Takeshi Urano

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.

Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

Ral proteins (RalA and RalB) comprise a distinct family of Ras‐related GTPases (Feig and Emkey, 1993). Recently, Ral‐GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R‐Ras and Rap1A, in the yeast two‐hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral‐GDS in mammalian cells, only RasH activates Ral‐GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral‐GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.

Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.

Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

Heterochromatin and RNAi Are Required to Establish CENP-A Chromatin at Centromeres

Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)–related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)–directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-ACnp1 and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-ACnp1 chromatin on naïve templates. Once assembled, CENP-ACnp1 chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified.

A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions—centromeres, the mating‐type region, and telomeres—and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1+ gene causes centromere‐specific decreases in Swi6 localization and histone H3‐K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating‐type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1‐mediated establishment steps differently than do other heterochromatic regions.

Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis.

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

Telomere Binding Protein Taz1 Establishes Swi6 Heterochromatin Independently of RNAi at Telomeres

BACKGROUND The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). RESULTS We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. CONCLUSION The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Tyrosine Phosphorylation of Crk-associated Substrates by Focal Adhesion Kinase A PUTATIVE MECHANISM FOR THE INTEGRIN-MEDIATED TYROSINE PHOSPHORYLATION OF Crk-ASSOCIATED SUBSTRATES

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.

Targeted Disruption of Gb3/CD77 Synthase Gene Resulted in the Complete Deletion of Globo-series Glycosphingolipids and Loss of Sensitivity to Verotoxins

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 μg of VT-2 or 1.0 μg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.