Notch regulates the angiogenic response via induction of VEGFR-1

Notch is a critical regulator of angiogenesis and arterial specification. We show that ectopic expression of activated Notch1 induces endothelial morphogenesis in human umbilical vein endothelial cells (HUVEC) in a VEGFR-1-dependent manner. Notch1-mediated upregulation of VEGFR-1 in HUVEC increased their responsiveness to the VEGFR-1 specific ligand, Placental Growth Factor (PlGF). In mice and human endothelial cells, inhibition of Notch signaling resulted in decreased VEGFR-1 expression during VEGF-A-induced neovascularization. In summary, we show that Notch1 plays a role in endothelial cells by regulating VEGFR-1, a function that may be important for physiological and pathological angiogenesis

The Role of P300-Dependent MYC Acetylation in MYC Functions

ABSTRACT OF THE DISSERTATIONThe Role of P300-Dependent MYC Acetylation in MYC FunctionsbyMarina VorontchikhinaDoctor of Philosophy, Graduate Program in Cell, Molecular and Developmental BiologyUniversity of California, Riverside, December 2011Dr. Ernest Martinez, ChairpersonThe MYC oncoprotein regulates transcription of a multitude of downstream target genes triggering various biological outcomes, such as the induction of cellular proliferation, apoptosis, and oncogenic transformation. While MYC protein levels and activity are tightly controlled in normal cells, MYC is deregulated in most human malignancies. Since cancer is one of the leading causes of death worldwide, it is vital to elucidate the molecular and biochemical mechanisms underlying the modification and regulation of the MYC protein, whose overexpression contributes to the development of most malignant tumors. Posttranslational modifications are implicated in the regulation of MYC stability and function. For instance, several co-activators/histone acetyltransferases (HATs) have been shown to bind and acetylate the MYC protein affecting its turnover by the proteasome. Co-activator/HAT p300 interacts with MYC increasing its stability and transactivation functions. However, once p300 acetylates the oncoprotein at seven lysine residues, MYC becomes more unstable due to induced degradation via the proteasome pathway. While MYC acetylation has been established, the role of acetylation in MYC biological functions has not been determined. Here, I report that by using site-directed mutagenesis I have identified lysine 158 in MYC as the major residue acetylated by the p300. I also demonstrate that acetylation of K158 reduces MYC-activated apoptosis which could be related to MYC-dependent regulation of certain pro-apoptotic genes associated with mitochondrial function, as it was shown by Real-time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis. Furthermore, I demonstrate that MYC transcriptionally activates p300 in mammalian cells and, by using Luciferase reporter assays, I further show that MYC and co-activator p300 synergistically activate the promoter of the Cyclin D2 (CCND2), a well-known cell cycle regulatory gene. Moreover, by utilizing immunoprecipitation methods, I establish a link between MYC acetylation by p300 and its interaction with the co-activator/HAT, TIP60. Other cell and molecular biology procedures, such as immunofluorescence and RNA interference, were used in this study as well. These findings begin to uncover a role of co-activator/HAT p300 in MYC biological functions and are important because they suggest potential new targets for the treatment of MYC-dependent cancers

STAGA Recruits Mediator to the MYC Oncoprotein To Stimulate Transcription and Cell Proliferation▿

Activation of eukaryotic gene transcription involves the recruitment by DNA-binding activators of multiprotein histone acetyltransferase (HAT) and Mediator complexes. How these coactivator complexes functionally cooperate and the roles of the different subunits/modules remain unclear. Here we report physical interactions between the human HAT complex STAGA (SPT3-TAF9-GCN5-acetylase) and a “core” form of the Mediator complex during transcription activation by the MYC oncoprotein. Knockdown of the STAF65γ component of STAGA in human cells prevents the stable association of TRRAP and GCN5 with the SPT3 and TAF9 subunits; impairs transcription of MYC-dependent genes, including MYC transactivation of the telomerase reverse transcriptase (TERT) promoter; and inhibits proliferation of MYC-dependent cells. STAF65γ is required for SPT3/STAGA interaction with core Mediator and for MYC recruitment of SPT3, TAF9, and core Mediator components to the TERT promoter but is dispensable for MYC recruitment of TRRAP, GCN5, and p300 and for acetylation of nucleosomes and loading of TFIID and RNA polymerase II on the promoter. These results suggest a novel STAF65γ-dependent function of STAGA-type complexes in cell proliferation and transcription activation by MYC postloading of TFIID and RNA polymerase II that involves direct recruitment of core Mediator

MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival

The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities

Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis