Noga Guttmann-Raviv

Semaphorin-3A and Semaphorin-3F Work Together to Repel Endothelial Cells and to Inhibit Their Survival by Induction of Apoptosis

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF165) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF165 do not inhibit VEGF165-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF121, 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 VEGF165 activity does not depend on competition with VEGF165 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.

Ime2, a meiosis-specific kinase in yeast, is required for destabilization of its transcriptional activator, Ime1.

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.

Segregation of arterial and venous markers in subpopulations of blood islands before vessel formation

The neuropilin‐1 (np1) and the neuropilin‐2 (np2) receptors bind vascular endothelial growth factor (VEGF) and class‐3 semaphorins. They form complexes with VEGF tyrosine‐kinase receptors or alternatively with type‐A plexins to transduce respective VEGF or semaphorin signals. We have compared the expression patterns of np1, np2, plexin‐A1, and plexin‐A2 in the emerging vasculature of chick embryos. Double in situ hybridization reveals that six‐somite embryos contain intermingled extraembryonic blood island (BI) subpopulations that express np1 or np2 as well as a BI subpopulation that coexpresses both neuropilins. In 13‐somite embryos, which already contain an extraembryonic vascular plexus, the expression of np1 and np2 is segregated between the arterial and venous parts of the plexus, despite the absence of blood flow. However, the arterial marker ephrin‐B2 was not yet expressed in the plexus at this stage. In 26‐somite embryos, which possess a functional vascular system, np1 and np2 are differentially expressed in arteries and veins as previously reported. At this stage, posterior BIs expressing np2 appear to undergo fusion to form the posterior sinus vein and its tributaries, suggesting that the venous identity of these veins may be established before their formation. The neuropilin coreceptor plexin‐A2 was expressed in extraembryonic veins but not in extraembryonic arteries. In contrast, within the embryo, plexin‐A2 expression was observed in the dorsal aorta as well as in the cardinal vein. Semaphorin‐3F (s3f), an np2 ligand, bound to np2‐expressing cells in 26‐somite embryos regardless of the presence or absence of plexin‐A1 or plexin‐A2. Of interest, even though s3f binds to np1 in vitro, np1‐expressing arteries fail to bind s3f in whole‐mount binding experiments. Developmental Dynamics 232:1047–1055, 2005.

PARP1-dependent recruitment of KDM4D histone demethylase to DNA damage sites promotes double-strand break repair

Significance Sophisticated DNA damage repair mechanisms are required to fix DNA lesions and preserve the integrity of the genome. This manuscript provides characterization of KDM4D role in promoting the repair of double-strand breaks (DSBs). Our findings show that KDM4D lysine demethylase is swiftly recruited to DNA breakage sites via its C-terminal region in a PARP1-dependent manner. Further, we have uncovered an exciting function of KDM4D in regulating the association of the DNA damage response master kinase, ATM, with chromatin, thus explaining the defective phosphorylation of ATM substrates found in KDM4D-depleted cells. Altogether, this study advances our understanding of the molecular mechanisms that regulate the repair of DSBs, a critical pathway that is essential for maintaining genome integrity. 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.

Heat shock protein 90 (Hsp90) selectively regulates the stability of KDM4B/JMJD2B histone demethylase

Background: The levels of the KDM4B histone demethylase are elevated in different types of cancer cells, and its depletion suppresses tumor formation. Results: Pharmacological inhibition of Hsp90 promotes ubiquitin-dependent proteasomal degradation of KDM4B, hence altering the histone code. Conclusion: KDM4B is a bona fide client protein of Hsp90. Significance: Hsp90 inhibitors may be useful for the treatment of tumors driven by KDM4B overexpression. The family of KDM4A-D histone demethylases selectively demethylates H3K9 and H3K36 and is implicated in key cellular processes including DNA damage response, transcription, cell cycle regulation, cellular differentiation, senescence, and carcinogenesis. Various human cancers exhibit elevated protein levels of KDM4A-D members, and their depletion impairs tumor formation, suggesting that their enhanced activity promotes carcinogenesis. However, the mechanisms regulating the KDM4 protein stability remain largely unknown. Here, we show that the molecular chaperon Hsp90 interacts with and stabilizes KDM4B protein. Pharmacological inhibition of Hsp90 with geldanamycin resulted in ubiquitin-dependent proteasomal degradation of KDM4B, but not of KDM4C, suggesting that the turnover of these demethylases is regulated by distinct mechanisms. This degradation was accompanied by increased methylation of H3K9. We further show that KDM4B is ubiquitinated on lysines 337 and 562; simultaneous substitution of these residues to arginine suppressed the geldanamycin-induced degradation of KDM4B, suggesting that the ubiquitination of Lys-337 and Lys-562 targets KDM4B for proteasomal degradation upon Hsp90 inhibition. These findings constitute a novel pathway by which Hsp90 activity alters the histone code via regulation of KDM4B stability. This pathway may prove a druggable target for the treatment of tumors driven by enhanced KDM4B activity.

KDM4C (GASC1) lysine demethylase is associated with mitotic chromatin and regulates chromosome segregation during mitosis

Various types of human cancers exhibit amplification or deletion of KDM4A-D members, which selectively demethylate H3K9 and H3K36, thus implicating their activity in promoting carcinogenesis. On this basis, it was hypothesized that dysregulated expression of KDM4A-D family promotes chromosomal instabilities by largely unknown mechanisms. Here, we show that unlike KDM4A-B, KDM4C is associated with chromatin during mitosis. This association is accompanied by a decrease in the mitotic levels of H3K9me3. We also show that the C-terminal region, containing the Tudor domains of KDM4C, is essential for its association with mitotic chromatin. More specifically, we show that R919 residue on the proximal Tudor domain of KDM4C is critical for its association with chromatin during mitosis. Interestingly, we demonstrate that depletion or overexpression of KDM4C, but not KDM4B, leads to over 3-fold increase in the frequency of abnormal mitotic cells showing either misaligned chromosomes at metaphase, anaphase–telophase lagging chromosomes or anaphase–telophase bridges. Furthermore, overexpression of KDM4C demethylase-dead mutant has no detectable effect on mitotic chromosome segregation. Altogether, our findings implicate KDM4C demethylase activity in regulating the fidelity of mitotic chromosome segregation by a yet unknown mechanism.

A role of human RNase P subunits, Rpp29 and Rpp21, in homology directed-repair of double-strand breaks

DNA damage response (DDR) is needed to repair damaged DNA for genomic integrity preservation. Defective DDR causes accumulation of deleterious mutations and DNA lesions that can lead to genomic instabilities and carcinogenesis. Identifying new players in the DDR, therefore, is essential to advance the understanding of the molecular mechanisms by which cells keep their genetic material intact. Here, we show that the core protein subunits Rpp29 and Rpp21 of human RNase P complex are implicated in DDR. We demonstrate that Rpp29 and Rpp21 depletion impairs double-strand break (DSB) repair by homology-directed repair (HDR), but has no deleterious effect on the integrity of non-homologous end joining. We also demonstrate that Rpp29 and Rpp21, but not Rpp14, Rpp25 and Rpp38, are rapidly and transiently recruited to laser-microirradiated sites. Rpp29 and Rpp21 bind poly ADP-ribose moieties and are recruited to DNA damage sites in a PARP1-dependent manner. Remarkably, depletion of the catalytic H1 RNA subunit diminishes their recruitment to laser-microirradiated regions. Moreover, RNase P activity is augmented after DNA damage in a PARP1-dependent manner. Altogether, our results describe a previously unrecognized function of the RNase P subunits, Rpp29 and Rpp21, in fine-tuning HDR of DSBs.

NELF‐E is recruited to DNA double‐strand break sites to promote transcriptional repression and repair

Double‐strand breaks (DSBs) trigger rapid and transient transcription pause to prevent collisions between repair and transcription machineries at damage sites. Little is known about the mechanisms that ensure transcriptional block after DNA damage. Here, we reveal a novel role of the negative elongation factor NELF in blocking transcription activity nearby DSBs. We show that NELF‐E and NELF‐A are rapidly recruited to DSB sites. Furthermore, NELF‐E recruitment and its repressive activity are both required for switching off transcription at DSBs. Remarkably, using I‐SceI endonuclease and CRISPR‐Cas9 systems, we observe that NELF‐E is preferentially recruited, in a PARP1‐dependent manner, to DSBs induced upstream of transcriptionally active rather than inactive genes. Moreover, the presence of RNA polymerase II is a prerequisite for the preferential recruitment of NELF‐E to DNA break sites. Additionally, we demonstrate that NELF‐E is required for intact repair of DSBs. Altogether, our data identify the NELF complex as a new component in the DNA damage response.

Total chemical synthesis of methylated analogues of histone 3 revealed KDM4D as a potential regulator of H3K79me3

Histone H3 methylation plays an important role in regulating gene expression. In histones in general, this mark is dynamically regulated via various demethylases, which found to control cell fate decisions as well as linked to several diseases, including neurological and cancer. Despite major progress in studying methylation mark at various positions in H3 histone proteins, less is known about the regulation of methylated H3 at Lys79. Methylation at this site is known to have direct cross-talk with monoubiquitination of histone H2B at positions Lys120 and 34, as well as with acetylated H3 at Lys9. Herein we applied convergent total chemical protein synthesis to prepare trimethylated H3 at Lys79 to perform initial studies related to the regulation of this mark. Our study enabled us to identify KDM4D lysine demethylase as a potential regulator for trimethylated H3 at Lys79.