| 1. |
- Andrae, Johanna, et al.
(författare)
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Analysis of Mice Lacking the Heparin-Binding Splice Isoform of Platelet-Derived Growth Factor A
- 2013
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 33:20, s. 4030-4040
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Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factor A-chain (PDGF-A) exists in two evolutionarily conserved isoforms, PDGF-Along and PDGF-Ashort, generated by alternative RNA splicing. They differ by the presence (in PDGF-Along) or absence (in PDGF-Ashort) of a carboxyterminal heparin/heparan sulfate proteoglycan-binding motif. In mice, similar motifs present in other members of the PDGF and vascular endothelial growth factor (VEGF) families have been functionally analyzed in vivo, but the specific physiological importance of PDGF-A(long) has not been explored previously. Here, we analyzed the absolute and relative expression of the two PDGF-A splice isoforms during early postnatal organ development in the mouse and report on the generation of a Pdgfa allele (Pdgfa(Delta ex6) incapable of producing PDGF-A(long) due to a deletion of the exon 6 splice acceptor site. In situations of limiting PDGF-A signaling through PDGF receptor alpha (PDGFR alpha), or in mice lacking PDGF-C, homozygous carriers of Pdgfa(Delta ex6) showed abnormal development of the lung, intestine, and vertebral column, pinpointing developmental processes where PDGF-A(long) may play a physiological role.
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| 2. |
- Banyai, Gabor, et al.
(författare)
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Mediator Can Regulate Mitotic Entry and Direct Periodic Transcription in Fission Yeast
- 2014
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 34:21, s. 4008-4018
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Tidskriftsartikel (refereegranskat)abstract
- Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events.
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| 3. |
- Björk Grimberg, Kristian, et al.
(författare)
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Basic Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome
- 2011
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 31:4, s. 897-909
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Tidskriftsartikel (refereegranskat)abstract
- While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.
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| 4. |
- Björkblom, Benny, et al.
(författare)
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c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells
- 2012
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 32:17, s. 3513-3526
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Tidskriftsartikel (refereegranskat)abstract
- Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1S120D,T148D,T183D) inhibits whereas dephospho-MARCKSL1S120A,T148A,T183A induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.
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| 5. |
- Björkblom, Benny, et al.
(författare)
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Reactive oxygen species-mediated DJ-1 monomerization modulates intracellular trafficking involving karyopherin beta 2
- 2014
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 34:16, s. 3024-3040
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin beta 2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.
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| 6. |
- Carlsten, Jonas O P, et al.
(författare)
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Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres
- 2012
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Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 32:19, s. 4035-4043
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Tidskriftsartikel (refereegranskat)abstract
- At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20 Delta cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
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| 7. |
- Culver, Carolyn, et al.
(författare)
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Mechanism of Hypoxia-Induced NF-kappa B
- 2010
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 30:20, s. 4901-4921
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Tidskriftsartikel (refereegranskat)abstract
- NF-kappa B activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, I kappa B kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)dependent pathway distinct from that for other common inducers of NF-kappa B. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-kappa B, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-kappa B inducers, IKK-mediated phosphorylation of I kappa B alpha does not result in its degradation. We show that this results from I kappa B alpha sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from I kappa B alpha and activate NF-kappa B target genes. These results define a novel pathway regulating NF-kappa Bactivation, important to its physiological role in human health and disease.
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| 8. |
- Hoch, Nicolas C, et al.
(författare)
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Molecular basis of the essential s phase function of the rad53 checkpoint kinase
- 2013
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 33:16, s. 3202-3213
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Tidskriftsartikel (refereegranskat)abstract
- The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.
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| 9. |
- Hogan, C. J., et al.
(författare)
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Fission yeast Iec1-Ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism
- 2010
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 30:3, s. 657-674
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Tidskriftsartikel (refereegranskat)abstract
- Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenineresponsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.
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| 10. |
- Johansson, Anna-Mia, 1978-, et al.
(författare)
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POF Regulates the Expression of Genes on the Fourth Chromosome in Drosophila melanogaster by Binding to Nascent RNA
- 2012
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 32:11, s. 2121-2134
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. Here, we show that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. We also show that genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment.
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