Showing papers in "Genes to Cells in 2010"
TL;DR: It is shown that Parkin‐catalyzed K63‐linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin.
Abstract: PINK1 and Parkin were first identified as the causal genes responsible for familial forms of early-onset Parkinson’s disease (PD), a prevalent neurodegenerative disorder. PINK1 encodes a mitochondrial serine/threonine protein kinase, whereas Parkin encodes an ubiquitin-protein ligase. PINK1 and Parkin cooperate to maintain mitochondrial integrity; however, the detailed molecular mechanism of how Parkin-catalyzed ubiquitylation results in mitochondrial integrity remains an enigma. In this study, we show that Parkin-catalyzed K63-linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin. In addition, p62/SQSTM1 (hereafter referred to as p62) was recruited to depolarized mitochondria after Parkin-directed ubiquitylation. Intriguingly, deletion of p62 in mouse embryonic fibroblasts resulted in a gross loss of mitochondrial perinuclear clustering but did not hinder mitochondrial degradation. Thus, p62 is required for ubiquitylation-dependent clustering of damaged mitochondria, which resembles p62-mediated ‘aggresome’ formation of misfolded/unfolded proteins after ubiquitylation.
387 citations
TL;DR: The role of selective autophagy in sorting vacuolar enzymes and removal of aggregate-prone proteins and unwanted organelles is discussed in this paper, where the authors discuss the physiological roles of selective auto-ophagy and their molecular mechanisms.
Abstract: Autophagy is a self-eating system conserved among eukaryotes, in which cellular components including organelles are entrapped into a double membrane structure called the autophagosome and then degraded by lysosomal hydrolases. In addition to its role in supplying amino acids in response to nutrient starvation, autophagy is involved in quality control to maintain cell health. Thus, inactivation of autophagy causes the formation of cytoplasmic protein inclusions, which comprise misfolded proteins and the accumulation of many degenerated organelles, resulting in liver injury, diabetes, myopathy and neurodegeneration. Furthermore, although autophagy has been considered nonselective, increasing evidence points to the selectivity of autophagy in sorting vacuolar enzymes and removal of aggregate-prone proteins and unwanted organelles. Such selectivity allows diverse cellular regulation, similar to the ubiquitin proteasome pathway. In this review, we discuss the physiological roles of selective autophagy and their molecular mechanisms.
156 citations
TL;DR: Over‐expression as well as repression of SOX2 expression in human ESCs resulted in their differentiation into the trophectoderm lineage, and the data show thatSOX2 plays an important role in the maintenance of pluripotency ofhuman ESCs and possibly, trophoblast development.
Abstract: Human embryonic stem cell (ESC) pluripotency is thought to be regulated by several key transcription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4 and NANOG in human ESCs are well defined, that of SOX2 has not been fully characterized. To investigate the role of SOX2, we modulated the level of SOX2 expression in human ESCs. Reduction of SOX2 expression in human ESCs induced trophectodermal and partial endodermal differentiation. Interestingly, CDX2, a typical trophectoderm-associated gene, was not up-regulated. In contrast, using the Tet-on gene inducible system, SOX2 over-expression in human ESCs induced trophectoderm differentiation accompanied by increased CDX2 expression. Additionally, SOX2 over-expression resulted in an increase in CGalpha-positive cells, which marks later stage trophectoderm development, rather than placental lactogen-positive cells. Thus, over-expression as well as repression of SOX2 expression in human ESCs resulted in their differentiation into the trophectoderm lineage. Our data show that SOX2 plays an important role in the maintenance of pluripotency of human ESCs and possibly, trophoblast development.
145 citations
TL;DR: Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers, which provides a powerful preclinical model for studies in regenerative medicine.
Abstract: Although embryonic stem (ES) cell–like induced pluripotent stem (iPS) cells have potential therapeutic applications in humans, they are also useful for creating genetically modified human disease models in nonhuman primates. In this study, we generated common marmoset iPS cells from fetal liver cells via the retrovirus-mediated introduction of six human transcription factors: Oct-3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28. Four to five weeks after introduction, several colonies resembling marmoset ES cells were observed and picked for further expansion in ES cell medium. Eight cell lines were established, and validation analyses of the marmoset iPS cells followed. We detected the expression of ES cell–specific surface markers. Reverse transcription-PCR showed that these iPS cells expressed endogenous Oct-3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes, whereas all of the transgenes were silenced. Karyotype analysis showed that two of three iPS cell lines retained a normal karyotype after a 2-month culture. Both embryoid body and teratoma formation showed that marmoset iPS cells had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. In summary, we generated marmoset iPS cells via the transduction of six transcription factors; this provides a powerful preclinical model for studies in regenerative medicine.
124 citations
TL;DR: DNA methylation analysis in the mutant oocytes and embryos found that hypomethylation of imprinted genes in Dnmt3a‐deficient oocytes was directly inherited to the embryos, but repetitive elements were re‐methylated during development.
Abstract: In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap-Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.
95 citations
TL;DR: The results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.
Abstract: Embryonic stem (ES) cells display heterogeneous responses upon induction of differentiation. Recent analysis has shown that Hes1 expression oscillates with a period of about 3-5 h in mouse ES cells and that this oscillating expression contributes to the heterogeneous responses: Hes1-high ES cells are prone to the mesodermal fate, while Hes1-low ES cells are prone to the neural fate. These outcomes of Hes1-high and Hes1-low ES cells are very similar to those of inactivation and activation of Notch signaling, respectively. These results suggest that Hes1 and Notch signaling lead to opposite outcomes in ES cell differentiation, although they work in the same direction in most other cell types. Here, we found that Hes1 acts as an inhibitor but not as an effector of Notch signaling in ES cell differentiation. Our results indicate that sustained Hes1 expression delays the differentiation of ES cells and promotes the preference for the mesodermal rather than the neural fate by suppression of Notch signaling.
81 citations
TL;DR: Results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus, which has an HPF homolog but no RMF homologue.
Abstract: In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.
80 citations
TL;DR: Elevated ATPase activities may be a hidden primary defect causing IBMPFD pathological phenotypes, which would be revealed when abnormal proteins are accumulated, as typically observed in aging.
Abstract: Valosin-containing protein (VCP) has been shown to colocalize with abnormal protein aggregates, such as nuclear inclusions of Huntington disease and Machado-Joseph disease, Lewy bodies in Parkinson disease. Several mis-sense mutations in the human VCP gene have been identified in patients suffering inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). Recently, we have shown that VCP possesses both aggregate-forming and aggregate-clearing activities. Here, we showed that in cells treated with proteasome inhibitors VCP first appeared as several small aggregates throughout the cells; and then, these small aggregates gathered together into a single big aggregate. Subcellular localization and ATPase activity of VCP clearly influenced the localization of the aggregates. Furthermore, all tested IBMPFD-causing mutant VCPs, possessed elevated ATPase activities and enhanced aggregate-forming activities in cultured cells. In Drosophila, these mutants and VCP(T761E), a super active VCP, did not appear to spontaneously induce eye degeneration, but worsened the phenotype when co-expressed with polyglutamines. Unexpectedly, these VCPs did not apparently change sizes and the amounts of polyglutamine aggregates in Drosophila eyes. Elevated ATPase activities, thus, may be a hidden primary defect causing IBMPFD pathological phenotypes, which would be revealed when abnormal proteins are accumulated, as typically observed in aging.
80 citations
TL;DR: It is shown that engineered zinc‐finger nucleases (ZFNs), which consist of a zinc‐ finger DNA‐binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo.
Abstract: We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.
80 citations
TL;DR: Results suggest that human NAP1‐like proteins play overlapping roles in transport and deposition of histone H2A‐H2B orH2A variants‐H 2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly.
Abstract: Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A-H2B hetero-dimer. However, the exact mechanism by which NAP1-like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1-like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A-H2B and various histone H2A variants-H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1-like proteins play overlapping roles in transport and deposition of histone H2A-H2B or H2A variants-H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly.
73 citations
TL;DR: Analysis showed that CES5/OOEP was directly associated with the CPLs, which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo.
Abstract: In a search for genes specifically expressed in mouse embryonic stem cells, we identified one we called Ces5. We found that it corresponded to the Ooep gene, which was recently reported to be expressed specifically in oocytes. Mouse Ces5/Ooep, also called Moep19 or Floped, encoded a 164-amino acid protein, which was detected in the cytoplasm of developing and mature oocytes and in embryos throughout the preimplantation period. To examine its function, we carried out targeted disruption of this gene. The Ces5/Ooep-null mice were grossly normal, but the females were infertile. Although the ovaries and ovulation appeared normal, the embryos from Ces5/Ooep-null females mated with wild-type males showed developmental arrest at the two- or four-cell stage. In addition, their first cleavage was considerably delayed and often asymmetrical. Thus, Ces5/Ooep is a maternal-effect gene. By electron microscopy, we found that the eggs from Ces5/Ooep-null females lacked oocyte cytoplasmic lattices (CPLs), which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo. Further analysis showed that CES5/OOEP was directly associated with the CPLs. These results indicate that CES5/OOEP is an essential component of the CPLs and is required for embryonic development at the maternal-zygotic stage transition.
TL;DR: It is established that CFIm not only plays a general role in 3′‐end processing but also plays a regulatory role in poly(A) site selection.
Abstract: Recent estimates indicate that ∼60% of human genes include alternative polyadenylation sites. Hence, control of alternative polyadenylation can have a great impact on gene expression and cellular function. Cleavage factor (CF) Im is a 3′-end processing factor that is essential for in vitro processing. CFIm purified from HeLa cells is associated with three polypeptides of 25, 59 and 68 kD, and it is generally thought to be a heterodimer composed of the 25-kD subunit and one of the larger subunits. Previously, we serendipitously discovered that knockdown of CFIm25 causes an upstream shift in the utilization of alternative polyadenylation sites. Here, we investigated whether this is because of an inherent property of the CFIm complex and, if so, what structural elements are important for its function. The major conclusions of this study are that (i) contrary to previous assumptions, CFIm forms stable heterotetramers through dimerization of CFIm25 and (ii) the CFIm complex per se is responsible for the control of alternative polyadenylation. (iii) However, the structurally related CFIm68 and CFIm59 are functionally redundant and (iv) CFIm68 appears to have a higher specific activity. Thus, this study establishes that CFIm not only plays a general role in 3′-end processing but also plays a regulatory role in poly(A) site selection.
TL;DR: It is revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase‐8 induced by DEC2 siRNA with or without TNF‐α, which indicates that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro‐apoptosis effect, which are involved in the balance of survival of human breast cancer MCF‐7 cells.
Abstract: DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-alpha (TNF-alpha) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-alpha treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-alpha. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells.
TL;DR: The growth of stable PER2 transformants in vivo was significantly and dose‐dependently suppressed according to the amount of PER2 expressed, indicating that PER2 plays a role in the growth suppression of sarcoma cells.
Abstract: Some reports have indicated that the core clock gene, Per2 regulates the cell cycle, immune system and neural functions. To understand the effects of PER2 on tumor growth in vivo, stable transformants of murine sarcoma 180 (S-180) cell lines expressing different levels of PER2 were established. The growth of stable PER2 transformants in vivo was significantly and dose-dependently suppressed according to the amount of PER2 expressed, indicating that PER2 plays a role in the growth suppression of sarcoma cells. The anchorage-dependent and -independent growth in vitro and expression of the clock controlled cell-cycle related genes, wee1, myc, and VEGF were not altered in stable PER2 transformants. In contrast, susceptibility to murine natural killer (NK) cell cytolytic activity was enhanced in PER2 transformants. Furthermore, PER2 transformants suppressed cell motility and reduced fibronectin expression, but the expression of integrin receptors was not affected. These results suggest that sarcoma cells overexpressing PER2 suppress tumors in vivo by changing the nature of tumor cell adhesion.
TL;DR: A structural mechanism that links ATP hydrolysis with the TA‐protein insertion mediated by the conserved DTAPTGH motif is proposed.
Abstract: Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C-terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP-bound and nucleotide-free forms at 3.0 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn2+-mediated homodimer in a head-to-head orientation, representing an open dimer conformation. Our cross-link experiments indicated the closed dimer-stimulating ATP hydrolysis, which might be coupled with TA-protein release. Further, our coexpression-based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA-protein insertion mediated by the conserved DTAPTGH motif.
TL;DR: The phosphorylation of Ser386 was found to be essential for its dimerization and binding with CBP/p300 using mutational analysis and mass spectrometry, and it is concluded that the phosphorylated of Ser 386 is essential for activation of IRF‐3.
Abstract: The transcription factor IRF-3 is activated by microbial invasions and produces a variety of cytokines including type-I interferon. Upon microbial infection, IRF-3 is phosphorylated at its C-terminal regulatory domain, then oligomerized, translocated into the nucleus, and here it binds to CBP/p300. Although a number of studies have been reported investigating the activation mechanism of IRF-3, there are a number of unresolved issues, especially on the phosphorylation sites, the oligomerization process and the binding mechanism with CBP/p300. In this report, the phosphorylated IRF-3 regulatory domain (IRF-3 RD) was prepared using the kinase IKK-i, and the active form of phosphorylated IRF-3 RD was identified. The paper also reports the crystal structure of the active form of the phosphorylated IRF-3 RD. Furthermore, the phosphorylation of Ser386 was found to be essential for its dimerization and binding with CBP/p300 using mutational analysis and mass spectrometry. Thus, we conclude that the phosphorylation of Ser386 is essential for activation of IRF-3.
TL;DR: This review focuses on Nup98 function and discusses how this nucleoporin has evolved in eukaryotic kingdoms, and finds that in ciliates, a unicellular organism having two functionally distinct nuclei, GLFG‐Nup98 is present in one of the nuclei and a distinct N up98 ortholog isPresent in the other nucleus, and these different Nup 98s participate in a nucleus‐selective transport mechanism.
Abstract: The nucleoporin Nup98 is an essential component of the nuclear pore complex. This peripheral nucleoporin with its Gly-Leu-Phe-Gly (GLFG) repeat domain contributes to nuclear-cytoplasmic trafficking, including mRNA export. In addition, accumulating studies indicate that Nup98 plays roles in several important biological events such as gene expression, mitotic checkpoint, and pathogenesis. Nup98 is well conserved among organisms belonging to the fungi and animal kingdoms. These kingdoms belong to the eukaryotic supergroup Opisthokonta. However, there is considerable diversity in the Nup98 orthologs expressed in organisms belonging to other eukaryotic supergroups. Intriguingly, in ciliates, a unicellular organism having two functionally distinct nuclei, GLFG-Nup98 is present in one of the nuclei and a distinct Nup98 ortholog is present in the other nucleus, and these different Nup98s participate in a nucleus-selective transport mechanism. In this review, we focus on Nup98 function and discuss how this nucleoporin has evolved in eukaryotic kingdoms.
TL;DR: It is proposed that PTIP promotes double‐strand break repair through a direct role in HR, and this role is associated with PTIP’s role in transcriptional regulation.
Abstract: PTIP (Pax2 transactivation domain-interacting protein) is a large nuclear protein containing six BRCT (BRCA1 C-Terminal) domains. PTIP is recruited to DNA-damage sites through its BRCT domains and thus has been implicated in the DNA damage response. To define the function of PTIP in DNA repair, we disrupted the PTIP gene of the chicken DT40 B cell line. PTIP mutant (PTIP−/−/−) cells displayed phenotypes frequently observed in cells with defective homologous recombination (HR), i.e. a marked increase in the number of spontaneously arising DNA lesions as well as sensitivity to ionizing radiation (IR) and the topoisomerase I inhibitor, camptothecin. Accordingly, analysis of HR efficiency by using artificial recombination substrates showed that the HR efficiency was reduced in the PTIP-deficient chicken and the PTIP-depleted HeLa cells. As microarray analysis showed no apparent difference between wild-type and PTIP−/−/− cells in the expression of known HR factors, it is unlikely that this reduction in HR efficiency in PTIP-deficient cells is associated with PTIP’s role in transcriptional regulation. We thus propose that PTIP promotes double-strand break repair through a direct role in HR.
TL;DR: It is demonstrated that RNA‐binding motif protein 24 (Rbm24) interacts with the 3′‐untranslated region of myogenin mRNA and affects its half‐life in C2C12 myogenesis, and suggested that Rbm24 binds to myogen in mRNA and regulates its stability in C 2C12 cells.
Abstract: The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12 RNA-binding proteins bind to specific target RNA sequences and regulate gene expression in a post-transcriptional manner This study demonstrates that RNA-binding motif protein 24 (Rbm24) interacts with the 3′-untranslated region of myogenin mRNA and affects its half-life in C2C12 myogenesis Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24-knockdown cells Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin-dependent post-transcriptional regulatory pathway
TL;DR: It is demonstrated that in vitro casein kinase 2 (CK2) specifically interacts with human 9‐1‐1 and phosphorylates serines 341 and 387 (Ser‐341 and Ser‐387) in the C‐terminal tail of Rad9, suggesting that CK2 plays a crucial role in the ATR‐dependent checkpoint pathway.
Abstract: The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded by the Rad17–RFC complex onto chromatin after DNA damage and plays a key role in the ATR-dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9-1-1 and phosphorylates serines 341 and 387 (Ser-341 and Ser-387) in the C-terminal tail of Rad9. Interestingly, phosphorylated Ser-387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9-1-1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over-expressed in HeLa cells, a mutant Rad9 harboring phospho-deficient substitutions at both Ser-341 and Ser-387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR-dependent checkpoint pathway through its ability to phosphorylate Ser-341 and Ser-387 of the Rad9 subunit of the 9-1-1 complex.
TL;DR: Use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia‐neuron interactions in vivo, and targeted and stable expressions of transgenes in glia were obtained.
Abstract: In utero electroporation is widely used to study neuronal development and function by introducing plasmid DNA into neural progenitors during embryogenesis. This is an effective and convenient method of introducing plasmid DNA into neural precursors and is suitable for manipulating gene expression in cells of the CNS. However, the applicability of this technique is comparatively limited to neuronal research, as the plasmid DNA introduced into neural progenitors during embryogenesis is diluted by cell proliferation and is not stably maintained in glial cells generated around and after birth. To overcome this limitation, we applied the Tol2 transposon system, which integrates a transgene into the genome of the host cell, to in utero electroporation. With this system, we confirmed that the transgene was effectively maintained in the progeny of embryonic neural precursors, astrocytes and oligodendrocytes. Using the glial promoters GFAP and S100β, targeted and stable expressions of transgenes in glia were obtained, which enabled the expression of different transgenes simultaneously in neurons and glia. Glia-targeted expression of the transgene that causes neuronal migration defect was achieved without the defect. Thus, use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia-neuron interactions in vivo.
TL;DR: It is found that PSF1 was expressed at relatively high levels in breast tumor cells, but at low levels in normal breast cells, and Knockdown ofPSF1 expression using small interfering RNA slowed the growth of breast cancer cell lines by delaying DNA replication but did not affect proliferation of normal human mammary epithelial cells.
Abstract: PSF1 is a subunit of the GINS complex that functions along with the MCM2-7 complex and Cdc45 in eukaryotic DNA replication. Although mammalian PSF1 is predominantly expressed in highly proliferating cells and organs, little is known about the roles of PSF1 in mature cells or cancer cells. We found that PSF1 was expressed at relatively high levels in breast tumor cells, but at low levels in normal breast cells. Knockdown of PSF1 expression using small interfering RNA (siRNA) slowed the growth of breast cancer cell lines by delaying DNA replication but did not affect proliferation of normal human mammary epithelial cells. Reduced PSF1 expression also inhibited anchorage-independent growth in breast cancer cell lines. These results suggest that PSF1 over-expression is specifically involved in breast cancer cell growth. Therefore, PSF1 inhibition might provide new therapeutic approaches for breast cancer.
TL;DR: It is found that the rate of DNA synthesis in thymine‐starved cells decreases exponentially, indicating replication fork stalling, and argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double‐strand breaks, whereas UvrD counteracts RecAFO‐catalyzed toxic single-strand gap processing.
Abstract: Starvation for DNA precursor dTTP, known as ‘thymineless death’ (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing.
TL;DR: The results suggest that Fox‐1, expressed specifically in the neural cell stage, promoted Mef2c exon β inclusion via the GCAUG.
Abstract: Mef2c protein is one of the MADS-box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon β specifically appeared in the neural cell stage. Because most transcripts contain exon β in the neural cell stage and in the brain, this suggests that the alternative splicing of exon β is highly regulated. Among known regulators, Fox-1 was specifically expressed in the neural cell stage in correlation with Mef2c exon β. Fox-1 promoted exon β inclusion in transfection experiments using Mef2cβ minigene. Moreover, we found that the promotion required RNA-binding activity of Fox-1 and GCAUG sequence located in adjacent intron of exon β. Taken together, our results suggest that Fox-1, expressed specifically in the neural cell stage, promoted Mef2c exon β inclusion via the GCAUG.
TL;DR: It is suggested that UBR1 and UBR2 are negative regulators of the leucine‐mTOR signaling pathway in part through inhibition of their ubiquitin ligase activity.
Abstract: Of 20 natural amino acids, leucine is particularly important for promoting cellular protein synthesis. The effect of leucine involves mammalian target of rapamycin (mTOR), a key protein kinase controlling cell growth. Leucine enhances mTOR-mediated phosphorylation of S6K1 and 4E-BP, thereby promoting protein synthesis. However, how the presence of leucine is sensed and transmitted to mTOR is poorly understood. Here, we show evidence that UBR1 and UBR2 might be cellular targets of leucine. UBR1 and UBR2 are E3 ubiquitin ligases that recognize the identity of N-terminal residues and contribute to selective destabilization of target proteins according to the N-end rule. Using leucine-immobilized affinity beads, we identified UBR1 and UBR2 as leucine-binding proteins from leucine-responsive rat hepatoma H4IIE cells. Over-expression of UBR1 or UBR2 resulted in a reduction in mTOR-dependent S6K1 phosphorylation, whereas knockdown of UBR1 or UBR2 increased S6K1 phosphorylation in amino acid-starved human 293T cells. We also found that leucine binds to the substrate-recognition domain of UBR2 and inhibits degradation of N-end rule substrates in vitro. These findings suggest that UBR1 and UBR2 are negative regulators of the leucine-mTOR signaling pathway. Leucine might activate this pathway in part through inhibition of their ubiquitin ligase activity.
TL;DR: In this article, structural and mechanistic similarities in the disulfide bond formation pathways have been uncovered, highlighting the molecular basis of the elaborate oxidative systems operating in the Escherichia coli periplasm, endoplasmic reticulum lumen and the mitochondrial intermembrane space.
Abstract: The formation of protein disulfide bonds is an oxidative reaction that is crucial for the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells possess various disulfide oxidoreductases and redox-active cofactors to accelerate this oxidative reaction in a correct manner. Crystal or solution structures have been solved for some of the oxidoreductases in the past 10 years, leading to remarkable progress in the field of thiol-based redox cell biology. Consequently, structural and mechanistic similarities in the disulfide bond formation pathways have been uncovered. This review highlights the molecular basis of the elaborate oxidative systems operating in the Escherichia coli periplasm, the endoplasmic reticulum lumen and the mitochondrial intermembrane space. The accumulated knowledge provides important insights into how protein and redox homeostasis are maintained in the cell.
TL;DR: Diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively are found, highlighting an active role ofTIEG1 in the normal clock oscillation and GC box‐mediated regulation of Bmal1 transcription.
Abstract: The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site The GC box/TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERBalpha, a well-known repressor of Bmal1 gene In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription
TL;DR: Functional overlaps between ERK1 and ERK2 are demonstrated and indicate that extracellular signal‐regulated kinase signaling plays a crucial role in cortical development, as well as suggesting that radial glial population expansion and maintenance are essential for the expansion of the radialglial population and for the maintenance of radial glia scaffolding.
Abstract: ERK1/2 is involved in a variety of cellular processes during development, but the functions of these isoforms in brain development remain to be determined. Here, we generated double knockout (DKO) mice to study the individual and combined roles of ERK1 and ERK2 during cortical development. Mice deficient in Erk2, and more dramatically in the DKOs, displayed proliferation defects in late radial glial progenitors within the ventricular zone, and a severe disruption of lamination in the cerebral cortex. Immunohistochemical analyses revealed that late-generated cortical neurons were misplaced and failed to migrate the upper cortical layers in DKO mice. Moreover, these mice displayed fewer radial glial fibers, which provide architectural guides for radially migrating neurons. These results suggest that extracellular signal-regulated kinase signaling is essential for the expansion of the radial glial population and for the maintenance of radial glial scaffolding. Tangential migration of interneurons and oligodendrocytes from the ganglionic eminences (GE) to the dorsal cortex was more severely impaired in DKO mice than in mice deficient for Erk2 alone, because of reduced progenitor proliferation in the GE of the ventral telencephalon. These data demonstrate functional overlaps between ERK1 and ERK2 and indicate that extracellular signal-regulated kinase signaling plays a crucial role in cortical development.
TL;DR: Results suggest that phosphorylation of serine residue occurs in a selective manner, being influenced by the types of modifications and the nature of neighboring lysine residues.
Abstract: Post-translational modifications of histones play important roles in regulating chromatin dynamics and epigenetic inheritance during mitosis. The epigenetic significance and stability of histone H3-lysine 9 (H3K9) modifications have been well studied in interphase cells, whereas not as much in mitotic cells. Here, we inspected mitosis-coupled alterations in the global modifications of H3K9. Signals for H3K9 mono-, di-methylation and acetylation became invisible as cells entered mitosis in contrast to the pattern observed for H3-serine 10 phosphorylation (H3S10ph). Treatment with the aurora-B inhibitor ZM447439 or expression of the dominant negative mutant Aur-B(K106R) resulted in prometaphase chromosomes that lacked signals for H3S10ph but were positive for H3K9 modifications. Trimethylation was the sole K9 modification that remained consistently detectable throughout the cell cycle. This phenomenon was specific for H3K9-S10, as this pattern was not observed at H3K27-S28. Methylated H3K27 remained detectable throughout the cell cycle, despite phosphorylation of the adjacent H3S28. Contrastingly, our dot-blot experiment using synthetic peptides showed that phosphorylation of serine residue basically kept adjacent lysine from antibody access. Together, these results suggest that phosphorylation of serine residue occurs in a selective manner, being influenced by the types of modifications and the nature of neighboring lysine residues.
TL;DR: It is shown that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I, and results suggest that CDK‐dependent phosphorylation regulates meiotic chromosome morphogenesis.
Abstract: Cyclin-dependent protein kinases (CDKs) are required for various cell cycle events both in mitosis and in meiosis During the meiotic prophase of Saccharomyces cerevisiae, only one CDK, Cdc28, which forms a complex with B-type cyclins, Clb5 or Clb6, promotes not only the onset of premeiotic DNA replication but also the formation of meiotic double-strand breaks (DSBs) In this study, we showed that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I Chromosomal localization of Cdc28, dependent on Clb5 and/or Clb6, is frequently observed in zygotene and pachytene, when formation of the synaptonemal complex (SC) occurs Interestingly, the CDK localization is independent of DSB formation, but rather dependent on meiosis-specific chromosome components such as Red1, Hop1 and a cohesin subunit Rec8 Compromised CDK activity in meiotic prophase leads to defective SC formation without affecting DSB formation These results suggest that CDK-dependent phosphorylation regulates meiotic chromosome morphogenesis