Showing papers in "Molecular and Cellular Biology in 2007"

Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation.

Abstract: Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.

A MicroRNA Signature of Hypoxia

Abstract: Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a well-documented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxia-induced microRNAs are also overexpressed in a variety of human tumors.

P-Body Formation Is a Consequence, Not the Cause, of RNA-Mediated Gene Silencing

Abstract: P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

Abstract: Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression

Abstract: microRNAs (miRNAs) are abundant, approximately 21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G(0)/G(1). Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.

Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1.

Abstract: Hypoxia is a pervasive microenvironmental factor that affects normal development as well as tumor progression. In most normal cells, hypoxia stabilizes hypoxia-inducible transcription factors (HIFs), particularly HIF-1, which activates genes involved in anaerobic metabolism and angiogenesis. As hypoxia signals a cellular deprivation state, HIF-1 has also been reported to counter the activity of MYC, which encodes a transcription factor that drives cell growth and proliferation. Since many human cancers express dysregulated MYC, we sought to determine whether HIF-1 would in fact collaborate with dysregulated MYC rather countering its function. Here, using the P493-6 Burkitt's lymphoma model with an inducible MYC, we demonstrate that HIF-1 cooperates with dysregulated c-Myc to promote glycolysis by induction of hexokinase 2, which catalyzes the first step of glycolysis, and pyruvate dehydrogenase kinase 1, which inactivates pyruvate dehydrogenase and diminishes mitochondrial respiration. We also found the collaborative induction of vascular endothelial growth factor (VEGF) by HIF-1 and dysregulated c-Myc. This study reports the previously unsuspected collaboration between HIF-1 and dysregulated MYC and thereby provides additional insights into the regulation of VEGF and the Warburg effect, which describes the propensity for cancer cells to convert glucose to lactate.

Wnt/β-Catenin Is Essential for Intestinal Homeostasis and Maintenance of Intestinal Stem Cells

Abstract: The Wnt signaling pathway is deregulated in over 90% of human colorectal cancers. β-Catenin, the central signal transducer of the Wnt pathway, can directly modulate gene expression by interacting with transcription factors of the TCF/LEF family. In the present study we investigate the role of Wnt signaling in the homeostasis of intestinal epithelium by using tissue-specific, inducible β-catenin gene ablation in adult mice. Block of Wnt/β-catenin signaling resulted in rapid loss of transient-amplifying cells and crypt structures. Importantly, intestinal stem cells were induced to terminally differentiate upon deletion of β-catenin, resulting in a complete block of intestinal homeostasis and fatal loss of intestinal function. Transcriptional profiling of mutant crypt mRNA isolated by laser capture microdissection confirmed those observations and allowed us to identify genes potentially responsible for the functional preservation of intestinal stem cells. Our data demonstrate an essential requirement of Wnt/β-catenin signaling for the maintenance of the intestinal epithelium in the adult organism. This challenges attempts to target aberrant Wnt signaling as a new therapeutic strategy to treat colorectal cancer.

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Abstract: Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.

Knockdown of ALR (MLL2) Reveals ALR Target Genes and Leads to Alterations in Cell Adhesion and Growth

Abstract: ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci

Abstract: Senescence was initially described as a stable cell proliferation arrest resulting from the progression of primary human fibroblasts through a finite number of population doublings in vitro (35). However, activated oncogenes, oxidative stress, DNA damage, and drug-like inhibitors of specific enzymatic activities also induce senescence (14, 37, 82). In addition, senescence occurs in other cell types, such as primary human epithelial cells. In vivo, senescence is an important tumor suppression mechanism that restrains the proliferation of cells that harbor activated oncogenes (12, 16, 17, 51). Also, by limiting the self-renewal capacity of adult tissue stem cells, senescence is thought to contribute to tissue aging of many multicellular adult animals (38, 42, 53). Senescent cells are typically characterized by a large flat morphology and the expression of a senescence-associated β-galactosidase (SA β-gal) activity of unknown function (16, 21). In the nucleus of senescent cells, the chromatin undergoes dramatic remodeling through the formation of domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) (56, 57, 86). Cytologically, SAHF appear as compacted punctate DAPI (4,6-diamidino-2-phenylindole)-stained foci of DNA in senescent cell nuclei. The formation of SAHF is also reflected in a general increase in the resistance of nuclear chromatin to digestion by nucleases (57). SAHF contain modifications and associated proteins characteristic of transcriptionally silent heterochromatin, such as methylated lysine 9 of histone H3 (H3K9Me), heterochromatin protein 1 (HP1), and the histone H2A variant macroH2A. In addition, Narita et al. recently showed that high-mobility group A (HMGA) proteins, a family of abundant non-histone chromatin proteins, are essential structural components of SAHF (56). Proliferation-promoting genes, such as E2F target genes (e.g., cyclin A), are recruited into SAHF, dependent on the pRB tumor suppressor protein, thereby irreversibly silencing expression of those genes. Recently, we showed that two chromatin regulators, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), drive the formation of SAHF in human cells (86). HIRA and ASF1a are the human orthologs of proteins known to create transcriptionally silent heterochromatin in yeasts, flies, and plants (9, 29, 39, 54, 63, 70-73, 78). In Saccharomyces cerevisiae, Hir1 and Hir2 are required for heterochromatin-mediated silencing of histone genes, telomeres, and mating loci, and the formation of pericentromeric chromatin structure (39, 70-73). Likewise, yeast Asf1p is required for heterochromatin-mediated silencing of telomeres, mating loci, and histone genes (40, 50, 70, 73, 75, 78) but also mediates nucleosome disassembly (2, 3, 68). Both HIRA and ASF1a bind to histones and exhibit histone chaperone activity in vitro (28, 64, 70, 78, 79). The HIRA/ASF1a-containing complex preferentially deposits the histone variant histone H3.3 into nucleosomes (46, 65, 76). Canonical human histone H3.1 and histone H3.3 differ in their primary amino acid sequences by only five amino acids. However, histone H3.1 is expressed periodically in the S phase of the cell cycle and is incorporated into chromatin during replication-coupled chromatin assembly (5, 36, 76). In contrast, histone H3.3 is expressed throughout the cell cycle and is incorporated into chromatin by the HIRA/ASF1a complex in a DNA replication- and repair-independent manner (5, 36, 76). Consistent with their partially overlapping biological and biochemical properties, yeast Asf1p and Hir proteins physically interact, and this interaction is necessary for telomeric silencing (19, 70). Likewise, the formation of SAHF in human cells by HIRA and ASF1a depends upon a physical interaction between these two proteins (76, 77, 86). A previous careful kinetic analysis of SAHF formation from our laboratory indicated that formation of SAHF is likely a multistep process (87). In the earliest defined step, the histone chaperone proteins HIRA and HP1 are both recruited to a specific subnuclear organelle, the acute promyelocytic leukemia (PML) nuclear body (10, 67). Most human cells contain 20 to 30 PML nuclear bodies, which are typically 0.1 to 1 μm in diameter and are enriched in the protein PML, as well as many other nuclear regulatory proteins (10, 67). PML bodies have been previously implicated in various cellular processes, including tumor suppression and cellular senescence (20, 23, 61). At a molecular level, they have been proposed as sites of assembly of macromolecular regulatory complexes and protein modification (24, 31, 61). After HIRA's translocation into PML bodies, chromatin condensation occurs, as defined by the appearance of DAPI-stained foci. Finally, H3K9Me accumulates, and HP1 and macroH2A proteins are recruited to SAHF. In this study, we set out to understand the series of events that contribute to the formation of SAHF in more detail and, in particular, to identify the molecular requirements for the different steps that were previously temporally defined. Here we report that during SAHF formation, each chromosome condenses into a single DAPI focus. Chromosome condensation mediated by the histone chaperone ASF1a depends on its binding to histone H3, as well as HIRA. Interestingly, HP1γ, but not HP1α and HP1β, is phosphorylated on serine 93 in senescent cells. This phosphorylation is not required for the protein's localization to PML bodies, but is required for its binding to SAHF. Remarkably, a large reduction in the amount of chromatin-bound HP1 proteins does not affect chromosome condensation, recruitment of histone variant macroH2A to SAHF, expression of SA β-gal, or senescence-associated cell cycle exit. Based on these data, we propose a multistep model of dependent and independent steps that culminate in the formation of mature SAHF.

DNA Damage-Dependent Acetylation and Ubiquitination of H2AX Enhances Chromatin Dynamics

Abstract: Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.

Degradation of Mcl-1 by β-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization

Abstract: Apoptosis is critical for embryonic development, tissue homeostasis, and tumorigenesis and is determined largely by the Bcl-2 family of antiapoptotic and prosurvival regulators. Here, we report that glycogen synthase kinase 3 (GSK-3) was required for Mcl-1 degradation, and we identified a novel mechanism for proteasome-mediated Mcl-1 turnover in which GSK-3β associates with and phosphorylates Mcl-1 at one consensus motif (155STDG159SLPS163T; phosphorylation sites are in italics), which will lead to the association of Mcl-1 with the E3 ligase β-TrCP, and β-TrCP then facilitates the ubiquitination and degradation of phosphorylated Mcl-1. A variant of Mcl-1 (Mcl-1-3A), which abolishes the phosphorylations by GSK-3β and then cannot be ubiquitinated by β-TrCP, is much more stable than wild-type Mcl-1 and able to block the proapoptotic function of GSK-3β and enhance chemoresistance. Our results indicate that the turnover of Mcl-1 by β-TrCP is an essential mechanism for GSK-3β-induced apoptosis and contributes to GSK-3β-mediated tumor suppression and chemosensitization.

Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response.

Abstract: Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.

BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy.

Abstract: Hypoxia and nutrient deprivation are environmental stresses governing the survival and adaptation of tumor cells in vivo. We have identified a novel role for the Rb tumor suppressor in protecting against nonapoptotic cell death in the developing mouse fetal liver, in primary mouse embryonic fibroblasts, and in tumor cell lines. Loss of pRb resulted in derepression of BNip3, a hypoxia-inducible member of the Bcl-2 superfamily of cell death regulators. We identified BNIP3 as a direct target of pRB/E2F-mediated transcriptional repression and showed that pRB attenuates the induction of BNIP3 by hypoxia-inducible factor to prevent autophagic cell death. BNIP3 was essential for hypoxia-induced autophagy, and its ability to promote autophagosome formation was enhanced under conditions of nutrient deprivation. Knockdown of BNIP3 reduced cell death, and remaining deaths were necrotic in nature. These studies identify BNIP3 as a key regulator of hypoxia-induced autophagy and suggest a novel role for the RB tumor suppressor in preventing nonapoptotic cell death by limiting the extent of BNIP3 induction in cells.

Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells.

Abstract: Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH- or FH-deficient cells, HIF activation is due to HIF1α stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIFα prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, α-ketoglutarate. We designed and synthesized cell-permeating α-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate- or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular α-ketoglutarate. Introduction of α-ketoglutarate derivatives restores normal PHD activity and HIF1α levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction.

Sequential Activation of Poly(ADP-Ribose) Polymerase 1, Calpains, and Bax Is Essential in Apoptosis-Inducing Factor-Mediated Programmed Necrosis

Abstract: Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

NRF2 modulates aryl hydrocarbon receptor signaling: influence on adipogenesis.

Abstract: The NF-E2 p45-related factor 2 (NRF2) and the aryl hydrocarbon receptor (AHR) are transcription factors controlling pathways modulating xenobiotic metabolism. AHR has recently been shown to affect Nrf2 expression. Conversely, this study demonstrates that NRF2 regulates expression of Ahr and subsequently modulates several downstream events of the AHR signaling cascade, including (i) transcriptional control of the xenobiotic metabolism genes Cyp1a1 and Cyp1b1 and (ii) inhibition of adipogenesis in mouse embryonic fibroblasts (MEFs). Constitutive expression of AHR was affected by Nrf2 genotype. Moreover, a pharmacological activator of NRF2 signaling, CDDO-IM {1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole}, induced Ahr, Cyp1a1, and Cyp1b1 transcription in Nrf2+/+ MEFs but not in Nrf2-/- MEFs. Reporter analysis and chromatin immunoprecipitation assay revealed that NRF2 directly binds to one antioxidant response element (ARE) found in the -230-bp region of the promoter of Ahr. Since AHR negatively controls adipocyte differentiation, we postulated that NRF2 would inhibit adipogenesis through the interaction with the AHR pathway. Nrf2-/- MEFs showed markedly accelerated adipogenesis upon stimulation, while Keap1-/- MEFs (which exhibit higher NRF2 signaling) differentiated slowly compared to their congenic wild-type MEFs. Ectopic expression of Ahr and dominant-positive Nrf2 in Nrf2-/- MEFs also substantially delayed differentiation. Thus, NRF2 directly modulates AHR signaling, highlighting bidirectional interactions of these pathways.

Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro.

Abstract: Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.

Sox17 and Sox4 Differentially Regulate β-Catenin/T-Cell Factor Activity and Proliferation of Colon Carcinoma Cells

Abstract: The canonical Wnt signaling pathway is involved in many biological processes, ranging from embryonic development to stem cell maintenance in adult tissues, while the dysregulation of Wnt signaling is implicated in human tumorigenesis. The key effector of the canonical Wnt pathway is β-catenin, which forms complexes with T-cell factor (TCF)/lymphoid enhancer factor (LEF) high-mobility-group (HMG) box transcription factors to stimulate the transcription of Wnt-responsive genes (7). While numerous studies have shown that β-catenin is regulated at many levels, less is known about the regulation of TCF/LEF transcription factors. In the absence of a Wnt signal, levels of cytosolic β-catenin are kept low via the interaction of β-catenin with a protein complex including glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC), and Axin. The phosphorylation of β-catenin by the kinase GSK3β allows β-catenin to be ubiquitinated and targeted for degradation by the proteasome (1). The binding of a canonical Wnt ligand to the frizzled-lipoprotein receptor-related protein 5/6 receptor complex results in the repression of GSK3β and the stabilization of β-catenin. Stabilized β-catenin accumulates in the nucleus, where it acts as a cofactor with the HMG box family of TCF/LEF transcription factors to regulate the expression of Wnt target genes, such as cyclin D1 and Cdx-1 (17, 22). Although the formation of a TCF-β-catenin complex is required for the activation of all Wnt target genes (36), Wnt signaling is involved in a wide array of biological processes, including cell proliferation, cellular transformation (14), and embryonic development (24), demonstrating that the output of this pathway is highly influenced by the cellular context. Given that aberrant activation of the canonical Wnt pathway can lead to unrestricted cell division and tumor formation (12, 26, 28, 31, 40), it is not surprising that this pathway is antagonized by several different mechanisms. For example, several extracellular antagonists that inhibit ligand-receptor interactions have been described previously, including Dickkopf (Dkk), Cerberus, and the secreted frizzled-related proteins (10, 21, 34, 35). In many instances, Wnt signaling is kept in check by a negative-feedback loop in which β-catenin/TCF activity induces the transcription of its own negative regulators, Axin and Dkk1 (4, 20, 39). Finally, in the absence of activated β-catenin, TCF/LEF transcription factors keep Wnt target genes off via their interaction with members of the Grouch family of transcriptional repressors (4, 20, 39). Structurally related to TCF/LEFs, several members of the Sox family of HMG box transcription factors, including Sox17, Sox3, Sox7, and Sox9, have also been implicated in repressing β-catenin activity by a mechanism that is not well understood (2, 48, 54, 55). In addition to acting as an antagonist, Sox17 cooperates with β-catenin to activate the transcription of its endoderm target genes in Xenopus laevis (44). These findings suggest that, dependent on the context, Sox proteins can utilize β-catenin as a cofactor or can antagonize β-catenin/TCF function. While the mechanism by which Sox proteins antagonize Wnt signaling is unknown, one possibility is that they compete with TCFs for binding to β-catenin (55). Here, we report that Sox proteins expressed in normal and neoplastic gut epithelia can modulate canonical Wnt signaling and the proliferation of gastrointestinal tumor cells. While several Sox factors, including Sox17, Sox2, and Sox9, are antagonists of canonical Wnt signaling, others, such as Sox4 and Sox5, promote Wnt signaling activity. Gain- and loss-of-function analyses demonstrate that the Wnt antagonist Sox17 represses colon carcinoma cell proliferation while the agonist Sox4 promotes proliferation. In contrast to a proposed model in which Sox17 protein antagonizes Wnt signaling by competing with TCFs for β-catenin binding, we found that Sox17 interacts with both TCF/LEF and β-catenin and that Sox17 and TCF/LEF proteins interact via their respective HMG domains. Binding experiments suggest that Sox17, TCF, and β-catenin cooperatively interact to form a complex. In contrast, Sox4 can bind to either TCF/LEF or β-catenin alone but does not appear to cooperatively bind both proteins. Structure-function analyses indicate that Sox17 must bind directly to both β-catenin and TCF in order to antagonize Wnt signaling and that Sox17 DNA binding activity is not required. Lastly, functional studies show that Sox17 promotes the degradation of TCF/LEF and β-catenin proteins via a GSK3β-independent mechanism that can be blocked by proteasome inhibitors. In contrast, Sox4 may function to stabilize β-catenin protein. Together, these findings suggest that Sox transcription factors act in a novel pathway to modulate the stability of β-catenin/TCF proteins and regulate the proliferation of colon carcinoma cells. These results have important implications for how Sox proteins regulate the transcriptional output of Wnt signaling in many developmental and pathological processes.

Luteinizing Hormone-Dependent Activation of the Epidermal Growth Factor Network Is Essential for Ovulation

Abstract: In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.

DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity

Abstract: The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity.

Integration of estrogen and Wnt signaling circuits by the polycomb group protein EZH2 in breast cancer cells.

Abstract: Essential for embryonic development, the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in breast and prostate cancers and is implicated in the growth and aggression of the tumors. The tumorigenic mechanism underlying EZH2 overexpression is largely unknown. It is believed that EZH2 exerts its biological activity as a transcription repressor. However, we report here that EZH2 functions in gene transcriptional activation in breast cancer cells. We show that EZH2 transactivates genes that are commonly targeted by estrogen and Wnt signaling pathways. We demonstrated that EZH2 physically interacts directly with estrogen receptor alpha and beta-catenin, thus connecting the estrogen and Wnt signaling circuitries, functionally enhances gene transactivation by estrogen and Wnt pathways, and phenotypically promotes cell cycle progression. In addition, we identified the transactivation activity of EZH2 in its two N-terminal domains and demonstrated that these structures serve as platforms to connect transcription factors and the Mediator complex. Our experiments indicated that EZH2 is a dual function transcription regulator with a dynamic activity, and we provide a mechanism for EZH2 in tumorigenesis.

Toll and IMD Pathways Synergistically Activate an Innate Immune Response in Drosophila melanogaster

Abstract: The inducible expression of antimicrobial peptide genes in Drosophila melanogaster is regulated by the conserved Toll and peptidoglycan recognition protein LC/immune deficiency (PGRP-LC/IMD) signaling pathways. It has been proposed that the two pathways have independent functions and mediate the specificity of innate immune responses towards different microorganisms. Scattered evidence also suggests that some antimicrobial target genes can be activated by both Toll and IMD, albeit to different extents. This dual activation can be mediated by independent stimulation or by cross-regulation of the two pathways. We show in this report that the Toll and IMD pathways can interact synergistically, demonstrating that cross-regulation occurs. The presence of Spatzle (the Toll ligand) and gram-negative peptidoglycan (the PGRP-LC ligand) together caused synergistic activation of representative target genes of the two pathways, including Drosomycin, Diptericin, and AttacinA. Constitutive activation of Toll and PGRP-LC/IMD could mimic the synergistic stimulation. RNA interference assays and promoter analyses demonstrate that cooperation of different NF-kappaB-related transcription factors mediates the synergy. These results illustrate how specific ligand binding by separate upstream pattern recognition receptors can be translated into a broad-spectrum host response, a hallmark of innate immunity.

Bmp2 Is Critical for the Murine Uterine Decidual Response

Abstract: The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2 ± 0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.

Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention.

Abstract: Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lalpha, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.

Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2

Abstract: The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase.

Abstract: Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.

Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

Abstract: Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-Lα and effectors modulating peroxisome proliferator-activated receptor γ (PPARγ) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-Lα expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPARγ enhances Ero1-Lα expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-Lα through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-Lα in Ero1-Lα-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.

Peroxisome Proliferator-Activated Receptor α Regulates a MicroRNA-Mediated Signaling Cascade Responsible for Hepatocellular Proliferation

Abstract: Activation of peroxisome proliferator-activated receptor alpha (PPARalpha) leads to hepatocellular proliferation and liver carcinomas. The early events mediating these effects are unknown. A novel mechanism by which PPARalpha regulates gene expression and hepatocellular proliferation was uncovered. MicroRNA (miRNA) expression profiling demonstrated that activated PPARalpha was a major regulator of hepatic miRNA expression. Of particular interest, let-7C, an miRNA important in cell growth, was inhibited following 4-h treatment and 2-week and 11-month sustained treatment with the potent PPARalpha agonist Wy-14,643 in wild-type mice. let-7C was shown to target c-myc via direct interaction with the 3' untranslated region of c-myc. The PPARalpha-mediated induction of c-myc via let-7C subsequently increased expression of the oncogenic mir-17-92 cluster; these events did not occur in Pparalpha-null mice. Overexpression of let-7C decreased c-myc and mir-17 and suppressed the growth of Hepa-1 cells. Furthermore, using the human PPARalpha-expressing mouse model, which is responsive to Wy-14,643 effects on beta-oxidation and serum triglycerides but resistant to hepatocellular proliferation and tumorigenesis, we demonstrated a critical role for let-7C in liver oncogenesis. Wy-14,643 treatment did not inhibit let-7C or induce c-myc and mir-17 expression. These observations reveal a let-7C signaling cascade critical for PPARalpha agonist-induced liver proliferation and tumorigenesis.

LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex

Abstract: LINE-1 retrotransposons constitute one-fifth of human DNA and have helped shape our genome. A full-length L1 encodes a 40-kDa RNA-binding protein (ORF1p) and a 150-kDa protein (ORF2p) with endonuclease and reverse transcriptase activities. ORF1p is distinctive in forming large cytoplasmic foci, which we identified as cytoplasmic stress granules. A phylogenetically conserved central region of the protein is critical for wild-type localization and retrotransposition. Yeast two-hybrid screens revealed several RNA-binding proteins that coimmunoprecipitate with ORF1p and colocalize with ORF1p in foci. Two of these proteins, YB-1 and hnRNPA1, were previously reported in stress granules. We identified additional proteins associated with stress granules, including DNA-binding protein A, 9G8, and plasminogen activator inhibitor RNA-binding protein 1 (PAI-RBP1). PAI-RBP1 is a homolog of VIG, a part of the Drosophila melanogaster RNA-induced silencing complex (RISC). Other RISC components, including Ago2 and FMRP, also colocalize with PAI-RBP1 and ORF1p. We suggest that targeting ORF1p, and possibly the L1 RNP, to stress granules is a mechanism for controlling retrotransposition and its associated genetic and cellular damage.