Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF 165 ) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF 165 do not inhibit VEGF 165 -induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF 121 , a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF 165 activity does not depend on competition with VEGF 165 for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.

Members of the lysine (K)-specific demethylase 4 (KDM4) A-D family of histone demethylases are dysregulated in several types of cancer. Here, we reveal a previously unrecognized role of KDM4D in the DNA damage response (DDR). We show that the C-terminal region of KDM4D mediates its rapid recruitment to DNA damage sites. Interestingly, this recruitment is independent of the DDR sensor ataxia telangiectasia mutated (ATM), but dependent on poly (ADP-ribose) polymerase 1 (PARP1), which ADP ribosylates KDM4D after damage. We demonstrate that KDM4D is required for efficient phosphorylation of a subset of ATM substrates. We note that KDM4D depletion impairs the DNA damage-induced association of ATM with chromatin, explaining its effect on ATM substrate phosphorylation. Consistent with an upstream role in DDR, KDM4D knockdown disrupts the damage-induced recombinase Rad51 and tumor protein P53 binding protein foci formation. Consequently, the integrity of homology-directed repair and nonhomologous end joining of DNA breaks is impaired in KDM4D-deficient cells. Altogether, our findings implicate KDM4D in DDR, furthering the links between the cancer-relevant networks of epigenetic regulation and genome stability.histone demethylation | chromosome instability | PARylation

No abstract

In the budding yeast Saccharomyces cerevisiae, entry into meiosis and its successful completion depend on two positive regulators, Ime1 and Ime2. Ime1 is a transcriptional activator that is required for transcription of IME2, a serine/threonine protein kinase. We show that in vivo Ime2 associates with Ime1, that in vitro Ime2 phosphorylates Ime1, and that in living cells the stability of Ime1 depends on Ime2. Diploid cells with IME2 deleted show an increase in the level of Ime1, whereas haploid cells overexpressing IME2 show a decrease in the stability of Ime1. Furthermore, the level of Ime1 depends on the kinase activity of Ime2. Using a mutation in one of the ATPase subunits of the proteasome, RPT2, we demonstrate that Ime1, amino acids 270 to 360, is degraded by the 26S proteasome. We also show that Ime2 itself is an extremely unstable protein whose expression in vegetative cultures is toxic. We propose that a negative-feedback loop ensures that the activity of Ime1 will be restricted to a narrow window.Successful progression and completion of the mitotic cell cycle depends on transcriptional and proteolytic regulation. These two processes determine the availability of cyclins and cyclin-dependent kinase (CDK) inhibitors that govern the sequential activation of CDKs (18,28,31,35). Initiation and progression through the meiotic cycle should also be subjected to transcriptional and proteolytic regulation. Indeed, in budding yeast a transcriptional cascade governs initiation and progression through the meiotic cell cycle (4). Yet there is no direct evidence concerning proteolysis of either positive or negative meiotic regulators. This report focuses on the regulated degradation of one of the two positive regulators of meiosis in Saccharomyces cerevisiae, Ime1, by the other, Ime2.IME1 encodes a transcriptional activator (30, 47) that is necessary for the transcription of meiosis-specific genes (48). Ime1 is tethered to promoters of early meiosis-specific genes, such as IME2, by a specific DNA-binding protein, Ume6 (39). Diploid cells with deletions of IME1 arrest at G 1 prior to the initiation of premeiotic DNA replication (22). The transcription of IME1 is regulated by nutrients. In vegetative cultures with glucose as the sole carbon source, IME1 is silent, but in the presence of acetate, low levels of IME1 mRNA are observed (22). Under meiotic conditions, i.e., nitrogen depletion and the presence of a nonfermentable carbon source such as acetate, transcription of IME1 is induced transiently in MATa/ MAT␣ diploids (22). It is not known whether this transient transcription reflects transient availability of the Ime1 protein.In addition, the IME1 promoter is subject to positive autoregulation (40,43,44), as well as negative-feedback regulation by both Ime1 and Ime2 (43,48,49).Another important regulator of meiosis and sporulation is the serine/threonine protein kinase Ime2 (12,24,34,48,49). Diploid cells with deletions of IME2 show a 5-to 12-h delay in the transcription of early meiosis-specific genes, a reduction...