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1.	Andrae, Johanna, et al. (författare) Analysis of Mice Lacking the Heparin-Binding Splice Isoform of Platelet-Derived Growth Factor A 2013 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 33:20, s. 4030-4040 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.
2.	Annicotte, Jean-Sébastien, et al. (författare) Pancreatic-duodenal homeobox 1 regulates expression of liver receptor homolog 1 during pancreas development. 2003 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 23:19, s. 6713-6124 Tidskriftsartikel (refereegranskat)abstract Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1(-/-) mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.
3.	Antonsson, Åsa, et al. (författare) Regulation of c-Rel Nuclear Localization by Binding of Ca2+/Calmodulin 2003 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 23:4, s. 1418-1427 Tidskriftsartikel (refereegranskat)abstract The NF-κB/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory IκB proteins sequester NF-κB/Rel in the cytoplasm. Cellular stimulation results in the degradation of IκB and modification of NF-κB/Rel proteins, allowing NF-κB/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca2+ binding protein that serves as a key mediator of intracellular Ca2+ signals. Here we report that two members of the NF-κB/Rel family, c-Rel and RelA, interact directly with Ca2+-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of IκB. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca2+ signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from IκB and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-κB/Rel proteins following stimulation.
4.	Arvidsson, Ann-Kristin, et al. (författare) Tyr-716 in the platelet-derived growth factor beta-receptor kinase insertis involved in GRB2 binding and Ras activation 1994 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 14:10, s. 6715-6726 Tidskriftsartikel (refereegranskat)abstract Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.
5.	Banyai, Gabor, et al. (författare) Mediator Can Regulate Mitotic Entry and Direct Periodic Transcription in Fission Yeast 2014 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 34:21, s. 4008-4018 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.
6.	Bao, W J, et al. (författare) Cell attachment to the extracellular matrix induces proteasomal degradation of p21(CIP1) via Cdc42/Rac1 signaling 2002 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 22:13, s. 4587-4597 Tidskriftsartikel (refereegranskat)abstract The cyclin-dependent kinase 2 (Cdk2) inhibitors p21(CIP1) and p27(KIP1) are negatively regulated by anchorage during cell proliferation, but it is unclear how integrin signaling may affect these Cdk2 inhibitors. Here, we demonstrate that integrin ligation led to rapid reduction of p21(CIp1) and p27(KIP1) protein levels in three distinct cell types upon attachment to various extracellular matrix (ECM) proteins, including fibronectin (FN), or to immobilized agonistic anti-integrin monoclonal antibodies. Cell attachment to FN did not rapidly influence p21(CIp1) mRNA levels, while the protein stability of p21(CIp1) was decreased. Importantly, the down-regulation of p21(CIP1) and p27(KIP1) was completely blocked by three distinct proteasome inhibitors, demonstrating that integrin ligation induced proteasomal degradation of these Cdk2 inhibitors. Interestingly, ECM-induced proteasomal proteolysis of a ubiquitination-deficient p21(CIP1) mutant (p21K6R) also occurred, showing that the proteasomal degradation of p21(CIP1) was ubiquitin independent. Concomitant with our finding that the small GTPases Cdc42 and Rac1 were activated by attachment to FN, constitutively active (ca) Cdc42 and ca Rac1 promoted down-regulation of p21(CIP1). However, dominant negative (dn) Cdc42 and do Rac1 mutants blocked the anchorage-induced degradation of p21(CIP1), suggesting that an integrin-induced Cdc42/Rac1 signaling pathway activates proteasomal degradation of p21(CIP1). Our results indicate that integrin-regulated proteasomal proteolysis might contribute to anchorage-dependent cell cycle control.
7.	Bjerling, Pernilla, et al. (författare) Functional divergence between histone deacetylases in fission yeast by distinct cellular localization and in vivo specificity 2002 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 22:7, s. 2170-2181 Tidskriftsartikel (refereegranskat)abstract Histone deacetylases (HDACs) are important for gene regulation and the maintenance of heterochromatin in eukaryotes. Schizosaccharomyces pombe was used as a model system to investigate the functional divergence within this conserved enzyme family. S. pombe has three HDACs encoded by the hda1(+), clr(3+), and clr6(+) genes. Strains mutated in these genes have previously been shown to display strikingly different phenotypes when assayed for viability, chromosome loss, and silencing. Here, conserved differences in the substrate binding pocket identify Clr6 and Hda1 as class I HDACs, while Clr3 belongs in the class II family. Furthermore, these HDACs were shown to have strikingly different subcellular localization patterns. Hda1 was localized to the cytoplasm, while most of Clr3 resided throughout the nucleus. Finally, Clr6 was localized exclusively on the chromosomes in a spotted pattern. Interestingly, Clr3, the only HDAC present in the nucleolus, was required for ribosomal DNA (rDNA) silencing. Clr3 presumably acts directly on heterochromatin, since it colocalized with the centromere, mating-type region, and rDNA as visualized by in situ hybridization. In addition, Clr3 could be cross-linked to mat3 in chromatin immunoprecipitation experiments. Western analysis of bulk histone preparations indicated that Hda1 (class I) had a generally low level of activity in vivo and Clr6 (class 1) had a high level of activity and broad in vivo substrate specificity, whereas Clr3 (class II) displayed its main activity on acetylated lysine 14 of histone H3. Thus, the distinct functions of the S. pombe HDACs are likely explained by their distinct cellular localization and their different in vivo specificities.
8.	Björk Grimberg, Kristian, et al. (författare) Basic Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome 2011 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 31:4, s. 897-909 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.
9.	Björkblom, Benny, et al. (författare) c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells 2012 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 32:17, s. 3513-3526 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.
10.	Björkblom, Benny, et al. (författare) Reactive oxygen species-mediated DJ-1 monomerization modulates intracellular trafficking involving karyopherin beta 2 2014 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 34:16, s. 3024-3040 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.
11.	Björkman, Lena, 1965, et al. (författare) The Neutrophil Response Induced by an Agonist for Free Fatty Acid Receptor 2 (GPR43) Is Primed by Tumor Necrosis Factor Alpha and by Receptor Uncoupling from the Cytoskeleton but Attenuated by Tissue Recruitment 2016 Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 36:20, s. 2583-2595 Tidskriftsartikel (refereegranskat)abstract Ligands with improved potency and selectivity for free fatty acid receptor 2 (FFA2R) have become available, and we here characterize the neutrophil responses induced by one such agonist (Cmp1) and one antagonist (CATPB). Cmp1 triggered an increase in the cytosolic concentration of Ca2+, and the neutrophils were then desensitized to Cmp1 and to acetate, a naturally occurring FFA2R agonist. The antagonist CATPB selectively inhibited responses induced by Cmp1 or acetate. The activated FFA2R induced superoxide anion secretion at a low level in naive blood neutrophils. This response was largely increased by tumor necrosis factor alpha (TNF-alpha) in a process associated with a recruitment of easily mobilizable granules, but neutrophils recruited to an aseptic inflammation in vivo were nonresponding. Superoxide production induced by Cmp1 was increased in latrunculin A-treated neutrophils, but no reactivation of desensitized FFA2R was induced by this drug, suggesting that the cytoskeleton is not directly involved in terminating the response. The functional and regulatory differences between the receptors that recognize short-chain fatty acids and formylated peptides, respectively, imply different roles of these receptors in the orchestration of inflammation and confirm the usefulness of a selective FFA2R agonist and antagonist as tools for the exploration of the precise role of the FFA2R.
12.	Carlsten, Jonas O P, et al. (författare) Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres 2012 Ingår i: Molecular and Cellular Biology. - : Informa UK Limited. - 0270-7306 .- 1098-5549. ; 32:19, s. 4035-4043 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.
13.	Chaudhari, Aditi, et al. (författare) p110alpha hot spot mutations E545K and H1047R exert metabolic reprogramming independently of p110alpha kinase activity : Kinase-independent signaling of p110 alpha mutants 2015 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 35:19, s. 3258-3273 Tidskriftsartikel (refereegranskat)abstract The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.
14.	Chaudhari, Aditi, et al. (författare) p110α hot spot mutations E545K and H1047R exert metabolic reprogramming independently of p110α kinase activity 2015 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 35:19, s. 3258-3273 Tidskriftsartikel (refereegranskat)abstract The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.
15.	Chernukhin, Igor, et al. (författare) CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide 2007 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 27:5, s. 1631-1648 Tidskriftsartikel (refereegranskat)abstract CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.
16.	Crabtree, Judy S, et al. (författare) Of mice and MEN1 : Insulinomas in a conditional mouse knockout. 2003 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 23:17, s. 6075-6085 Tidskriftsartikel (refereegranskat)abstract Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.
17.	Culver, Carolyn, et al. (författare) Mechanism of Hypoxia-Induced NF-kappa B 2010 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 30:20, s. 4901-4921 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.
18.	Dahlén, Maria, 1965, et al. (författare) Replication proteins influence the maintenance of telomere length and telomerase protein stability 2003 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 23, s. 3031-3042 Tidskriftsartikel (refereegranskat)abstract We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability
19.	Dever, Thomas E, et al. (författare) Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes 1995 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 15:11, s. 6351-6363 Tidskriftsartikel (refereegranskat)abstract To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.
20.	DiRenzo, J, et al. (författare) Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors 1997 Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 17:4, s. 2166-2176 Tidskriftsartikel (refereegranskat)abstract As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation.
21.	Edlund, Sofia, et al. (författare) Interaction between Smad7 and beta-catenin : importance for transforming growth factor beta-induced apoptosis 2005 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 25:4, s. 1475-1488 Tidskriftsartikel (refereegranskat)abstract Members of the transforming growth factor beta (TGF-beta) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with beta-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-beta-dependent manner. Smad7 was found to be required for TGF-beta1-induced accumulation of beta-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-beta-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3beta phosphorylated on Ser9 was prevented, as well as the TGF-beta-induced association between beta-catenin and LEF1. Notably, the observed physical association of Smad7 and beta-catenin was found to be important for TGF-beta-induced apoptosis, since suppression of beta-catenin expression by small interfering RNA decreased the apoptotic response to TGF-beta.
22.	Fagerström-Billai, Fredrik, et al. (författare) Functional comparison of the Tup11 and Tup12 transcriptional corepressors in fission yeast 2005 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 25:2, s. 716-727 Tidskriftsartikel (refereegranskat)abstract Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11(-) and tup12(-) mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11(-) and tup12- mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes. Many of these genes are differentially derepressed in tup11(-) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12(-) mutants require the Ssn6 protein for their repression. As for Tupl.2, Ssn6 is also required for efficient adaptation to KCI- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.
23.	Fagerström-Billai, Fredrik, et al. (författare) Individual Subunits of the Ssn6-Tup11/12 corepressor are selectively required for repression of different target genes 2007 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 27:3, s. 1069-1082 Tidskriftsartikel (refereegranskat)abstract The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (CIr3) and III (Hst4 and Sir2) HDACs.
24.	Fleenor, Courtney J., et al. (författare) Zinc Finger Protein 521 Regulates Early Hematopoiesis through Cell-Extrinsic Mechanisms in the Bone Marrow Microenvironment 2018 Ingår i: Molecular and Cellular Biology. - : AMER SOC MICROBIOLOGY. - 0270-7306 .- 1098-5549. ; 38:17 Tidskriftsartikel (refereegranskat)abstract Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Kruppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated Zfp521 genes (Zfp521(-/-)) possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521s role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies.
25.	Friant, S, et al. (författare) Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo 1998 Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 18:2, s. 799-806 Tidskriftsartikel (refereegranskat)abstract Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).

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