MCB

Phosphorylation of MNAR Promotes Estrogen Activation of Phosphatidylinositol 3-Kinase.

Publication Date: 2010 Mar PMID: 20194629
Authors: Greger, J. G. - Fursov, N. - Cooch, N. - McLarney, S. - Freedman, L. P. - Edwards, D. P. - Cheskis, B. J.
Journal: Mol Cell Biol

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Articles of significant interest selected from this issue by the editors.

Publication Date: 2010 Mar PMID: 20194628
Authors:
Journal: Mol Cell Biol

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p27kip1 controls cell morphology and motility by regulating microtubule-dependent lipid raft recycling.

Publication Date: 2010 Mar 1 PMID: 20194624
Authors: Belletti, B. - Pellizzari, I. - Berton, S. - Fabris, L. - Wolf, K. - Lovat, F. - Schiappacassi, M. - D'Andrea, S. - Nicoloso, M. S. - Lovisa, S. - Sonego, M. - Defilippi, P. - Vecchione, A. - Colombatti, A. - Friedl, P. - Baldassarre, G.
Journal: Mol Cell Biol

p27(kip1) (p27) is an inhibitor of cyclin/CDK complexes whose nuclear loss indicates poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/Stathmin (Stathmin), a microtubule (MT) destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three dimensional (3D)-matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D-environments and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility and MT network composition and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and Stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D-contexts.

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MED14 tethers Mediator to the N-terminal domain of PPAR{gamma} and is required for full transcriptional activity and adipogenesis.

Publication Date: 2010 Mar 1 PMID: 20194623
Authors: Grontved, L. - Madsen, M. S. - Boergesen, M. - Roeder, R. G. - Mandrup, S.
Journal: Mol Cell Biol

The Mediator subunit MED1/TRAP220/DRIP205/PBP interacts directly with many nuclear receptors and was long thought to be responsible for tethering the Mediator to peroxisome proliferator activated receptor (PPAR) responsive promoters. However, recently it was demonstrated that PPARgamma can recruit the Mediator by MED1 independent mechanisms. Here we show that target gene activation by ectopically expressed PPARgamma and PPARalpha is independent of MED1. Consistent with this finding, recruitment of PPARgamma, MED6, MED8, TBP and RNA PII to the enhancer and proximal promoter of the PPARgamma target gene Fabp4 is also independent of MED1. Using an siRNA-based approach, we identify MED14 as a novel critical Mediator component for PPARgamma-dependent transactivation, and we demonstrate that MED14 interacts directly with the N-terminus of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect recruitment of PPARgamma, MED6 and MED8 to the Fabp4 enhancer, but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show that knockdown of MED14 impairs adipogenesis of 3T3-L1 cells. Thus, MED14 constitutes a novel anchoring point between the Mediator and the N-terminal domain of PPARgamma that is necessary for functional PPARgamma-mediated recruitment of Mediator and transactivation of PPARgamma subtype specific target genes.

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MOLECULAR BASIS FOR LYSINE SPECIFICITY IN THE YEAST UBIQUITIN-CONJUGATING ENZYME CDC34.

Publication Date: 2010 Mar 1 PMID: 20194622
Authors: Sadowski, M. - Suryadinata, R. - Lai, X. - Heierhorst, J. - Sarcevic, B.
Journal: Mol Cell Biol

Ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of ubiquitin (Ub) to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for generating diverse substrate-Ub structures, which provides versatility to this pathway in targeting proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that ubiquitination site/s are selected by E2/E3-mediated positioning of lysine/s toward the E2/E3 active site. By studying polyubiquitination of Sic1 by the E2, Cdc34, and the RING E3, Skp1/Cul1/F box protein (SCF), we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified if this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

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Human mitochondrial leucyl-tRNA synthetase corrected mitochondrial dysfunctions due to the MELAS and diabetes associated tRNALeu(UUR) A3243G mutation.

Publication Date: 2010 Mar 1 PMID: 20194621
Authors: Li, R. - Chomyn, A. - Guan, M. X.
Journal: Mol Cell Biol

Mutations in mitochondrial tRNA genes are associated with a wide spectrum of human diseases. In particular, the tRNA(Leu(UUR)) A3243G mutation causes mitochondrial encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS) and 2% cases of type 2 diabetes. The primary defect in this mutation was an inefficient aminoacylation of the tRNA(Leu(UUR)). In the present study, we have investigated the molecular mechanism of the A3243G mutation and if the overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) in the cybrid cells carrying the A3243G mutation corrects the mitochondrial dysfunctions. Human LARS2 localizes exclusively to mitochondria and LARS2 is expressed ubiquitously but most abundantly in tissues with high metabolic rates. We showed that the alteration of aminoacylation tRNA(Leu(UUR)) caused by the A3243G mutation led to mitochondrial translational defects and thereby reduced the aminoacylated efficiencies of tRNA(Leu(UUR)) as well as tRNA(Ala) and tRNA(Met). We demonstrated that the transfer of human mitochondrial leucyl-tRNA synthetase into the cybrid cells carrying the A3243G mutation improved the efficiency of aminoacylation and stability of mitochondrial tRNAs, then increased the rates of mitochondrial translation and respiration, consequently correcting the mitochondrial dysfunction. These findings provide new insights into the molecular mechanism of maternally inherited diseases and a step towards therapeutic interventions for these disorders.

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Control of Adipogenesis by the SUMO-Specific Protease SENP2.

Publication Date: 2010 Mar 1 PMID: 20194620
Authors: Chung, S. S. - Ahn, B. Y. - Kim, M. - Choi, H. H. - Park, H. S. - Kang, S. A. - Park, S. G. - Kim, Y. B. - Cho, Y. M. - Lee, H. K. - Chung, C. H. - Park, K. S.
Journal: Mol Cell Biol

Here, we demonstrate that SENP2, a desumoylating enzyme, plays a critical role in the control of adipogenesis. SENP2 expression was markedly increased upon the induction of adipocyte differentiation, and this increase was dependent on PKA activation. Remarkably, knockdown of SENP2 led to a dramatic attenuation of adipogenesis with a marked decrease in the mRNA levels of PPARgamma and C/EBPalpha. Knockdown of SENP2 also caused a marked reduction in the level of C/EBPbeta protein, but not that of its mRNA. Interestingly, sumoylation of C/EBPbeta dramatically increased its ubiquitination and destabilization, and this increase could be reversed by SENP2. In addition, over-expression of C/EBPbeta could overcome the inhibitory effect of SENP2 knockdown on adipogenesis. Furthermore, SENP2 was absolutely required for adipogenesis of preadipocytes implanted into mice. These results establish a critical role of SENP2 in the regulation of adipogenesis by desumoylation and stabilization of C/EBPbeta and in turn by promoting the expression of its downstream effectors, such as PPARgamma and C/EBPalpha.

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The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.

Publication Date: 2010 Mar 1 PMID: 20194619
Authors: Dey, S. - Curtis, D. J. - Jane, S. M. - Brandt, S. J.
Journal: Mol Cell Biol

Monocytopoiesis involves the step-wise differentiation in the bone marrow (BM) of common myeloid precursor (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and, while reported as expressed by BM-derived macrophages, its role in monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal through G1 and S phases. In conjunction, steady state levels of p16(Ink4a) mRNA were increased and Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during monocytopoiesis and the requirement for direct DNA binding in these processes.

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HMGA1a Trapping of U1 snRNP at Authentic 5' Splice Site Induces Aberrant Exon Skipping in Sporadic Alzheimer's Disease.

Publication Date: 2010 Mar 1 PMID: 20194618
Authors: Ohe, K. - Mayeda, A.
Journal: Mol Cell Biol

Overexpression of high-mobility group A protein 1a (HMGA1a) causes aberrant exon 5 skipping of the Presenilin-2 (PS2) pre-mRNA, which is almost exclusively detected in patients with sporadic Alzheimer's disease. An electrophoretic mobility shift assay confirmed aberrant U1 snRNP-HMGA1a complex formation (via the U1-70K component) with RNA containing a specific HMGA1a-binding site and an adjacent 5' splice site. Psoralen crosslinking analysis demonstrated that the binding of HMGA1a adjacent to the 5' splice site induces unusually extended association of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site. As a result, spliceosome assembly across either the intron or the exon is arrested at an early ATP-independent stage. We conclude that the HMGA1a-induced aberrant exon skipping is caused by impaired dissociation of U1 snRNP from the 5' splice site, leading to a defect in exon definition. The proposed molecular mechanism has profound implications for other known post-transcriptional modulation strategies in various organisms, all of which are triggered by aberrant U1 snRNP binding.

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Par1b/MARK2 phosphorylates kinesin-like motor protein GAKIN/KIF13B to regulate axon formation.

Publication Date: 2010 Mar 1 PMID: 20194617
Authors: Yoshimura, Y. - Terabayashi, T. - Miki, H.
Journal: Mol Cell Biol

Here we report that Par1b/MARK2 regulates axon formation via phosphorylation of a kinesin superfamily protein GAKIN/KIF13B. Accumulating evidence indicated the importance of the evolutionarily conserved kinase Par1b in the regulation of cell polarity. Using hippocampal neurons in culture, it has been shown that Par1b regulates axon specification, but the underlining mechanism remains uncharacterized. We identify GAKIN/KIF13B as a novel Par1b-binding protein and reveal that GAKIN/KIF13B is a physiological substrate for Par1b, and the phosphorylation sites are conserved from Drosophila. In hippocampal neurons, GAKIN/KIF13B accumulates at the distal part of the microtubules in the tips of axons, but not of dendrites. Overexpression of GAKIN/KIF13B by itself can induce the formation of extra axons, which is inhibited by the co-expression of Par1b in a manner dependent on its kinase activity. In contrast, siRNA-mediated knockdown of GAKIN/KIF13B severely retards neurite extension and promotes axonless phenotype. The extra axon phenotype caused by Par1b-siRNA is suppressed by co-introduction of GAKIN/KIF13B-siRNA, thus placing the GAKIN/KIF13B function at the downstream of Par1b. We also find that GAKIN/KIF13B acts downstream of the PI3K signaling via Par1b-phosphorylation. These results reveal that GAKIN/KIF13B is a key intermediate linking Par1b to the regulation of axon formation.

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A comprehensive model that explains the regulation of Phospholipase D2 (PLD2) activity by phosphorylation-dephosphorylation.

Publication Date: 2010 Feb 22 PMID: 20176813
Authors: Henkels, K. M. - Peng, H. J. - Frondorf, K. - Gomez-Cambronero, J.
Journal: Mol Cell Biol

We report here that the enzymatic activity of PLD2 is regulated by phosphorylation-dephosphorylation. Phosphatase treatment of PLD2-overexpressing cells indicated a biphasic nature of changes in activity that indicated the existence "activator" and "inhibitory" sites. We have identified three kinases capable of phosphorylating PLD2 in vitro: EGF-R, JAK3 and Src (with JAK3 reported for the first time in this study), that phosphorylate an inhibitory, an activator, and an ambivalent (one that can yield either effect) site, respectively. Mass spectrometry analyses indicated the target of each of these kinases as Y(296) for EGFR, Y(415) for JAK3 and Y(511) for Src. The extent to which each site is activated or inhibited depends on the cell type considered. In COS-7, cells that show the highest level of PLD2 activity, the Y(415) is a prominent site and JAK3 compensates the negative modulation by EGFR on Y(296). In MCF-7, cells that show the lowest level of PLD2 activity, the converse is the case, with Y(296) unable to compensate the positive modulation by Y(415). MTLn3, with medium/low levels of lipase activity, show an intermediate pattern of regulation but closer to MCF-7 than to COS-7 cells. The negative effect of EGFR on the two cancer cell lines MTLn3 and MCF-7 is further proven by RNA silencing experiments that yield COS-7 showing lower PLD2 activity, and MTLn3 and MCF-7 cells showing an elevated activity. MCF-7 is a cancer cell line derived from a low-aggressive/invasive form of breast cancer that has relatively low levels of PLD activity. We propose that PLD2 activity is low in the breast cancer cell line MCF-7 because it is kept downregulated by tyrosyl phosphorylation of Y(296) by EGFR kinase. Thus, phosphorylation of PLD2-Y(296) could be the signal for lowering the level of PLD2 activity, in transformed cells with low invasive capabilities.

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Repression of transcriptional activity of C/EBP{alpha} by E2F-DP complexes.

Publication Date: 2010 Feb 22 PMID: 20176812
Authors: Zaragoza, K. - Begay, V. - Schuetz, A. - Heinemann, U. - Leutz, A.
Journal: Mol Cell Biol

The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) coordinates proliferation arrest and differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, cells of the lung, and placenta. C/EBPalpha transactivates lineage specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPalpha BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPalpha mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. Data presented here show that, independently of pocket proteins, C/EBPalpha interacts with the dimerization partner (DP) of E2F and that C/EBPalpha-E2F/DP interaction prevents both, binding of C/EBPalpha to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity towards DNA yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPalpha, E2F/DP and pocket proteins in the control of proliferation, differentiation and tumorigenesis.

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The Molecular Architecture of the Human Prp19/CDC5L Complex.

Publication Date: 2010 Feb 22 PMID: 20176811
Authors: Grote, M. - Wolf, E. - Will, C. L. - Lemm, I. - Agafonov, D. E. - Schomburg, A. - Fischle, W. - Urlaub, H. - Luhrmann, R.
Journal: Mol Cell Biol

Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like the yeast NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease resistant complex comprised of SPF27, the C-terminus of CDC5L, and the N-terminus of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of approximately 20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex, but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.

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The SUMO E3 ligase activity of Pc2 is coordinated through a SUMO-interaction motif.

Publication Date: 2010 Feb 22 PMID: 20176810
Authors: Yang, S. H. - Sharrocks, A. D.
Journal: Mol Cell Biol

Protein modification by SUMO conjugation has emerged to be an important regulatory event. Recently the mechanisms through which SUMO elicits its effects on target proteins have been elucidated. One of these is the non-covalent association between SUMO and coregulatory proteins via SUMO interaction motifs (SIMs). We therefore searched for additional binding proteins to elucidate how SUMO acts as a signal to potentiate novel non-covalent interactions with SUMO-binding proteins. We identified an E3 ligase, Pc2, as a SUMO-binding protein with two functionally distinct SIMs. Here, we focus on the role of SIM2 and demonstrate that it is crucial for many of the documented Pc2 functions which converge on determining its E3 ligase activity. One role of SUMO binding in this context is the subnuclear partitioning of the active form of Ubc9 (SUMO approximately Ubc9) by Pc2. The significance of the SIM2-dependent functions of Pc2 is demonstrated in the control of the precise expression of lineage-specific genes during embryonic stem cell differentiation.

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PRH/HHex controls cell survival through coordinate transcriptional regulation of VEGF signalling.

Publication Date: 2010 Feb 22 PMID: 20176809
Authors: Noy, P. - Williams, H. - Sawasdichai, A. - Gaston, K. - Jayaraman, P. S.
Journal: Mol Cell Biol

The Proline Rich Homeodomain protein (PRH) plays multiple roles in the control of gene expression during embryonic development and in the adult. Vascular Endothelial Growth Factor (VEGF) is a mitogen that stimulates cell proliferation and survival via cell surface receptors including VEGFR-1 and VEGFR-2. VEGF signalling is of critical importance in angiogenesis and hematopoiesis and is elevated in many tumors. Here we show that PRH binds directly to the promoter regions of Vegf, Vegfr-1 and Vegfr-2 genes and that in each case PRH represses transcription. We demonstrate that over-expression or knockdown of PRH directly impinges on the survival of both leukemic and tumor cells, and that the modulation of VEGF and VEGF receptor signalling by PRH mediates these effects. Our findings demonstrate that PRH is a key regulator of the VEGF signalling pathway and describe a mechanism whereby PRH plays an important role in tumorigenesis and leukemogenesis.

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TEX14 Interacts with CEP55 to Block Cell Abscission.

Publication Date: 2010 Feb 22 PMID: 20176808
Authors: Iwamori, T. - Iwamori, N. - Ma, L. - Edson, M. A. - Greenbaum, M. P. - Matzuk, M. M.
Journal: Mol Cell Biol

In somatic cells, abscission, the physical separation of daughter cells at the completion of cytokinesis, requires CEP55, ALIX, and TSG101. In contrast, cytokinesis is arrested prior to abscission in differentiating male germ cells that are interconnected by TEX14-positive intercellular bridges. We have previously shown that targeted deletion of TEX14 disrupts intercellular bridges in all germ cells and causes male sterility. Although these findings demonstrate that intercellular bridges are essential for spermatogenesis, it remains to be shown how TEX14 and other proteins come together to prevent abscission and form stable intercellular bridges. Using a biochemical enrichment of male germ cell intercellular bridges, we identified additional bridge proteins including CEP55. Although CEP55 is highly expressed in testes at the RNA level, there is no report of the presence of CEP55 in germ cells. Herein, we show that CEP55 becomes a stable component of the intercellular bridge, and an evolutionarily-conserved GPPX3Y motif of TEX14 binds strongly to CEP55 to block similar GPPX3Y motifs of ALIX and TSG101 from interacting and localizing to the midbody. Thus, TEX14 prevents the completion of cytokinesis by altering the destiny of CEP55 from a nidus for abscission to an integral component of the intercellular bridge.

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Drosophila RB proteins repress differentiation-specific genes via two different mechanisms.

Publication Date: 2010 Feb 22 PMID: 20176807
Authors: Lee, H. - Ohno, K. - Voskoboynik, Y. - Ragusano, L. - Martinez, A. - Dimova, D. K.
Journal: Mol Cell Biol

RB and E2F proteins play important roles in the regulation of cell division, cell death and development, by controlling the expression of genes involved in these processes. The mechanisms of repression by pRB have been extensively studied at cell cycle regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here we have taken advantage of the simplicity of the E2F/RB pathway in flies, and inspected the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex -dREAM/MMB in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional repression mechanism by the Polycomb group (PcG) protein, Enhancer of zeste - E(Z), which is involved in silencing of these genes trough the di-methylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).

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Cell-Specific Determinants of PPAR{gamma} Function in Adipocytes and Macrophages.

Publication Date: 2010 Feb 22 PMID: 20176806
Authors: Lefterova, M. I. - Steger, D. J. - Zhuo, D. - Qatanani, M. - Mullican, S. E. - Tuteja, G. - Manduchi, E. - Grant, G. R. - Lazar, M. A.
Journal: Mol Cell Biol

The nuclear receptor Peroxisome Proliferator Activator Receptor gamma (PPARgamma) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARgamma is required for adipocyte differentiation, but is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARgamma binding in macrophages occurs at the same or different genomic locations compared to adipocytes. Here, utilizing chromatin immunoprecipitation and high throughput sequencing (ChIP-seq), we demonstrate that PPARgamma cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycollate-elicited macrophages, PPARgamma colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARgamma, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell-specificity of PPARgamma function is regulated by cell-specific transcription factors, chromatin accessibility, and histone mark. Our data support the existence of an epigenomic hierarchy in which PPARgamma binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks including histone acetylation.

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Microtubule stabilization by BMP receptor-mediated scaffolding of JNK promotes dendrite formation.

Publication Date: 2010 Feb 22 PMID: 20176805
Authors: Podkowa, M. - Zhao, X. - Chow, C. W. - Coffey, E. T. - Davis, R. J. - Attisano, L.
Journal: Mol Cell Biol

Neuronal outgrowth occurs via coordinated remodelling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone Morphogenetic Proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics and we show that JNKs associate with the carboxy-terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodelling, and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.

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PPAR{gamma} dances with different partners in macrophage and adipocytes.

Publication Date: 2010 Feb 22 PMID: 20176804
Authors: Scotti, E. - Tontonoz, P.
Journal: Mol Cell Biol

The peroxisome proliferator-activated receptors (PPARalpha, -gamma and -beta/delta) are ligand-activated nuclear receptors that influence metabolism, differentiation, and immune response (4, 16)....

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The RasGAP Proteins Ira2/Neurofibromin Are Negatively Regulated by Gpb1 in Yeast and ETEA in Humans.

Publication Date: 2010 Feb 16 PMID: 20160012
Authors: Phan, V. T. - Ding, V. W. - Li, F. - Chalkley, R. J. - Burlingame, A. - McCormick, F.
Journal: Mol Cell Biol

The Neurofibromatosis type 1 (NF1) gene encodes the GTPase activating protein (GAP) neurofibromin which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin dependent proteolysis. We extended our findings to show that in mammalian cells the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 in yeast and neurofibromin in humans.

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Functional Interplay between Acetylation and Methylation of the RelA Subunit of NF-{kappa}B.

Publication Date: 2010 Feb 16 PMID: 20160011
Authors: Yang, X. D. - Tajkhorshid, E. - Chen, L. F.
Journal: Mol Cell Biol

Posttranslational modifications of the RelA subunit of NF-kappaB, including acetylation and methylation, play a key role in controlling the strength and duration of its nuclear activity. Whether these modifications are functionally linked is largely unknown. Here, we show that acetylation of lysine 310 of RelA impairs the Set9-mediated methylation of lysines 314/315, which is important for the ubiquitination and degradation of chromatin-associated RelA. Abolishing the acetylation of lysine 310 either by deacetylase SIRT1 or by mutating lysine 310 to arginine enhances methylation. Conversely, enhancing the acetylation of lysine 310 by depleting SIRT1 or by substituting lysine 310 with acetyl-mimetic glutamine inhibits the methylation, therefore decreasing ubiquitination, prolonging the stability of chromatin-associated RelA, and enhancing the transcriptional activity of NF-kappaB. Acetylation of lysine 310 of RelA interferes with its interaction with Set9. Based on structural modeling of the SET domain of Set9 with RelA, we propose that the positive charge of lysine 310 is critical for the binding of RelA to a negatively charged "exosite" within the SET domain of Set9. Together, these findings demonstrate for the first time an interplay between RelA acetylation and methylation and also provide a novel mechanism for the regulation of lysine methylation by acetylation.

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CENP-A reduction induces a p53-dependent cellular senescence response to protect cells from executing defective mitoses.

Publication Date: 2010 Feb 16 PMID: 20160010
Authors: Maehara, K. - Takahashi, K. - Saitoh, S.
Journal: Mol Cell Biol

Cellular senescence is an irreversible growth arrest and is presumed to be a natural barrier to tumor development. Like telomere shortening, certain defects in chromosome integrity can trigger senescence, however, the roles of centromere proteins in regulating commitment to the senescent state remains to be established. We examined chromatin structure in senescent human primary fibroblasts, and found that CENP-A protein levels are diminished in senescent cells. Senescence-associated reduction of CENP-A is caused by transcriptional and posttranslational control. Surprisingly, forced reduction of CENP-A by short-hairpin RNA was found to cause premature senescence in human primary fibroblasts. This premature senescence is dependent on a tumor suppressor, p53, but not on p16(INK4a)-Rb; the depletion of CENP-A in p53-deficient cells results in aberrant mitosis with chromosome missegregation. We propose that p53-dependent senescence that arises from CENP-A reduction acts as a "self-defense mechanism" to prevent centromere-defective cells from undergoing mitotic proliferation that potentially leads to massive generation of aneuploid cells.

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Cadherins and Pak1 control contact inhibition of proliferation by Pak1-{beta}PIX-complex dependent regulation of cell-matrix signaling.

Publication Date: 2010 Feb 12 PMID: 20154149
Authors: Liu, F. - Jia, L. - Thompson-Baine, A. M. - Puglise, J. M. - Ter Beest, M. B. - Zegers, M. M.
Journal: Mol Cell Biol

It is crucial for organ homeostasis that epithelia have effective mechanism to restrict motility and cell proliferation in order to maintain tissue architecture. On the other hand, epithelial cells need to be able to rapidly and transiently acquire a more mesenchymal phenotype with high levels of cell motility and proliferation in order to repair epithelia upon injury. Crosstalk between cell-cell and cell-matrix signaling is crucial for regulating these transitions. The Pak1-betaPIX-GIT complex, is an effector complex downstream of the small GTPase Rac1. We previously showed that translocation of this complex from cell-matrix to cell-cell adhesion sites was required for the establishment of contact inhibition of proliferation. In this study we provide evidence that this translocation depends on cadherin function. Cadherins do not recruit the complex by direct interaction. Rather, we find that inhibition of the normal function of cadherin or Pak1 leads to defects in focal adhesion turnover and increased signaling by phosphatidyl inositol-3-kinase. We propose that cadherins are involved in regulation of contact inhibition by controlling the function of the Pak1-betaPIX-GIT complex at focal contacts.

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Mechanisms of recombination between diverged sequences in wild-type and BLM-deficient mouse and human cells.

Publication Date: 2010 Feb 12 PMID: 20154148
Authors: Larocque, J. R. - Jasin, M.
Journal: Mol Cell Biol

Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence templates repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged ("homeologous") sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells, and are generally
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The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membrane.

Publication Date: 2010 Feb 12 PMID: 20154147
Authors: Gilquin, B. - Taillebourg, E. - Cherradi, N. - Hubstenberger, A. - Gay, O. - Merle, N. - Assard, N. - Fauvarque, M. O. - Tomohiro, S. - Kuge, O. - Baudier, J.
Journal: Mol Cell Biol

Dynamic interactions between components of the outer (OM) and inner (IM) membrane control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA(+) ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central trans-membrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA+ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using, dominant negative mutants, including a defective ATP-binding mutant and a truncated 50 amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.

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OGFOD1: A Novel Modulator of eIF2{alpha} Phosphorylation and the Cellular Response to Stress.

Publication Date: 2010 Feb 12 PMID: 20154146
Authors: Wehner, K. A. - Schutz, S. - Sarnow, P.
Journal: Mol Cell Biol

Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of translation initiation factor eIF2alpha. We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Co-immunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1 and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knock down of OGFOD1 resulted in lower amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted both with eIF2alpha and the heme-regulated inhibitor eIF2alpha kinase HRI, which was identified as a novel stress granule resident. These findings argue that OGOFD1 plays important pro-apoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

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The chaperone-like protein HYPK acts together with NatA in cotranslational N-terminal acetylation and prevention of Huntingtin aggregation.

Publication Date: 2010 Feb 12 PMID: 20154145
Authors: Arnesen, T. - Starheim, K. K. - Van Damme, P. - Evjenth, R. - Dinh, H. - Betts, M. - Ryningen, A. - Vandekerckhove, J. - Gevaert, K. - Anderson, D.
Journal: Mol Cell Biol

The human NatA protein N(alpha)-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser-, Ala-, Thr-, Gly-, and Val- N-termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry we identified endogenous HYPK, a Huntingtin (Htt) interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p and hNaa15p were associated with polysome fractions indicating a function of HYPK when associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G0/G1 phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-EGFP fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and N-terminal acetylation.

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Uncoupling of Expression of an Intronic microRNA and its Myosin Host Gene by Exon Skipping.

Publication Date: 2010 Feb 12 PMID: 20154144
Authors: Bell, M. L. - Buvoli, M. - Leinwand, L. A.
Journal: Mol Cell Biol

The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that down-regulates MYH7b protein production without affecting microRNA expression. Among others, endogenous miR-499 targets the 3' UTR of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted transcription and alternative splicing uncouple the level of expression of MYH7b and miR-499 when their co-expression is not required.

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Kinetics of Hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation.

Publication Date: 2010 Feb 12 PMID: 20154143
Authors: Wen, X. - Lai, C. K. - Evangelista, M. - Hongo, J. A. - de Sauvage, F. J. - Scales, S. J.
Journal: Mol Cell Biol

Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. Generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitor their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilia tips is full-length and likely not PKA-phosphorylated. We also confirm that phosphorylation and betaTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than Gli3 repressor, the latter not requiring SPOP/Cul3 or betaTrCP/Cul1. This may contribute to the increased Gli3 activator:repressor ratios found in IFT mutants.

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Notch Exhibits Ligand bias and Manoeuvres Stage Specific Steering of Neural Differentiation in ESCs.

Publication Date: 2010 Feb 12 PMID: 20154142
Authors: Ramasamy, S. K. - Lenka, N.
Journal: Mol Cell Biol

Notch dictates multiple developmental events including stem cells maintenance and differentiation through intercellular communication. However, its temporal influence during early development and of particular interest, its regulation of binary fate decision at different stages during neurogenesis is least explored. Here using embryonic stem cells (ESCs) model, we have deciphered Notch ligand preference during ESCs commitment to different germ layers and determined the stage specific temporal effect of Notch during neural differentiation. ESCs during maintenance remain impervious to Notch inhibition. However, Notch activation promotes differentiation even in presence of LIF displaying ligand preference associated lineage discrimination; where Jagged-1 favours neural commitment and Delta-like-4, the mesoderm. This differential ligands action involves a combination of Notch receptors influencing specific downstream target genes expression. Though Notch activation during early neural differentiation specifically promotes neural stem cells or early neural progenitors and delays their maturation; its inhibition promotes late neural progenitors and expedites neurogenesis preferring neurons to glia. However, gliogenesis is promoted upon Notch activation only when executed in combination with ciliary neurotrphic factor. Thus, our investigation underscores a multifaceted role of Notch demonstrating the interdependency of ligand usage and lineage specification and Notch acting as a master switch displaying stage specific influence on neurogenesis.

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Alternative chromatin structures of the 35S ribosomal RNA genes in S. cerevisiae provide a molecular basis for the selective recruitment of RNA polymerases I and II.

Publication Date: 2010 Feb 12 PMID: 20154141
Authors: Goetze, H. - Wittner, M. - Hamperl, S. - Hondele, M. - Merz, K. - Stoeckl, U. - Griesenbeck, J.
Journal: Mol Cell Biol

In all eukaryotes a specialized enzyme, RNA polymerase I (Pol I), is dedicated to transcribe the 35S rRNA gene from a multicopy gene cluster, the ribosomal DNA (rDNA). In certain yeast mutants 35S rRNA genes can be transcribed by RNA polymerase II (Pol II). In these mutants rDNA silencing of Pol II transcription is impaired. It has been speculated that upstream activating factor (UAF), which binds to a specific DNA element within the Pol I promoter, plays a crucial role in forming chromatin structures responsible for polymerase specificity and silencing at the rDNA locus. We therefore performed an in depth analysis of chromatin structure and composition in different mutant backgrounds. We demonstrate that chromatin architecture of the entire Pol I transcribed region is substantially altered upon UAF deletion allowing RNA polymerases II and III to access DNA elements flanking a promoter-proximal Reb1 binding site. Furthermore, lack of UAF leads to the loss of Sir2 from rDNA correlating with impaired Pol II silencing. This analysis of rDNA chromatin provides a molecular basis explaining many phenotypes observed in previous genetic analyses.

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The Nematode eIF4E/G Complex works with a Trans-spliced Leader Stem Loop to Enable Efficient Translation of Trimethylguanosine-capped RNAs.

Publication Date: 2010 Feb 12 PMID: 20154140
Authors: Wallace, A. - Filbin, M. E. - Veo, B. - McFarland, C. - Stepinski, J. - Jankowska-Anyszka, M. - Darzynkiewicz, E. - Davis, R. E.
Journal: Mol Cell Biol

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader RNA trans-splicing adds a trimethylguanosine (TMG) cap and sequence, the spliced leader (SL), to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the spliced leader sequence. Here we define a core set of nucleotides and stem loop within the 22 nt nematode SL that stimulates translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode spliced leaders and correspond to regions of SL1 required for early C. elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem loop structures are predicted in phylogenetically diverse SLs. We show that the nematode eIF4E/G complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase eIF4E/G's affinity for capped RNA. These studies suggest the mRNA 5' UTR can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes, and raises the question of whether eIF4E/G-RNA interactions may play a role in the translation of other eukaryotic mRNAs.

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A novel conserved phosphotyrosine motif in the Drosophila FGF-signaling adaptor Dof with a redundant role in signal transmission.

Publication Date: 2010 Feb 12 PMID: 20154139
Authors: Csiszar, A. - Vogelsang, E. - Beug, H. - Leptin, M.
Journal: Mol Cell Biol

Fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila, the FGFR specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to MAPK activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and Src kinases contribute to FGFR dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events.

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REGULATION OF THE COPPER CHAPERONE CCS BY XIAP-MEDIATED UBIQUITINATION.

Publication Date: 2010 Feb 12 PMID: 20154138
Authors: Brady, G. F. - Galban, S. - Liu, X. - Basrur, V. - Gitlin, J. D. - Elenitoba-Johnson, K. S. - Wilson, T. E. - Duckett, C. S.
Journal: Mol Cell Biol

In order to balance the cellular requirements for copper with its toxic properties, an elegant set of mechanisms has evolved to regulate and buffer intracellular copper. The X-linked inhibitor of apoptosis (XIAP) protein was recently identified as a copper binding protein and regulator of copper homeostasis, although the mechanism by which XIAP binds copper in the cytosol is unclear. Here we describe the identification of the copper chaperone for superoxide dismutase (CCS) as a mediator of copper delivery to XIAP in cells. We also find that CCS is a target of the E3 ubiquitin ligase activity of XIAP, although interestingly ubiquitination of CCS by XIAP was found to lead to enhancement of its chaperone activity toward its physiologic target superoxide dismutase-1 (SOD1) rather than proteasomal degradation. Collectively, our results reveal a novel link between apoptosis, copper metabolism, and redox regulation through the XIAP-CCS complex.

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p32 (C1QBP) and Cancer Cell Metabolism: Is the Warburg Effect a Lot of Hot Air?

Publication Date: 2010 Mar PMID: 20100868
Authors: Dang, C. V.
Journal: Mol Cell Biol

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Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via Maintenance of Oxidative Phosphorylation.

Publication Date: 2010 Mar PMID: 20100866
Authors: Fogal, V. - Richardson, A. D. - Karmali, P. P. - Scheffler, I. E. - Smith, J. W. - Ruoslahti, E.
Journal: Mol Cell Biol

p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.

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Identification and Analysis of the Interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae.

Publication Date: 2010 Mar PMID: 20086104
Authors: Harigaya, Y. - Jones, B. N. - Muhlrad, D. - Gross, J. D. - Parker, R.
Journal: Mol Cell Biol

Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

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Tandem Phosphorylation of Serines 221 and 318 by Protein Kinase C{delta} Coordinates mRNA Binding and Nucleocytoplasmic Shuttling of HuR.

Publication Date: 2010 Mar PMID: 20086103
Authors: Doller, A. - Schlepckow, K. - Schwalbe, H. - Pfeilschifter, J. - Eberhardt, W.
Journal: Mol Cell Biol

Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cdelta (PKCdelta)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCdelta mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D(1), and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.

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RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei.

Publication Date: 2010 Mar PMID: 20086102
Authors: Aphasizheva, I. - Aphasizhev, R.
Journal: Mol Cell Biol

RNA uridylylation is critical for the expression of the mitochondrial genome in trypanosomes. Short U tails are added to guide RNAs and rRNAs, while long A/U heteropolymers mark 3' ends of most mRNAs. Three divergent mitochondrial terminal uridylyl transferases (TUTases) are known: RET1 catalyzes guide RNA (gRNA) uridylylation, RET2 executes U insertion mRNA editing, and MEAT1 associates with the editosome-like complex. However, the activities responsible for 3' uridylylation of rRNAs and mRNAs, and the roles of these modifications, are unclear. To dissect the functions of mitochondrial TUTases, we investigated the effects of their repression and overexpression on abundance, processing, 3'-end status, and in vivo stability of major mitochondrially encoded RNA classes. We show that RET1 adds U tails to gRNAs, rRNAs, and select mRNAs and contributes U's into A/U heteropolymers. Furthermore, RET1's TUTase activity is required for the nucleolytic processing of gRNA, rRNA, and mRNA precursors. The U tail's presence does not affect the stability of gRNAs and rRNAs, while transcript-specific uridylylation triggers 3' to 5' mRNA decay. We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors.

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Yeast exonuclease 5 is essential for mitochondrial genome maintenance.

Publication Date: 2010 Mar PMID: 20086101
Authors: Burgers, P. M. - Stith, C. M. - Yoder, B. L. - Sparks, J. L.
Journal: Mol Cell Biol

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.

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Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B.

Publication Date: 2010 Mar PMID: 20086100
Authors: Shahbazian, D. - Parsyan, A. - Petroulakis, E. - Topisirovic, I. - Martineau, Y. - Gibbs, B. F. - Svitkin, Y. - Sonenberg, N.
Journal: Mol Cell Biol

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5' untranslated region (5'UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5'UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.

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14-3-3{tau} Regulates Ubiquitin-Independent Proteasomal Degradation of p21, a Novel Mechanism of p21 Downregulation in Breast Cancer.

Publication Date: 2010 Mar PMID: 20086099
Authors: Wang, B. - Liu, K. - Lin, H. Y. - Bellam, N. - Ling, S. - Lin, W. C.
Journal: Mol Cell Biol

14-3-3 proteins regulate many cellular functions, including proliferation. However, the detailed mechanisms by which they control the cell cycle remain to be fully elucidated. We report that one of the 14-3-3 isoforms, 14-3-3tau, is required for the G(1)/S transition through its role in ubiquitin-independent proteasomal degradation of p21. 14-3-3tau binds to p21, MDM2, and the C8 subunit of the 20S proteasome in G(1) phase and facilitates proteasomal targeting of p21. This function of 14-3-3tau may be deregulated in cancer. The overexpression of 14-3-3tau is frequently found in primary human breast cancer and correlates with lower levels of p21 and shorter patient survival. Tenascin-C, an extracellular matrix protein involved in tumor initiation and progression and a known 14-3-3tau inducer, decreases p21 and abrogates adriamycin-induced G(1)/S arrest. It has been known that p21 is required for a proper tamoxifen response in breast cancer. We show that the overexpression of 14-3-3tau inhibits tamoxifen-induced p21 induction and growth arrest in MCF7 cells. Together, the findings of our studies strongly suggest a novel oncogenic role of 14-3-3tau by downregulating p21 in breast cancer. Therefore, 14-3-3tau may be a potential therapeutic target in breast cancer.

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HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK.

Publication Date: 2010 Mar PMID: 20086093
Authors: Lin, Q. - Wang, J. - Childress, C. - Sudol, M. - Carey, D. J. - Yang, W.
Journal: Mol Cell Biol

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.

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CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells.

Publication Date: 2010 Mar PMID: 20065043
Authors: Piao, X. - Zhang, X. - Wu, L. - Belasco, J. G.
Journal: Mol Cell Biol

MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of "off-target" mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

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Nuclear Receptor SHP, a Death Receptor That Targets Mitochondria, Induces Apoptosis and Inhibits Tumor Growth.

Publication Date: 2010 Mar PMID: 20065042
Authors: Zhang, Y. - Soto, J. - Park, K. - Viswanath, G. - Kuwada, S. - Abel, E. D. - Wang, L.
Journal: Mol Cell Biol

Small heterodimer partner (SHP) is an epigenetically regulated nuclear transcriptional repressor that suppresses the development of liver cancer by inhibiting cellular growth. Here we report a novel cytoplasmic function of SHP through its regulation of mitochondrial activity. SHP is a pivotal cell death receptor that targets mitochondria, where it binds with Bcl-2, disrupts Bcl-2/Bid interaction, and induces cytochrome c release. The apoptosis inducer AHPN {retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid} acts by regulating SHP gene expression and promotes the translocation of SHP from the nucleus to the mitochondria. Induction of apoptosis by SHP activation inhibits peritoneal pancreatic tumor growth. Our findings provide for the first time a mechanism by which SHP regulates cell survival, namely, by controlling mitochondrial function via modulating the activity of Bcl-2 through AHPN-mediated or AHPN-independent action. Thus, SHP regulates a mechanism by which apoptotic signals can mediate local control of mitochondrial function and apoptosis, which in turn may limit tumorigenesis.

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Rab35 Mediates Transport of Cdc42 and Rac1 to the Plasma Membrane during Phagocytosis.

Publication Date: 2010 Mar PMID: 20065041
Authors: Shim, J. - Lee, S. M. - Lee, M. S. - Yoon, J. - Kweon, H. S. - Kim, Y. J.
Journal: Mol Cell Biol

Phagocytosis of invading microbes requires dynamic rearrangement of the plasma membrane and its associated cytoskeletal actin network. The polarization of Cdc42 and Rac1 Rho GTPases to the site of plasma membrane protrusion is responsible for the remodeling of actin structures. However, the mechanism of Rho GTPase recruitment to these sites and the identities of accessory molecules involved in this process are not well understood. In this study, we uncovered several new components involved in innate immunity in Drosophila melanogaster. Our data demonstrate that Rab35 is a regulator of vesicle transport required specifically for phagocytosis. Moreover, recruitment of Cdc42 and Rac1 to the sites of filopodium and lamellipodium formation is Rab35 dependent and occurs by way of microtubule tracks. These results implicate Rab35 as the immune cell-specific regulator of vesicle transport within the actin-remodeling complex.

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Estrogen Receptors Recruit SMRT and N-CoR Corepressors through Newly Recognized Contacts between the Corepressor N Terminus and the Receptor DNA Binding Domain.

Publication Date: 2010 Mar PMID: 20065040
Authors: Varlakhanova, N. - Snyder, C. - Jose, S. - Hahm, J. B. - Privalsky, M. L.
Journal: Mol Cell Biol

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

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Mitochondrial DNA toxicity in forebrain neurons causes apoptosis, neurodegeneration, and impaired behavior.

Publication Date: 2010 Mar PMID: 20065039
Authors: Lauritzen, K. H. - Moldestad, O. - Eide, L. - Carlsen, H. - Nesse, G. - Storm, J. F. - Mansuy, I. M. - Bergersen, L. H. - Klungland, A.
Journal: Mol Cell Biol

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIalpha-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.

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Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates {gamma}-H2AX.

Publication Date: 2010 Mar PMID: 20065038
Authors: Douglas, P. - Zhong, J. - Ye, R. - Moorhead, G. B. - Xu, X. - Lees-Miller, S. P.
Journal: Mol Cell Biol

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.

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A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei.

Publication Date: 2010 Mar PMID: 20065037
Authors: Hines, J. C. - Ray, D. S.
Journal: Mol Cell Biol

Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3'-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

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Acetylation of H3 K56 Is Required for RNA Polymerase II Transcript Elongation through Heterochromatin in Yeast.

Publication Date: 2010 Mar PMID: 20065036
Authors: Varv, S. - Kristjuhan, K. - Peil, K. - Looke, M. - Mahlakoiv, T. - Paapsi, K. - Kristjuhan, A.
Journal: Mol Cell Biol

In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription.

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c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal.

Publication Date: 2010 Mar PMID: 20065035
Authors: Xu, P. - Davis, R. J.
Journal: Mol Cell Biol

The c-Jun NH(2)-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1(-/-) Jnk2(-/-) ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.

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The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation.

Publication Date: 2010 Mar PMID: 20065034
Authors: Chowdhury, A. - Liu, G. - Kemp, M. - Chen, X. - Katrangi, N. - Myers, S. - Ghosh, M. - Yao, J. - Gao, Y. - Bubulya, P. - Leffak, M.
Journal: Mol Cell Biol

Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

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Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation.

Publication Date: 2010 Mar PMID: 20065033
Authors: Thangavel, S. - Mendoza-Maldonado, R. - Tissino, E. - Sidorova, J. M. - Yin, J. - Wang, W. - Monnat, R. J. Jr - Falaschi, A. - Vindigni, A.
Journal: Mol Cell Biol

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G(1), after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.

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Distribution of Activator of G-Protein Signaling 3 within the Aggresomal Pathway: Role of Specific Residues in the Tetratricopeptide Repeat Domain and Differential Regulation by the AGS3 Binding Partners Gi{alpha} and Mammalian Inscuteable.

Publication Date: 2010 Mar PMID: 20065032
Authors: Vural, A. - Oner, S. - An, N. - Simon, V. - Ma, D. - Blumer, J. B. - Lanier, S. M.
Journal: Mol Cell Biol

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

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Upregulation of c-MYC in cis through a Large Chromatin Loop Linked to a Cancer Risk-Associated Single-Nucleotide Polymorphism in Colorectal Cancer Cells.

Publication Date: 2010 Mar PMID: 20065031
Authors: Wright, J. B. - Brown, S. J. - Cole, M. D.
Journal: Mol Cell Biol

Genome-wide association studies have mapped many single-nucleotide polymorphisms (SNPs) that are linked to cancer risk, but the mechanism by which most SNPs promote cancer remains undefined. The rs6983267 SNP at 8q24 has been associated with many cancers, yet the SNP falls 335 kb from the nearest gene, c-MYC. We show that the beta-catenin-TCF4 transcription factor complex binds preferentially to the cancer risk-associated rs6983267(G) allele in colon cancer cells. We also show that the rs6983267 SNP has enhancer-related histone marks and can form a 335-kb chromatin loop to interact with the c-MYC promoter. Finally, we show that the SNP has no effect on the efficiency of chromatin looping to the c-MYC promoter but that the cancer risk-associated SNP enhances the expression of the linked c-MYC allele. Thus, cancer risk is a direct consequence of elevated c-MYC expression from increased distal enhancer activity and not from reorganization/creation of the large chromatin loop. The findings of these studies support a mechanism for intergenic SNPs that can promote cancer through the regulation of distal genes by utilizing preexisting large chromatin loops.

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FKBP51 promotes assembly of the Hsp90 chaperone complex and regulates androgen receptor signaling in prostate cancer cells.

Publication Date: 2010 Mar PMID: 20048054
Authors: Ni, L. - Yang, C. S. - Gioeli, D. - Frierson, H. - Toft, D. O. - Paschal, B. M.
Journal: Mol Cell Biol

Prostate cancer progression to the androgen-independent (AI) state involves acquisition of pathways that allow tumor growth under low-androgen conditions. We hypothesized that expression of molecular chaperones that modulate androgen binding to AR might be altered in prostate cancer and contribute to progression to the AI state. Here, we report that the Hsp90 cochaperone FKBP51 is upregulated in LAPC-4 AI tumors grown in castrated mice and describe a molecular mechanism by which FKBP51 regulates AR activity. Using recombinant proteins, we show that FKBP51 stimulates recruitment of the cochaperone p23 to the ATP-bound form of Hsp90, forming an FKBP51-Hsp90-p23 superchaperone complex. In cells, FKBP51 expression promotes superchaperone complex association with AR and increases the number of AR molecules that undergo androgen binding. FKBP51 stimulates androgen-dependent transcription and cell growth, and FKBP51 is part of a positive feedback loop that is regulated by AR and androgen. Finally, depleting FKBP51 levels by short hairpin RNA reduces the transcript levels of genes regulated by AR and androgen. Because the superchaperone complex plays a critical role in determining the ligand-binding competence and transcription function of AR, it provides an attractive target for inhibiting AR activity in prostate cancer cells.

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Asf1 can promote trimethylation of H3 K36 by Set2.

Publication Date: 2010 Mar PMID: 20048053
Authors: Lin, L. J. - Minard, L. V. - Johnston, G. C. - Singer, R. A. - Schultz, M. C.
Journal: Mol Cell Biol

Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.

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Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan.

Publication Date: 2010 Mar PMID: 20048052
Authors: Zhang, J. - Graham, T. G. - Vivekanand, P. - Cote, L. - Cetera, M. - Rebay, I.
Journal: Mol Cell Biol

The ETS family transcriptional repressor Yan is an important downstream target and effector of the receptor tyrosine kinase (RTK) signaling pathway in Drosophila melanogaster. Structural and biochemical studies have shown that the N-terminal sterile alpha motif (SAM) of Yan is able to self associate to form a helical polymeric structure in vitro, although the extent and functional significance of self-association of full-length Yan remain unclear. In this study, we demonstrated that full-length Yan self associates via its SAM domain to form higher-order complexes in living cells. Introduction of SAM domain missense mutations that restrict Yan to a monomeric state reduces Yan's transcriptional repression activity and impairs its function during embryonic and retinal development. Coexpression of combinations of SAM domain mutations that permit the formation of Yan dimers, but not higher-order oligomers, increases activity relative to that of monomeric Yan, but not to the level obtained with wild-type Yan. Mechanistically, self-association directly promotes transcriptional repression of target genes independent of its role in limiting mitogen-activated protein kinase (MAPK)-mediated phosphorylation and nuclear export of Yan. Thus, we propose that the formation of higher-order Yan oligomers contributes to proper repression of target gene expression and RTK signaling output in developing tissues.

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The hsp90-FKBP52 complex links the mineralocorticoid receptor to motor proteins and persists bound to the receptor in early nuclear events.

Publication Date: 2010 Mar PMID: 20038533
Authors: Galigniana, M. D. - Erlejman, A. G. - Monte, M. - Gomez-Sanchez, C. - Piwien-Pilipuk, G.
Journal: Mol Cell Biol

In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.

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H19 imprinting control region methylation requires an imprinted environment only in the male germ line.

Publication Date: 2010 Mar PMID: 20038532
Authors: Gebert, C. - Kunkel, D. - Grinberg, A. - Pfeifer, K.
Journal: Mol Cell Biol

The 2.4-kb H19 imprinting control region (H19ICR) is required to establish parent-of-origin-specific epigenetic marks and expression patterns at the Igf2/H19 locus. H19ICR activity is regulated by DNA methylation. The ICR is methylated in sperm but not in oocytes, and this paternal chromosome-specific methylation is maintained throughout development. We recently showed that the H19ICR can work as an ICR even when inserted into the normally nonimprinted alpha fetoprotein locus. Paternal but not maternal copies of the ICR become methylated in somatic tissue. However, the ectopic ICR remains unmethylated in sperm. To extend these findings and investigate the mechanisms that lead to methylation of the H19ICR in the male germ line, we characterized novel mouse knock-in lines. Our data confirm that the 2.4-kb element is an autonomously acting ICR whose function is not dependent on germ line methylation. Ectopic ICRs become methylated in the male germ line, but the timing of methylation is influenced by the insertion site and by additional genetic information. Our results support the idea that DNA methylation is not the primary genomic imprint and that the H19ICR insertion is sufficient to transmit parent-of-origin-dependent DNA methylation patterns independent of its methylation status in sperm.

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RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.

Publication Date: 2010 Mar PMID: 20038531
Authors: Tian, X. - Jin, R. U. - Bredemeyer, A. J. - Oates, E. J. - Blazewska, K. M. - McKenna, C. E. - Mills, J. C.
Journal: Mol Cell Biol

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1(-)(/)(-) ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

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Evolutionarily conserved function of RRP36 in early cleavages of the pre-rRNA and production of the 40S ribosomal subunit.

Publication Date: 2010 Mar PMID: 20038530
Authors: Gerus, M. - Bonnart, C. - Caizergues-Ferrer, M. - Henry, Y. - Henras, A. K.
Journal: Mol Cell Biol

Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

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Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation.

Publication Date: 2010 Mar PMID: 20038529
Authors: Farrar, D. - Rai, S. - Chernukhin, I. - Jagodic, M. - Ito, Y. - Yammine, S. - Ohlsson, R. - Murrell, A. - Klenova, E.
Journal: Mol Cell Biol

Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.

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The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.

Publication Date: 2010 Mar PMID: 20038528
Authors: Hommel, A. - Hesse, D. - Volker, W. - Jaschke, A. - Moser, M. - Engel, T. - Bluher, M. - Zahn, C. - Chadt, A. - Ruschke, K. - Vogel, H. - Kluge, R. - Robenek, H. - Joost, H. G. - Schurmann, A.
Journal: Mol Cell Biol

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

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Splicing factor Spf30 assists exosome-mediated gene silencing in fission yeast.

Publication Date: 2010 Mar PMID: 20028739
Authors: Bernard, P. - Drogat, J. - Dheur, S. - Genier, S. - Javerzat, J. P.
Journal: Mol Cell Biol

Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways.

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Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links.

Publication Date: 2010 Mar PMID: 20028736
Authors: Hicks, J. K. - Chute, C. L. - Paulsen, M. T. - Ragland, R. L. - Howlett, N. G. - Gueranger, Q. - Glover, T. W. - Canman, C. E.
Journal: Mol Cell Biol

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Poleta), REV1, and zeta (Polzeta) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polzeta are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polzeta facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Poleta, whereas RAD18 plus Poleta, REV1, and Polzeta are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.

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The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation.

Publication Date: 2010 Mar PMID: 20028735
Authors: Zupkovitz, G. - Grausenburger, R. - Brunmeir, R. - Senese, S. - Tischler, J. - Jurkin, J. - Rembold, M. - Meunier, D. - Egger, G. - Lagger, S. - Chiocca, S. - Propst, F. - Weitzer, G. - Seiser, C.
Journal: Mol Cell Biol

Histone deacetylases (HDACs) are chromatin-modifying enzymes that are involved in the regulation of proliferation, differentiation and development. HDAC inhibitors induce cell cycle arrest, differentiation, or apoptosis in tumor cells and are therefore promising antitumor agents. Numerous genes were found to be deregulated upon HDAC inhibitor treatment; however, the relevant target enzymes are still unidentified. HDAC1 is required for mouse development and unrestricted proliferation of embryonic stem cells. We show here that HDAC1 reversibly regulates cellular proliferation and represses the cyclin-dependent kinase inhibitor p21 in embryonic stem cells. Disruption of the p21 gene rescues the proliferation phenotype of HDAC1(-/-) embryonic stem cells but not the embryonic lethality of HDAC1(-/-) mice. In the absence of HDAC1, mouse embryonic fibroblasts scarcely undergo spontaneous immortalization and display increased p21 expression. Chromatin immunoprecipitation assays demonstrate a direct regulation of the p21 gene by HDAC1 in mouse embryonic fibroblasts. Transformation with simian virus 40 large T antigen or ablation of p21 restores normal immortalization of primary HDAC1(-/-) fibroblasts. Our data demonstrate that repression of the p21 gene is crucial for HDAC1-mediated control of proliferation and immortalization. HDAC1 might therefore be one of the relevant targets for HDAC inhibitors as anticancer drugs.

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A new role for sterol regulatory element binding protein 1 transcription factors in the regulation of muscle mass and muscle cell differentiation.

Publication Date: 2010 Mar PMID: 20028734
Authors: Lecomte, V. - Meugnier, E. - Euthine, V. - Durand, C. - Freyssenet, D. - Nemoz, G. - Rome, S. - Vidal, H. - Lefai, E.
Journal: Mol Cell Biol

The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

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Rap1 in Candida albicans: an unusual structural organization and a critical function in suppressing telomere recombination.

Publication Date: 2010 Mar PMID: 20008550
Authors: Yu, E. Y. - Yen, W. F. - Steinberg-Neifach, O. - Lue, N. F.
Journal: Mol Cell Biol

Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.

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