The Ezh2 methyltransferase complex: actin up in the cytosol

Ezh2, a polycomb group protein, is known to function in histone methylation, thereby regulating gene expression. However, in a recent study by Su et al., the Ezh2-containing complex has been given an additional role in cellular regulation. Cytosolic Ezh2 methyltransferase complexes were shown to associate with Vav1 and control receptor-induced actin polymerization and proliferation in a methylation-dependent manner. Overall, these findings implicate lysine methylation as a posttranslational modification crucial for receptor-mediated signal transduction events.     

Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.     

Conditional ablation of Ezh2 in murine hearts reveals its essential roles in endocardial cushion formation, cardiomyocyte proliferation and survival

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.     

Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability

The H3K27 histone methyltransferase, Ezh2 (enhancer of zeste 2), is a Polycomb group protein that plays important roles in many biological processes including cellular differentiation, stem cell biology, and cancer development. Up-regulation of Ezh2 is observed in various human cancers consistent with its role in cell proliferation. Thus, understanding the regulation of Ezh2 may reveal how it contributes to the cellular proliferation process. Here, we demonstrate that Ezh2 can be regulated by the cyclin-dependent kinase, CDK1, which phosphorylates Ezh2 at threonines 345 and 487. Consistent with the cell cycle phase during which CDK1 exhibits peak activity, Ezh2 phosphorylation is enriched in cells arrested in mitosis when compared with S-phase. Phosphorylation of Thr-345 and Thr-487 promotes Ezh2 ubiquitination and subsequent degradation by the proteasome. Furthermore, expression of T345A/T487A confers a proliferative disadvantage when compared with cells expressing wild-type Ezh2, which suggests that phosphorylation of Ezh2 is important for cell proliferation. Collectively, these results establish a novel function for CDK1-mediated Ezh2 phosphorylation and provide a mechanism by which Ezh2 protein levels can be regulated in cells.     

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2-deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2-deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2-deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.     

Rac1 function during fucoid development

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.Key words: actin, Arp2/3 complex, manumycin A, NSC23766, Rac1, Rho GTPase, Scar/WAVE, Silvetia compressa     

Regulating the actin cytoskeleton during vesicular transport

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.     

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2''s role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.     

The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis

Guard cell actin reorganization has been observed in stomatal responses to a wide array of stimuli. However, how the guard cell signaling machinery regulates actin dynamics is poorly understood. Here, we report the identification of an allele of the Arabidopsis thaliana ACTIN-RELATED PROTEIN C2/DISTORTED TRICHOMES2 (ARPC2) locus (encoding the ARPC2 subunit of the ARP2/3 complex) designated high sugar response3 (hsr3). The hsr3 mutant showed increased transpirational water loss that was mainly due to a lesion in stomatal regulation. Stomatal bioassay analyses revealed that guard cell sensitivity to external stimuli, such as abscisic acid (ABA), CaCl(2), and light/dark transition, was reduced or abolished in hsr3. Analysis of a nonallelic mutant of the ARP2/3 complex suggested no pleiotropic effect of ARPC2 beyond its function in the complex in regard to stomatal regulation. When treated with ABA, guard cell actin filaments underwent fast disruption in wild-type plants, whereas those in hsr3 remained largely bundled. The ABA insensitivity phenotype of hsr3 was rescued by cytochalasin D treatment, suggesting that the aberrant stomatal response was a consequence of bundled actin filaments. Our work indicates that regulation of actin reassembly through ARP2/3 complex activity is crucial for stomatal regulation.     

Arabidopsis NAP1 is essential for Arp2/3-dependent trichome morphogenesis

The dynamic nature of the eukaryotic actin cytoskeleton is essential for the locomotion of animal cells and the morphogenesis of plant and fungal cells. The F-actin nucleating/branching activity of the Arp2/3 complex is a key function for all of these processes. The SCAR/WAVE family represents a group of Arp2/3 activators that are associated with lamellipodia formation. A protein complex of PIR121, NAP1, ABI, and HSPC300 is required for SCAR regulation by cell signaling pathways, but the exact nature of this interaction is controversial and represents a continually evolving model. The mechanism originally proposed was of a SCAR trans repressing complex supported by evidence from in vitro experiments. This model was reinforced by genetic studies in the Drosophila central nervous system and Dictyostelium, where the knockout of certain SCAR-complex components leads to excessive SCAR-mediated actin polymerization. Conflicting data have steadily accumulated from animal tissue culture experiments suggesting that the complex activates rather than represses in vivo SCAR activity. Recent biochemical evidence supports the SCAR-complex activator model. Here, we show that genetic observations in Arabidopsis are compatible with an activation model and provide one potential mechanism for the regulation of the newly identified Arabidopsis Arp2/3 complex.     

Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms

Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including survival.