1, a master regulator of metabolism, is in part modulated by Cited2. The role of Cited2 in murine embryonic stem cell (mESC) glucose metabolism remains unknown. Results: Deletion of Cited2 in mESCs results in impaired mitochondria morphology, reduced glucose oxidation, increased glycolysis, and defective mESC differentiation. Conclusion: Cited2 coordinates glucose metabolism to regulate mESC differentiation. Significance: Cited2 is a potential target for metabolic reprogramming in mESCs. CREB-binding protein (CBP)/p300 interacting transactivator with glutamic acid (Glu) and aspartic acid (Asp)-tail 2 (Cited2) was recently shown to be essential for gluconeogenesis in the adult mouse. The metabolic function of Cited2 in mouse embryonic stem cells (mESCs) remains elusive. In the current study, the metabolism of glucose was investigated in mESCs, which contained a deletion in the gene for Cited2 (Cited2Δ/−). Compared with its parental wild type counterpart, Cited2Δ/− ESCs have enhanced glycolysis, alternations in mitochondria morphology, reduced glucose oxidation, and decreased ATP content. Cited2 is recruited to the hexokinase 1 (HK1) gene promoter to regulate transcription of HK1, which coordinates glucose metabolism in wild type ESCs. Reduced glucose oxidation and enhanced glycolytic activity in Cited2Δ/− ESCs correlates with defective differentiation during hypoxia, which is reflected in an increased expression of pluripotency marker (Oct4) and epiblast marker (Fgf5) and decreased expression of lineage specification markers (T, Gata-6, and Cdx2). Knockdown of hypoxia inducible factor-1α in Cited2Δ/− ESCs re-initiates the expression of differentiation markers T and Gata-6. Taken together, a deletion of Cited2 in mESCs results in abnormal mitochondrial morphology and impaired glucose metabolism, which correlates with a defective cell fate decision.