scispace - formally typeset
Search or ask a question

Showing papers in "Genes & Development in 2010"


Journal ArticleDOI
TL;DR: The various features of cellular senescence are reviewed and their contribution to tumor suppression is discussed and the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo are highlighted.
Abstract: Almost half a century after the first reports describing the limited replicative potential of primary cells in culture, there is now overwhelming evidence for the existence of “cellular senescence” in vivo. It is being recognized as a critical feature of mammalian cells to suppress tumorigenesis, acting alongside cell death programs. Here, we review the various features of cellular senescence and discuss their contribution to tumor suppression. Additionally, we highlight the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo.

1,838 citations


Journal ArticleDOI
TL;DR: It is reported that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).
Abstract: The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).

1,131 citations


Journal ArticleDOI
TL;DR: New insights are provided into the Hippo signaling pathway, elucidating novel phosphorylation-dependent and independent mechanisms of YAP/Yki inhibition by the hippo pathway, new Hippo pathway components, novel YAP target transcription factors and target genes, and the three-dimensional structure of the YAP-TEAD complex, and providing further evidence for the involvement of Y AP and the Hippi pathway in tumorigenesis.
Abstract: The Hippo signaling pathway is gaining recognition as an important player in both organ size control and tumorigenesis, which are physiological and pathological processes that share common cellular signaling mechanisms. Upon activation by stimuli such as high cell density in cell culture, the Hippo pathway kinase cascade phosphorylates and inhibits the Yes-associated protein (YAP)/TAZ transcription coactivators representing the major signaling output of the pathway. Altered gene expression resulting from YAP/TAZ inhibition affects cell number by repressing cell proliferation and promoting apoptosis, thereby limiting organ size. Recent studies have provided new insights into the Hippo signaling pathway, elucidating novel phosphorylation-dependent and independent mechanisms of YAP/Yki inhibition by the Hippo pathway, new Hippo pathway components, novel YAP target transcription factors and target genes, and the three-dimensional structure of the YAP–TEAD complex, and providing further evidence for the involvement of YAP and the Hippo pathway in tumorigenesis.

1,016 citations


Journal ArticleDOI
TL;DR: An overview of major themes in prostate cancer research is presented, focusing on current knowledge of principal events in cancer initiation and progression, including new insights into the mechanisms of castration resistance, identification of stem cells and tumor-initiating cells, and development of mouse models for preclinical evaluation of novel therapuetics.
Abstract: Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Since early studies on the role of the androgen receptor that led to the advent of androgen deprivation therapy in the 1940s, there has long been intensive interest in the basic mechanisms underlying prostate cancer initiation and progression, as well as the potential to target these processes for therapeutic intervention. Here, we present an overview of major themes in prostate cancer research, focusing on current knowledge of principal events in cancer initiation and progression. We discuss recent advances, including new insights into the mechanisms of castration resistance, identification of stem cells and tumor-initiating cells, and development of mouse models for preclinical evaluation of novel therapuetics. Overall, we highlight the tremendous research progress made in recent years, and underscore the challenges that lie ahead.

915 citations


Journal ArticleDOI
TL;DR: In this paper, the authors sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns.
Abstract: MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

813 citations


Journal ArticleDOI
TL;DR: This review summarizes the progress that has been made in the iPSC field over the last 4 years, with an emphasis on understanding the mechanisms of cellular reprogramming and its potential applications in cell therapy.
Abstract: The generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the enforced expression of a few embryonic transcription factors. This discovery has raised fundamental questions about the mechanisms by which transcription factors influence the epigenetic conformation and differentiation potential of cells during reprogramming and normal development. In addition, iPSC technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders with autologous cells. In this review, we summarize the progress that has been made in the iPSC field over the last 4 years, with an emphasis on understanding the mechanisms of cellular reprogramming and its potential applications in cell therapy.

799 citations


Journal ArticleDOI
TL;DR: Emerging insights into this process indicate that pathways that are frequently deregulated in cancer often play important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.
Abstract: Alternative splicing of mRNA precursors is a nearly ubiquitous and extremely flexible point of gene control in humans. It provides cells with the opportunity to create protein isoforms of differing, even opposing, functions from a single gene. Cancer cells often take advantage of this flexibility to produce proteins that promote growth and survival. Many of the isoforms produced in this manner are developmentally regulated and are preferentially re-expressed in tumors. Emerging insights into this process indicate that pathways that are frequently deregulated in cancer often play important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.

754 citations


Journal ArticleDOI
TL;DR: This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications, on the regulation of t RNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions.
Abstract: tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis.

706 citations


Journal ArticleDOI
TL;DR: It is reported that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation, which establishes a critical role of YAP in maintaining stem cell pluripotency.
Abstract: Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.

675 citations


Journal ArticleDOI
TL;DR: It is demonstrated that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression and that interleukin-4 (IL-4) is responsible for inducing cat hepsin activity in macrophage in vitro and in vivo.
Abstract: Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis, and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin-4 (IL-4) is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs.

627 citations


Journal ArticleDOI
TL;DR: Results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for DnMT2 enzymes during the biogenesis of tRNA-derived small RNAs.
Abstract: The covalent modification of nucleic acids plays an important role in regulating the functions of DNA and RNA. DNA modifications have been analyzed in considerable detail, and the characterization of (cytosine-5) DNA methylation has been crucial for understanding the molecular basis of epigenetic gene regulation (Klose and Bird 2006). (Cytosine-5) methylation has also been documented in various RNA species, including tRNA, but the function of RNA methylation has not been firmly established yet (Motorin et al. 2010). Dnmt2 proteins were originally assigned to the DNA methyltransferase family, because of their strong sequence conservation of catalytic DNA methyltransferase motifs (Okano et al. 1998; Yoder and Bestor 1998). A recent study has suggested that Dnmt2-mediated DNA methylation is important for transposon silencing in Drosophila (Phalke et al. 2009). However, only a weak and distributive DNA methylation activity has been reported in various systems (Jeltsch et al. 2006). The ambiguities associated with the DNA methyltransferase activity of Dnmt2 have also prompted the search for alternative enzyme substrates, and resulted in the discovery of a tRNA methyltransferase activity of Dnmt2 (Goll et al. 2006). Purified recombinant human Dnmt2 methylated RNA preparations from Dnmt2 mutant mice, flies, and plants. Further experiments identified C38 in the anti-codon loop of tRNAAsp as the methylation target site of Dnmt2 (Goll et al. 2006). However, the functional relevance of the tRNA methyltransferase activity of Dnmt2 remains to be established. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile (Goll et al. 2006) under standard laboratory conditions. A distinct Dnmt2 mutant phenotype, caused by morpholino knockdown experiments, has so far been reported only in zebrafish, leading to lethal differentiation defects in the retina, liver, and brain (Rai et al. 2007). In addition, two studies have indicated increased stress tolerance in Dnmt2-overexpressing flies and amoebas (Lin et al. 2005; Fisher et al. 2006). However, the underlying molecular mechanisms have not been investigated yet.

Journal ArticleDOI
TL;DR: An emerging PYR/RCAR-PP2C-SnRK2 model of ABA signal transduction provides researchers with a framework through which early ABA signaling can be understood, and allow novel questions about the hormone response pathway and possible applications in stress resistance engineering of plants to be addressed.
Abstract: The plant hormone abscisic acid (ABA) regulates many key processes in plants, including seed germination and development and abiotic stress tolerance, particularly drought resistance. Understanding early events in ABA signal transduction has been a major goal of plant research. The recent identification of the PYRABACTIN (4-bromo-N-[pyridin-2-yl methyl]naphthalene-1-sulfonamide) RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family of ABA receptors and their biochemical mode of action represents a major breakthrough in the field. The solving of PYR/RCAR structures provides a context for resolving mechanisms mediating ABA control of protein-protein interactions for downstream signaling. Recent studies show that a pathway based on PYR/RCAR ABA receptors, PROTEIN PHOSPHATASE 2Cs (PP2Cs), and SNF1-RELATED PROTEIN KINASE 2s (SnRK2s) forms the primary basis of an early ABA signaling module. This pathway interfaces with ion channels, transcription factors, and other targets, thus providing a mechanistic connection between the phytohormone and ABA-induced responses. This emerging PYR/RCAR-PP2C-SnRK2 model of ABA signal transduction is reviewed here, and provides an opportunity for testing novel hypotheses concerning ABA signaling. We address newly emerging questions, including the potential roles of different PYR/RCAR isoforms, and the significance of ABA-induced versus constitutive PYR/RCAR-PP2C interactions. We also consider how the PYR/RCAR-PP2C-SnRK2 pathway interfaces with ABA-dependent gene expression, ion channel regulation, and control of small molecule signaling. These exciting developments provide researchers with a framework through which early ABA signaling can be understood, and allow novel questions about the hormone response pathway and possible applications in stress resistance engineering of plants to be addressed.

Journal ArticleDOI
TL;DR: The data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3, establishing a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure.
Abstract: The histone variant H3.3 marks active chromatin by replacing the conventional histone H3.1. In this study, we investigate the detailed mechanism of H3.3 replication-independent deposition. We found that the death domain-associated protein DAXX and the chromatin remodeling factor ATRX (α-thalassemia/mental retardation syndrome protein) are specifically associated with the H3.3 deposition machinery. Bacterially expressed DAXX has a marked binding preference for H3.3 and assists the deposition of (H3.3–H4)2 tetramers on naked DNA, thus showing that DAXX is a H3.3 histone chaperone. In DAXX-depleted cells, a fraction of H3.3 was found associated with the replication-dependent machinery of deposition, suggesting that cells adapt to the depletion. The reintroduced DAXX in these cells colocalizes with H3.3 into the promyelocytic leukemia protein (PML) bodies. Moreover, DAXX associates with pericentric DNA repeats, and modulates the transcription from these repeats through assembly of H3.3 nucleosomes. These findings establish a new link between the PML bodies and the regulation of pericentric DNA repeat chromatin structure. Taken together, our data demonstrate that DAXX functions as a bona fide histone chaperone involved in the replication-independent deposition of H3.3.

Journal ArticleDOI
TL;DR: It is concluded that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.
Abstract: Increasing survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 29-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord—a prerequisite for disease treatment—and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a microosmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.

Journal ArticleDOI
TL;DR: It is shown that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.
Abstract: Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.

Journal ArticleDOI
TL;DR: The studies reveal that the two homologous transcription factors, Hif-1alpha and HIF-2alpha, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.
Abstract: Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1α and HIF-2α. Previous studies have revealed that both HIF-α proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. We show here that the HIF-α isoforms can be differentially activated: HIF-1α is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2α is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF-α isoform-specific regulation of the inducible NO synthase gene by HIF-1α, and the arginase1 gene by HIF-2α. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1α versus HIF-2α, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1α and HIF-2α, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.

Journal ArticleDOI
TL;DR: It is found thatJarid2 associates with PRC2 and stimulates its enzymatic activity in vitro, and defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.
Abstract: The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.

Journal ArticleDOI
TL;DR: It is demonstrated that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo, and support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass.
Abstract: Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/ΔEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the ΔEGFR lesion. We hypothesized that the minor ΔEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by ΔEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf−/− astrocytes that express ΔEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Journal ArticleDOI
TL;DR: It is shown that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b, suggesting that recruitment of DN MT3b by DNA: RNA triplexes may be a common and generally used pathway in epigenetic regulation.
Abstract: Noncoding RNAs are important components of regulatory networks controlling the epigenetic state of chromatin. We analyzed the role of pRNA (promoter-associated RNA), a noncoding RNA that is complementary to the rDNA promoter, in mediating de novo CpG methylation of rRNA genes (rDNA). We show that pRNA interacts with the target site of the transcription factor TTF-I, forming a DNA:RNA triplex that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling new mechanism of RNA-dependent DNA methylation, suggesting that recruitment of DNMT3b by DNA:RNA triplexes may be a common and generally used pathway in epigenetic regulation.

Journal ArticleDOI
TL;DR: It is demonstrated here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components.
Abstract: Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors—LRP5/6 and Ror1/2, respectively—through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.

Journal ArticleDOI
TL;DR: It is proposed that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.
Abstract: The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.

Journal ArticleDOI
TL;DR: It is found that regenerating crypts express elevated Yes-associated protein (YAP) levels, and the YAP oncoprotein must be exquisitely controlled in tissue regeneration to allow compensatory proliferation and prevent the intrinsic oncogenic potential of a tissue regeneration program.
Abstract: Although a developmental role for Hippo signaling in organ size control is well appreciated, how this pathway functions in tissue regeneration is largely unknown. Here we address this issue using a dextran sodium sulfate (DSS)-induced colonic regeneration model. We find that regenerating crypts express elevated Yes-associated protein (YAP) levels. Inactivation of YAP causes no obvious intestinal defects under normal homeostasis, but severely impairs DSS-induced intestinal regeneration. Conversely, hyperactivation of YAP results in widespread early-onset polyp formation following DSS treatment. Thus, the YAP oncoprotein must be exquisitely controlled in tissue regeneration to allow compensatory proliferation and prevent the intrinsic oncogenic potential of a tissue regeneration program.

Journal ArticleDOI
TL;DR: It is concluded that the loss of Top1 enhances inherent R-loop formation, particularly over the 5' region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.
Abstract: Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5–Air1/Air2–Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5′ region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5′ region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5′ region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.

Journal ArticleDOI
TL;DR: Using high-throughput sequencing to map ORC binding and nucleosome positioning, it is shown that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS.
Abstract: The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

Journal ArticleDOI
TL;DR: This review considers how microRNAs contribute to gene regulatory networks that confer robustness and how this contributes to developmental and physiological processes that confer remarkable stability.
Abstract: Biological systems are continuously challenged by an environment that is variable. Yet, a key feature of developmental and physiological processes is their remarkable stability. This review considers how microRNAs contribute to gene regulatory networks that confer robustness.

Journal ArticleDOI
TL;DR: It is shown that Ezh2 interacts with HOTAIR and Xist and is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner.
Abstract: Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5′ end of Xist.

Journal ArticleDOI
TL;DR: It is demonstrated that metazoan CDK12 and CDK13 are CTD kinases, and that dCDK12 is orthologous to yeast Ctk1, and the following orthology relationships are suggested.
Abstract: The C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) is an intrinsically unstructured extension of the enzyme's largest subunit, RPB1. The CTD is composed of a tandem array of seven amino acid repeats with the consensus sequence Y1S2P3T4S5P6S7; the number of heptad repeats varies from organism to organism and appears to correlate with genomic complexity (Corden 1990). Although dispensable for polymerase activity in vitro, the CTD is conserved throughout evolution and is essential to life (Zehring et al. 1988). The CTD's function is to coordinate transcription with nuclear processes such as mRNA processing and chromatin modification; it fulfills this role by serving as a selective binding scaffold for nuclear factors (for review, see Phatnani and Greenleaf 2006). The binding specificity of the CTD for particular factors is determined by its phosphorylation state, which varies during the transcription cycle; Ser2, Ser5, and, more recently, Ser7 of the heptad repeat have been identified as the primary targets of phosphorylation (Phatnani and Greenleaf 2006; Chapman et al. 2007; Egloff et al. 2007). Initiated and early transcribing polymerases are thought to be phosphorylated at Ser5 positions in the CTD repeats, whereas actively elongating polymerases carry phosphates on both Ser2 and Ser5 residues. Toward the 3′ end of pre-mRNA transcription units, Ser2 phosphorylation often appears to predominate (cf. Phatnani and Greenleaf 2006; Rahl et al. 2010). CTD kinases in yeast and metazoa The phosphorylation of CTD Ser5 residues during initiation and early transcription is mediated by the TFIIH CTD kinase subunit: Kin28 in yeast, and CDK7 in metazoans (Komarnitsky et al. 2000; Schroeder et al. 2000). The majority of Ser2 phosphorylation on productively elongating RNAPII in Saccharomyces cerevisiae (Sc) appears to be catalyzed by CTDK-I (Buratowski 2009; Qiu et al. 2009), a three-subunit enzyme consisting of Ctk1 (a CDK homolog), Ctk2 (a cyclin homolog), and Ctk3 (function unknown) (Lee and Greenleaf 1991; Sterner et al. 1995). Although it is responsible for the bulk of Ser2 phosphorylation in vivo, the Ctk1-containing enzyme is not the only elongation phase kinase in yeast; it coexists with the essential Bur1 kinase (CDK/cyclin pair = Bur1/Bur2) (Yao et al. 2000). While it has been proposed that the Bur1 kinase phosphorylates the transcription elongation factor subunit SPT5 rather than the CTD of Rpb1 (Zhou et al. 2009), recent evidence suggests that the situation is more complex, and that, in addition to acting on SPT5, the Bur1 kinase phosphorylates Ser2 positions in the CTD (Liu et al. 2009; Qiu et al. 2009), possibly stimulating subsequent phosphorylation by Ctk1. A similar pair of Ser2 CTD kinases is also present in the fission yeast Schizzosaccharomyces pombe (Sp): Lsk1, the Ctk1 ortholog in Sp, phosphorylates Ser2 positions in the CTD (Karagiannis and Balasubramanian 2007; Coudreuse et al. 2010), whereas SpCDK9, the Bur1 ortholog, phosphorylates both Rpb1 (on its CTD) and Spt5 (Viladevall et al. 2009). With respect to the principal Ser2 kinase in metazoa, the situation has been less clear cut. It is currently thought that there is only one Ser2 CTD kinase in higher organisms: P-TEFb (consisting of CDK9 and a cyclin, usually cyclin T) (e.g., see Peterlin and Price 2006). P-TEFb is able to phosphorylate both the CTD and the elongation factor subunit SPT5 (Kim and Sharp 2001). The dual functionality of CDK9, coupled with its equal sequence similarity to both Bur1 and Ctk1, has led to the assumption that CDK9 reconstitutes the activities of both Bur1 and Ctk1 in higher eukaryotes (Wood and Shilatifard 2006). On the other hand, two independent molecular evolution studies concluded that Sc Bur1 is the closest Sc relative of metazoan CDK9 proteins, while Sc Ctk1 is closer to a set of metazoan CDK proteins that are largely unstudied (Liu and Kipreos 2000; Guo and Stiller 2004): the “Ctk1” group (Supplemental Fig. S1). The genomes of Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) each encode one member of the Ctk1 group (genes CG7597 and B0285.1, respectively), while the genome of Homo sapiens (Hs) encodes two (genes CRKRS and CDC2L5; see Supplemental Fig S1). The human “Ctk1” proteins have been renamed recently CDK12 and CDK13 (Chen et al. 2006, 2007). We refer to them as hCDK12 and hCDK13, and to the Dm protein as dCDK12. The evolutionary studies thus suggest that the metazoan ortholog of Ctk1 in Drosophila is dCDK12 and in humans is one or both of the isozyme pair, hCDK12 + hCDK13. Also, the evolutionary arguments and the recent experimental work mentioned above both suggest that the metazoan ortholog of Bur1 is CDK9. We tested these suggestions with a set of diverse experiments that lead us to conclude that, indeed, the Drosophila ortholog of yeast Ctk1 is dCDK12 (CG7597), and the human ortholog is most likely hCDK12. Conversely, we describe other experiments supporting the idea that the metazoan ortholog of yeast Bur1 is CDK9. Because the metazoan orthologs of yeast Ctk1 have not been studied previously as CTD kinases, our findings open the door to novel studies of a vast array of transcription-related events in metazoa that are critical to many aspects of proper gene expression and other nuclear functions.

Journal ArticleDOI
TL;DR: TulP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3-IFT-A interaction suggests how these proteins cooperate during neural tube patterning.
Abstract: Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3–IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

Journal ArticleDOI
TL;DR: Growing evidence argues for p53-independent effects, as well as the remarkable possibility that Mdm2 has tumor suppressor functions in the appropriate context, which is proving to be a key player in human cancer in its own right, and thus an important target for therapeutic intervention.
Abstract: Mdm2 has been well characterized as a negative regulator of the tumor suppressor p53. Recent studies have shown that Mdm2 is activated in response to a variety of oncogenic pathways independent of p53. Although its role as an oncogene via suppression of p53 function remains clear, growing evidence argues for p53-independent effects, as well as the remarkable possibility that Mdm2 has tumor suppressor functions in the appropriate context. Hence, Mdm2 is proving to be a key player in human cancer in its own right, and thus an important target for therapeutic intervention.

Journal ArticleDOI
TL;DR: A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.
Abstract: Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively poorly. Two conserved kinases called Cdc7 (cell division cycle 7) and cyclin-dependent kinase (CDK) are required to establish replication forks during the initiation of chromosome replication, and a key feature of this process is the activation of the replicative DNA helicase in situ at each origin of DNA replication. A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.