The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.

Vascular endothelial growth factor receptor-3 (VEGFR-3) plays a key role for the remodeling of the primary capillary plexus in the embryo and contributes to angiogenesis and lymphangiogenesis in the adult. However, VEGFR-3 signal transduction pathways remain to be elucidated. Here we investigated VEGFR-3 signaling in primary human umbilical vein endothelial cells (HUVECs) by the systematic mutation of the tyrosine residues potentially involved in VEGFR-3 signaling and identified the tyrosines critical for its function. Y1068 was shown to be essential for the kinase activity of the receptor. Y1063 signals the receptor-mediated survival by recruiting CRKI/II to the activated receptor, inducing a signaling cascade that, via mitogen-activated protein kinase kinase-4 (MKK4), activates c-Jun N-terminal kinase-1/2 (JNK1/2). Inhibition of JNK1/2 function either by specific peptide inhibitor JNKI1 or by RNA interference (RNAi) demonstrated that activation of JNK1/2 is required for a VEGFR-3-dependent prosurvival signaling. Y1230/Y1231 contributes, together with Y1337, to proliferation, migration, and survival of endothelial cells.Phospho-Y1230/Y1231 directly recruits growth factor receptor-bonus protein (GRB2) to the receptor, inducing the activation of both AKT and extracellular signal-related kinase 1/2 (ERK1/2) signaling. IntroductionVascular endothelial growth factor receptor-1 (VEGFR-1) (also known as Flt-1), VEGFR-2 (also known as KDR/Flk-1), and VEGFR-3 (also known as Flt-4) play a central role in vasculogenesis and angiogenesis. 1,2 They are activated by the family of angiogenic factors that is composed of several members with partially overlapping function due to the promiscuity of receptor recognition. Placental growth factor (PlGF) and vascular endothelial growth factor-B (VEGF-B) recognize VEGFR-1, VEGF is the ligand of both VEGFR-1 and VEGFR-2, while VEGF-C and VEGF-D each recognize and activate VEGFR-2 and VEGFR-3. These receptors are highly related, with an extracellular ligand binding site containing 7 immunoglobulin-homology segments, a single polypeptide chain transmembrane sequence, and an interrupted intracellular tyrosine kinase domain. Gene disruption experiments demonstrated that each of these receptors is essential for vascular development and angiogenesis. 2 Both VEGFR-1 and VEGFR-2 act early during endothelial differentiation although with different functions: while Vegfr-1 Ϫ/Ϫ embryos die for excessive endothelial proliferation, Vegfr-2 Ϫ/Ϫ die with no endothelial or hematopoietic cells. 3,4 The absence of Vegfr-3 does not impair the differentiation of endothelial cells or the formation of the primary vascular plexus; rather, it affects the remodeling of the primary vascular network. 5 In adults, VEGFR-3 is expressed mainly in lymphatic endothelial cells. 6,7 VEGFR-3 is also expressed in quiescent endothelial cells of fenestrated capillaries and microvessels of several tissues. [8][9][10] It is transiently induced in vessels undergoing active angiogenesis during wound healing and in tumors. [1...

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Key Words: cell adhesion Ⅲ endothelial cells Ⅲ angiogenesis Ⅲ biochemistry Ⅲ signaling C rosstalk between integrins and growth factor receptors plays an important role in vascular development and its maintenance. Several examples demonstrated that crosstalk between integrins and tyrosine kinase receptors is required for growth factor-induced biological processes to ensure cell growth, survival, and differentiation in normal and pathological processes. 1 The angiogenic processes require the coordination of signals from the extracellular environment to activates specific tyrosine kinase receptors and integrins. 1,2 Integrins associating with growth factor receptors regulate the capacity of the integrin/receptor complexes to propagate downstream signals. 3,4 Integrin-dependent activation of receptor tyrosine kinases is a general mechanism to enhance growth factor signals, the recruitment of transducing proteins to membrane cytoskeletal complexes as well as nuclear responses. This cooperation has been shown for several signaling pathways including insulin, epidermal growth factor (EGF), platelet-derived growth factor, fibroblast growth factor, and vascular endothelial growth factor (VEGF) signaling. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] Integrins are able to trigger ligand-independent EGF receptor autophosphorylation leading to activation of the downstream pathway. 16 Integrins, c-Src, p130Cas and EGF receptor associate in a macromolecular complex on the cell membrane and integrin-dependent adhesion induces phosphorylation of specific tyrosine residues of EGF receptor, distinct from those obtained by the soluble ligand EGF. 11,19 A large body of evidence demonstrates that the angiogenic process is initiated by mitogenic signals induced by growth factors such as VEGF and the interplay between the tyrosine 34 -36 VEGFR-3, in contrast to its highly related endothelial receptor VEGFR-2 (which, after VEGF stimulation, forms a complex with integrin ␤3 subunit 8,20,21,24,25,37 ), associates selectively with integrin ␤1. 10,12 Cell attachment to fibronectin or collagen induces the phosphorylation of VEGFR-3 in the absence of a ligand and significantly enhances the phosphorylation of the receptor induced by its ligand. 10,12 However, the mechanism of integrin activation of VEGFR-3 has not been clarified.Here, we demonstrate that collagen I-induced activation of VEGFR-3 is independent from the intrinsic catalytic activity of the receptor but it is a direct target of c-Src. c-Src phosphorylates VEGFR-3 at specific tyrosine residues with a pattern of phosphorylation that is distinct from the pattern induced by the ligand. Cell adhesion induces the phosphorylation of the tyrosine residues 1063 and 1337, which are known binding sites for CRKI/II and SHC. Pull-down assays demonstrated that integrin-mediated receptor phosphorylation induces the recruitment of CRKI/II and SHC second messengers to the receptor suggesting that the integrin/receptor complex can activate growth and survival signaling in the absence ...