MCB

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

Publication Date: 2012 Feb PMID: 22290684
Authors:
Journal: Mol Cell Biol

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A novel role for copper in Ras/MAPK signaling.

Publication Date: 2012 Jan 30 PMID: 22290441
Authors: Turski, M. L. - Brady, D. C. - Kim, H. J. - Kim, B. E. - Nose, Y. - Counter, C. M. - Winge, D. R. - Thiele, D. J.
Journal: Mol Cell Biol

Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the MAP kinase pathway. Ablation of the Ctr1 high affinity Cu transporter in flies and mouse cells, mutation of Ctr1, or Cu chelators all reduce the ability of the MAP kinase kinase, Mek1, to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-specific knock out of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. In vitro investigations reveal recombinant Mek1 binds two Cu atoms with high affinity and Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Co-immunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.

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MdmX is required for p53 interaction with and full induction of the Mdm2 promoter after cellular stress.

Publication Date: 2012 Jan 30 PMID: 22290440
Authors: Biderman, L. - Poyurovsky, M. V. - Assia, Y. - Manley, J. L. - Prives, C.
Journal: Mol Cell Biol

The activity of the tumor suppressor p53 is tightly controlled by its main negative regulator Mdm2, which inhibits p53's transcriptional activity and targets it for degradation via the proteasome pathway. The closely related Mdm2 homologue, MdmX, is also considered to be a general inhibitor of transactivation by p53, through binding to the p53 activation domain. We show here that, unexpectedly, upon DNA damage and ribosomal stress, MdmX plays a positive role in p53-mediated activation of the Mdm2 gene, but not of numerous other p53 target genes including p21. Down-regulation of MdmX results in lower levels of mature and nascent Mdm2 transcripts following cellular stress. This correlates with a longer p53 half-life following DNA damage. In vitro, Mdm2 inhibits the binding of p53 to DNA to a much greater extent than does MdmX, although MdmX does not stimulate p53 interaction with Mdm2 promoter DNA. Strikingly, however, MdmX is required for optimal p53 binding to the Mdm2 promoter in vivo. Thus, we have described a new mechanism by which MdmX can suppress p53, which is through transcriptional activation of p53's principal negative regulator, Mdm2.

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Twist-1 induces Ezh2 recruitment regulating histone methylation along the Ink4A/Arf locus in mesenchymal stem cells.

Publication Date: 2012 Jan 30 PMID: 22290439
Authors: Cakouros, D. - Isenmann, S. - Cooper, L. - Zannettino, A. - Anderson, P. - Glackin, C. - Gronthos, S.
Journal: Mol Cell Biol

The main impairment to tissue maintenance during ageing is the reduced capacity for stem cell self-renewal over-time due to senescence, the irreversible block in proliferation. We have previously described that the basic Helix-loop-Helix (betaHLH) transcription factors, Twist-1 can greatly enhance the life span of bone marrow derived mesenchymal stem/stromal cells (MSC). In the present study, we show that Twist-1 potently suppresses senescence and the the Ink4A/Arf locus with a dramatic decreased expression of p16 and to some extent, p14. Furthermore, the polycomb group protein and histone methyl transferase, Ezh2 which suppresses the Ink4A/Arf locus was found to be induced by Twist-1 resulting in an increase in H3K27me3 along the Ink4A/Arf locus repressing transcription of both p16/p14 and senescence of human MSCs. Furthermore, Twist-1 inhibits the expression of the betaHLH transcription factor, E47 which is normally expressed in senescent MSCs and induces transcription of the p16 promoter. Reduced Twist-1 wild type expression and function in bone cells derived from Saethre Chotzen patients also revealed an increase in senescence. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferases as well as the expression of other betaHLH factors.

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The Spt5 C-terminal region recruits yeast 3' -RNA cleavage factor I.

Publication Date: 2012 Jan 30 PMID: 22290438
Authors: Mayer, A. - Schreieck, A. - Lidschreiber, M. - Leike, K. - Martin, D. E. - Cramer, P.
Journal: Mol Cell Biol

During transcription elongation, RNA polymerase (Pol) II binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for co-transcriptional recruitment of the Paf1 complex (44, 88). Here we report a new role of the Spt5 CTR in the recruitment of 3'-RNA processing factors. Chromatin immunoprecipitation (ChIP) reveals that the Spt5 CTR is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3'-end of yeast genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 nucleotides downstream of the poly-adenylation (pA) site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo.

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YY1 Controls Immunoglobulin Class Switch Recombination and Nuclear AID Levels.

Publication Date: 2012 Jan 30 PMID: 22290437
Authors: Zaprazna, K. - Atchison, M. L.
Journal: Mol Cell Biol

Activation induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue-and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here we show that YY1 conditional knock-out in activated splenic B cells interferes with CSR. Knock-out of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germline transcripts. However, we show that YY1 physically interacts with AID and controls accumulation of nuclear AID at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels.

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Retina Restored and Brain Abnormalities Ameliorated by Single-Copy Knock in of Human NR2E1 in Null Mice.

Publication Date: 2012 Jan 30 PMID: 22290436
Authors: Schmouth, J. F. - Banks, K. G. - Mathelier, A. - Gregory-Evans, C. Y. - Castellarin, M. - Holt, R. - Gregory-Evans, K. - Wasserman, W. W. - Simpson, E. M.
Journal: Mol Cell Biol

Nr2e1 encodes a stem-cell-fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behaviour abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented for the first time, the expression pattern of the human BAC using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harbouring one functional copy of human NR2E1. These results correlated with absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatio-temporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate-genomic regions controlling expression in proliferating cells during neurogenesis were identified.

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The chromatin-remodeling enzymes BRG1 and CHD4 antagonistically regulate vascular Wnt signaling.

Publication Date: 2012 Jan 30 PMID: 22290435
Authors: Curtis, C. D. - Griffin, C. T.
Journal: Mol Cell Biol

Canonical Wnt signaling plays an important role in embryonic and postnatal blood vessel development. We previously reported that the chromatin-remodeling enzyme BRG1 promotes vascular Wnt signaling. Vascular deletion of Brg1 results in aberrant yolk sac blood vessel morphology, which is rescued by pharmacological stimulation of Wnt signaling with lithium chloride (LiCl). We have now generated embryos lacking the chromatin-remodeling enzyme Chd4 in vascular endothelial cells. Unlike Brg1 mutants, Chd4 mutant embryos had normal yolk sac vascular morphology. However, concomitant deletion of Chd4 and Brg1 rescued vascular abnormalities seen in Brg1 mutant yolk sacs to the same extent as LiCl treatment. We hypothesized that Wnt signaling was up-regulated in Chd4 mutant yolk sac vasculature. Indeed we found Chd4 deletion resulted in up-regulation of the Wnt-responsive transcription factor Tcf7 and an increase in Wnt target gene expression in endothelial cells. Furthermore, we identified one Wnt target gene, Pitx2, which was downregulated in Brg1 mutant endothelial cells but was rescued following LiCl treatment and in Brg1/Chd4 double-mutant vasculature, suggesting that PITX2 helps mediate the restoration of yolk sac vascular remodeling under both conditions. We conclude that BRG1 and CHD4 antagonistically modulate Wnt signaling in developing yolk sac vessels to mediate normal vascular remodeling.

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Phosphorylation of BRN2 modulates its interaction with the Pax3 promoter to control melanocyte migration and proliferation.

Publication Date: 2012 Jan 30 PMID: 22290434
Authors: Berlin, I. - Denat, L. - Steunou, A. L. - Puig, I. - Champeval, D. - Colombo, S. - Roberts, K. - Bonvin, E. - Bourgeois, Y. - Davidson, I. - Delmas, V. - Nieto, L. - Goding, C. R. - Larue, L.
Journal: Mol Cell Biol

MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues are replaced by alanines (BRN2TS-BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat colour phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by non-phosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.

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Hepatic deletion of SIRT1 decreases HNF1alpha/FXR signaling and induces formation of cholesterol gallstones in mice.

Publication Date: 2012 Jan 30 PMID: 22290433
Authors: Purushotham, A. - Xu, Q. - Lu, J. - Foley, J. F. - Yan, X. - Kim, D. H. - Kemper, J. K. - Li, X.
Journal: Mol Cell Biol

SIRT1, a highly conserved NAD(+)-dependent protein deacetylase, is a key metabolic sensor that directly links nutrient signals to animal metabolic homeostasis. Although SIRT1 has been implicated in a number of hepatic metabolic processes, the mechanisms by which hepatic SIRT1 modulates bile acid metabolism are still not well understood. Here we report that deletion of hepatic SIRT1 reduces the expression of farnesoid X receptor (FXR), a nuclear receptor that regulates bile acid homeostasis. We provide evidence that SIRT1 regulates the expression of FXR through hepatocyte nuclear factor 1alpha (HNF1alpha). SIRT1 deficiency in hepatocytes leads to decreased binding of HNF1alpha to the FXR promoter. Furthermore, we show that hepatocyte-specific deletion of SIRT1 leads to derangements in bile acid metabolism, predisposing the mice to development of cholesterol gallstones on a lithogenic diet. Taken together, our findings indicate that SIRT1 plays a vital role in the regulation of hepatic bile acid homeostasis through the HNF1alpha/FXR signaling pathway.

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Viral factors reveal a role for Ref/Aly in nuclear RNA stability.

Publication Date: 2012 Jan 30 PMID: 22290432
Authors: Stubbs, S. H. - Hunter, O. V. - Hoover, A. - Conrad, N. K.
Journal: Mol Cell Biol

TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In yeast, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV nuclear noncoding PAN RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA, but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independent of their export, and support a broader conservation of RNA quality control mechanisms from yeast to human.

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The E3 ubiquitin ligase TRAF6 intercedes starvation-induced skeletal muscle atrophy through multiple mechanisms.

Publication Date: 2012 Jan 30 PMID: 22290431
Authors: Paul, P. K. - Bhatnagar, S. - Mishra, V. - Srivastava, S. - Darnay, B. G. - Choi, Y. - Kumar, A.
Journal: Mol Cell Biol

Starvation, like many other catabolic conditions, induces loss of skeletal muscle mass by promoting fiber atrophy. In addition to the canonical processes, starvation-induced response employs many distinct pathways that make it a unique atrophic program. However, in the multiplex of the underlying mechanisms, several components of starvation-induced atrophy are yet to be fully understood and their role and interplay remain to be elucidated. Here, we unveiled the role of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), a unique E3 ubiquitin ligase and adaptor protein, in starvation-induced muscle atrophy. Targeted ablation of TRAF6 suppresses the expression of key regulators of atrophy including MAFBx, MuRF1, p62, LC3B, Beclin1, Atg12, and Fn14. Ablation of TRAF6 also improved the phosphorylation of Akt and FoxO3a and inhibited the activation of AMPK in skeletal muscle in response to starvation. In addition, our study provides the first evidence of involvement of endoplasmic stress (ER) stress and unfolding protein response (UPR) pathways in starvation-induced muscle atrophy and its regulation through TRAF6. Finally, our results also identify lysine-63-linked autoubiquitination of TRAF6 as an essential process for its regulatory role in starvation-induced muscle atrophy.

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HOXA9 methylation by PRMT5 is essential for endothelial cell expression of leukocyte adhesion molecules.

Publication Date: 2012 Jan 23 PMID: 22269951
Authors: Bandyopadhyay, S. - Harris, D. P. - Adams, G. N. - Lause, G. E. - McHugh, A. - Tillmaand, E. G. - Money, A. - Willard, B. - Fox, P. L. - Dicorleto, P. E.
Journal: Mol Cell Biol

The induction of proinflammatory proteins in stimulated endothelial cells (EC) requires activation of multiple transcription programs. The homeobox transcription factor HOXA9 has an important regulatory role in cytokine induction of the EC-leukocyte adhesion molecules (ELAM) E-selectin and VCAM-1. However, the mechanism underlying stimulus-dependent activation of HOXA9 is completely unknown. Here, we elucidate the molecular mechanism of HOXA9 activation by tumor necrosis factor (TNF)-alpha and show an unexpected requirement for arginine methylation by protein arginine methyltrans-ferase 5 (PRMT5). PRMT5 was identified as a TNF-alpha-dependent binding partner of HOXA9 by mass spectrometry. siRNA-mediated depletion of PRMT5 abrogated stimulus-dependent HOXA9 methylation with concomitant loss in E-selectin or VCAM-1 induction. Chromatin immunoprecipitation analysis revealed that PRMT5 is recruited to the E-selectin promoter following transient HOXA9 binding to its cognate recognition sequence. PRMT5 induces symmetric dimethylation of Arg140 on HOXA9, an event essential for E-selectin induction. In summary, PRMT5 is a critical co-activator component in a newly-defined, HOXA9-containing transcription complex. Moreover, stimulus-dependent methylation of HOXA9 is essential for ELAM expression during the EC inflammatory response.

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RYBP represses endogenous retroviruses, preimplantation- and germline-specific genes in mouse embryonic stem cells.

Publication Date: 2012 Jan 23 PMID: 22269950
Authors: Hisada, K. - Sanchez, C. - Endo, T. - Endoh, M. - Roman-Trufero, M. - Sharif, J. - Koseki, H. - Vidal, M.
Journal: Mol Cell Biol

Polycomb repressive complexes (PRCs) are important chromatin regulators of ES cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B, and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed KO ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP represses efficiently endogenous retroviruses (MuERV class), preimplantation-(including zygotic genome activation stage) and germline-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germline genes and PcG targets before formation of pluripotent epiblast cells.

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Berardinelli-Seip Congenital Lipodystrophy-2 (BSCL2)/Seipin is a Cell Autonomous Regulator of Lipolysis Essential for Adipocyte Differentiation.

Publication Date: 2012 Jan 23 PMID: 22269949
Authors: Chen, W. - Chang, B. - Saha, P. - Hartig, S. M. - Li, L. - Reddy, V. T. - Yang, Y. - Yechoor, V. - Mancini, M. A. - Chan, L.
Journal: Mol Cell Biol

Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2(-/-) mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance and hepatic steatosis. In vitro differentiation of both Bscl2(-/-) murine embryonic fibroblasts (MEFs) and stromal vascular cells (SVCs) reveals normal early-phase adipocyte differentiation but a striking failure in terminal differentiation due to unbridled cyclic AMP (cAMP)-dependent protein kinase A (PKA)-activated lipolysis, which leads to loss of lipid droplets and silencing of the expression of adipose-specific transcription factors. Importantly, such defects in differentiation can be largely rescued by inhibitors of lipolysis but not PPARgamma agonist. The residual epididymal WAT (EWAT) in Bscl2(-/-) mice displays enhanced lipolysis. It also assumes a "brown-like" phenotype with marked upregulation of UCP1 and other brown adipose specific markers. Together with decreased Pref1 but increased C/EBPbeta, these changes highlight a possible increased cAMP signaling which impairs terminal adipocyte differentiation in the EWAT of Bscl2(-/-) mice. Our study underscores the fundamental role of regulated cAMP/PKA-mediated lipolysis in adipose differentiation, and identifies Bscl2 as a novel cell-autonomous determinant of activated lipolysis essential for terminal adipocyte differentiation.

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Identification of an AAA-ATPase VPS4B-Dependent Pathway that Modulates EGFR Abundance and Signaling during Hypoxia.

Publication Date: 2012 Jan 17 PMID: 22252323
Authors: Lin, H. H. - Li, X. - Chen, J. L. - Sun, X. - Cooper, F. N. - Chen, Y. R. - Zhang, W. - Chung, Y. - Li, A. - Cheng, C. T. - Yang, L. - Deng, X. - Liu, X. - Yen, Y. - Johnson, D. L. - Shih, H. M. - Yang, A. - Ann, D. K.
Journal: Mol Cell Biol

VPS4B, an AAA-ATPase (ATPase associated with various cellular activities) protein, participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (3D spheroids) hypoxia induces VPS4B downregulation by ubiquitin-proteasome system. Inhibition of VPS4B function by sh-VPS4B or expression of dominant-negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126 or genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied with increased EGFR accumulation and altered intracellular compartmentalization, are observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1-promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a non-genomic mechanism for EGFR overproduction in human breast cancer.

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Dcp2 decapping protein modulates mRNA stability of the critical intereferon response factor, IRF-7.

Publication Date: 2012 Jan 17 PMID: 22252322
Authors: Li, Y. - Dai, J. - Song, M. - Fitzgerald-Bocarsly, P. - Kiledjian, M.
Journal: Mol Cell Biol

The mammalian Dcp2 mRNA decapping protein primarily functions on a subset of mRNAs in a transcript-specific manner. Here we show that Dcp2 is an important modulator of genes involved in the type-I IFN response, which is the initial line of anti-viral innate immune response elicited by viral challenge. Mouse embryonic fibroblast (MEF) cells with reduced Dcp2 levels (Dcp2(beta/beta)) contained significantly elevated levels of mRNAs encoding proteins involved in the type-I IFN response. In particular, analysis of a key type-I IFN transcription factor, IRF-7 revealed an increase of both IRF-7 mRNA and protein in Dcp2(beta/beta) cells. Importantly, the increase in IRF-7 mRNA within the background of reduced Dcp2 levels was attributed to a stabilization of the IRF-7 mRNA suggesting Dcp2 normally modulates IRF-7 mRNA stability. Moreover, Dcp2 expression was also induced upon viral infection consistent with a role in attenuating the anti-viral response by promoting IRF-7 mRNA degradation. The induction of Dcp2 levels following viral challenge and the specificity of Dcp2 in targeting the decay of IRF-7 mRNA suggests Dcp2 may negatively contribute to the innate immune response in a negative feedback mechanism to restore normal homeostasis following viral infection.

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Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes.

Publication Date: 2012 Jan 17 PMID: 22252321
Authors: Ott, C. - Ross, K. - Straub, S. - Thiede, B. - Gotz, M. - Goosmann, C. - Krischke, M. - Mueller, M. J. - Krohne, G. - Rudel, T. - Kozjak-Pavlovic, V.
Journal: Mol Cell Biol

Mitochondria possess an outer (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential and proteins in the IMM influence cristae organization. Here we show an essential role of the OMM protein Sam50 in the maintenance of cristae structure. Sam50 is a part of the sorting and assembly machinery (SAM), necessary for the assembly of beta-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins Mitofilin and CHCHD3, which we term the Mitochondrial Intermembrane Space Bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observe deficiency in the assembly of respiratory chain complexes. Long term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondria-encoded subunits, pointing to a connection between cristae structural integrity, assembly of respiratory complexes and/or the maintenance of mtDNA.

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snRNA 3' End Formation Requires Heterodimeric Association of Integrator Subunits.

Publication Date: 2012 Jan 17 PMID: 22252320
Authors: Albrecht, T. R. - Wagner, E. J.
Journal: Mol Cell Biol

The Integrator Complex is a group of proteins responsible for the endonucleolytic cleavage of primary snRNA transcripts within the nucleus. Integrator subunits 9 and 11 (IntS9/11) are thought to contain the catalytic activity based upon their high sequence similarity with CPSF100 and CPSF73, which have been shown to be components of both the poly(A)+ and histone pre-mRNA cleavage complex. Here we demonstrate that the specific heterodimeric interaction between IntS9 and IntS11 is mediated by a discrete domain present at the extreme C-terminus of IntS9 and within the C-terminus of IntS11, adjacent to the predicted active site of this endonuclease. This domain is highly conserved within IntS11 but conspicuously absent in CPSF73. Using a cellular-based complementation assay that measures Integrator activity, we determined that the IntS9 interaction domain within IntS11 is required for its ability to restore snRNA 3' end processing after RNAi-mediated depletion of IntS11. Moreover, overexpression of these interaction domains alone elicits snRNA misprocessing through a dominant-negative titration of endogenous Integrator subunits. These data collectively explain the mechanism of how the IntS11/9 and, by analogy, the CPSF73/100 heterodimeric cleavage factors distinguish themselves from each other and demonstrate that the heterodimeric interaction is functionally required for snRNA 3' end formation.

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Paf1 restricts Gcn4 occupancy and antisense transcription at the ARG1 promoter.

Publication Date: 2012 Jan 17 PMID: 22252319
Authors: Crisucci, E. M. - Arndt, K. M.
Journal: Mol Cell Biol

The conserved Paf1 complex negatively regulates the expression of numerous genes, yet the mechanisms by which it represses gene expression are not well understood. In this study, we use ARG1 as a model gene to investigate the repressive functions of the Paf1 complex in Saccharomyces cerevisiae. Our results indicate that Paf1 mediates repression of ARG1 independently of the gene-specific repressor, ArgR/Mcm1. Rather, by promoting histone H2B lysine 123 ubiquitylation, Paf1 represses ARG1 by negatively affecting Gcn4 occupancy at the promoter. Consistent with this observation, Gcn5 and its acetylation sites on histone H3 are required for full ARG1 derepression in paf1Delta cells, and the repressive effect of Paf1 is largely maintained when the ARG1 promoter directs transcription of a heterologous coding region. Derepression of ARG1 in paf1Delta cells is accompanied by small changes in nucleosome occupancy, although these changes are subtle in comparison to those that accompany gene activation through amino acid starvation. Additionally, conditions that stimulate ARG1 transcription, including PAF1 deletion, lead to increased antisense transcription across the ARG1 promoter. This promoter-associated antisense transcription positively correlates with ARG1 sense transcription. Finally, our results indicate that Paf1 represses other genes through mechanisms similar to those used at ARG1.

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Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB's deadenylation-enhancing and anti-proliferative activities.

Publication Date: 2012 Jan 17 PMID: 22252318
Authors: Ezzeddine, N. - Chen, C. Y. - Shyu, A. B.
Journal: Mol Cell Biol

The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3' end of mRNAs by simultaneously binding CAF1 and PABP. Yet, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' anti-proliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' anti-proliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate anti-proliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.

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Contribution of a TBK1-IRF7 pathway to IFN-gamma-induced gene expression.

Publication Date: 2012 Jan 17 PMID: 22252317
Authors: Farlik, M. - Rapp, B. - Marie, I. - Levy, D. E. - Jamieson, A. M. - Decker, T.
Journal: Mol Cell Biol

STATs (signal transducers and activators of transcription) and IRFs (interferon regulatory factors) share common target genes. Here we show that IRF is regulated by transcription factors STAT1 and IRF9 in response to the type II IFN, IFNgamma. IRF7 cooperated with STAT1 and IRF1 to stimulate the expression of a subset of IFNgamma-induced STAT1 target genes. IRF7-mediated control of the Gbp2 gene required the presence and basal activity of the S/T kinase TBK1 (TANK-binding kinase 1), whereas the binding of IRF7 to the Gbp2 promoter did not. Analysis of RNA polymerase II (Pol II) recruitment to the Gbp2 promoter revealed a role for IRF7 at later stages of the IFNgamma response. In support of the role of IRF7 in establishing an effective anti-bacterial response, IFNgamma-pretreated Irf7 -/- macrophages showed an increased bacterial burden after infection with Listeria monocytogenes. Our data thus describe a biologically relevant basal activity of TBK1 and identify IRF7 as a novel player in the IFNgamma response.

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Dynamic loss of H2B ubiquitylation without corresponding changes in H3K4 tri-methylation during myogenic differentiation.

Publication Date: 2012 Jan 17 PMID: 22252316
Authors: Vethantham, V. - Yang, Y. - Bowman, C. - Asp, P. - Lee, J. H. - Skalnik, D. G. - Dynlacht, B. D.
Journal: Mol Cell Biol

Ubiquitylation of H2B on lysine 120 (H2Bub) is associated with active transcriptional elongation. H2Bub has been implicated in histone cross-talk and is generally regarded to be a prerequisite for H3K4 and H3K79 tri-methylation in both yeast and mammalian cells. We performed a genome-wide analysis of epigenetic marks during muscle differentiation, and, strikingly, we observed a near-complete loss of H2Bub in the differentiated state. We examined the basis for global loss of this mark and found that the H2B ubiquitin E3 ligase, RNF20, was depleted from chromatin in differentiated myotubes, indicating that recruitment of this protein to genes substantially decreases upon differentiation. Remarkably, during the course of myogenic differentiation, we observed retention and acquisition of H3K4 tri-methylation on a large number of genes in the absence of detectable H2Bub. The Set1 H3K4 trimethylase complex was efficiently recruited to a subset of genes in myotubes in the absence of detectable H2Bub, accounting in part for H3K4 tri-methylation in myotubes. Our studies suggest that H3K4me3 deposition in the absence of detectable H2Bub in myotubes is mediated via Set1 and, perhaps, MLL complexes, whose recruitment does not require H2Bub. Thus, muscle cells represent a novel setting in which to explore mechanisms that regulate histone cross-talk.

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T-Cell Protein Tyrosine Phosphatase (TC-PTP) Regulates Bone Resorption and Whole-Body Insulin Sensitivity Through Its Expression in Osteoblasts.

Publication Date: 2012 Jan 17 PMID: 22252315
Authors: Zee, T. - Settembre, C. - Levine, R. L. - Karsenty, G.
Journal: Mol Cell Biol

Insulin signaling in osteoblasts contributes to whole body glucose homeostasis in the mouse and in humans by increasing the activity of osteocalcin. The osteoblast insulin signaling cascade is negatively regulated by ESP, a tyrosine phosphatase dephosphorylating the insulin receptor. Esp is one of many tyrosine phosphatases expressed in osteoblasts, and this observation suggests that other protein tyrosine phosphatases may contribute to the attenuation of insulin receptor phosphorylation in this cell type. In this study, we sought to identify additional PTP(s) that like ESP, would function in the osteoblast to regulate insulin signaling and thus affect activity of the insulin-sensitizing hormone osteocalcin. For that purpose, we used as criteria, expression in osteoblasts, regulation by isoproterenol, and ability to trap the insulin receptor in a substrate-trapping assay. Here we show that the T-cell protein tyrosine phosphatase (TC-PTP) regulates insulin receptor phosphorylation in the osteoblast, thus compromising bone resorption and bioactivity of osteocalcin. Accordingly, osteoblast-specific deletion of TC-PTP promotes insulin sensitivity in an osteocalcin-dependent manner. This study increases the number of genes involved in the bone regulation of glucose homeostasis.

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A Quantitative High Throughput in vitro Splicing Assay Identifies Inhibitors of Spliceosome Catalysis.

Publication Date: 2012 Jan 17 PMID: 22252314
Authors: Berg, M. G. - Wan, L. - Younis, I. - Diem, M. D. - Soo, M. - Wang, C. - Dreyfuss, G.
Journal: Mol Cell Biol

Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components, Y14 or eIF4AIII. Deploying EJIPT we performed high throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anti-cancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron-lariat and ligation of exons. We show that BN82685 inhibits activated spliceosome's elaborate structural rearrangements that are required for second step catalysis, allowing definition of spliceosomes stalled in mid-catalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.

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Loss of cadherin-binding proteins, beta-catenin and plakoglobin, in the heart leads to gap junction remodelling and arrhythmogenesis.

Publication Date: 2012 Jan 17 PMID: 22252313
Authors: Swope, D. - Cheng, L. - Gao, E. - Li, J. - Radice, G. L.
Journal: Mol Cell Biol

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart-muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome including plakoglobin (JUP). We previously reported that cardiac-specific plakoglobin (PG) knockout (CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, beta-catenin (CTNNB1), showed increased association with the gap junction protein, connexin43 (Cx43) in PG CKO hearts. To determine whether beta-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and beta-catenin specifically in the heart (i.e. double knockout, DKO). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin-linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory ECG monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and beta-catenin, are indispensable for maintaining mechano-electrical coupling in the heart.

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

Publication Date: 2012 Feb PMID: 22247540
Authors:
Journal: Mol Cell Biol

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RNA profiling and ChIP-Sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors.

Publication Date: 2012 Jan 9 PMID: 22232429
Authors: Thompson, N. - Gesina, E. - Scheinert, P. - Bucher, P. - Grapin-Botton, A.
Journal: Mol Cell Biol

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a bHLH transcription factor which controls pancreas exocrine cell differentiation, maintenance and functionality, is also needed for early specification of pancreas progenitors. We used RNA profiling and ChIP-sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1 and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV and P2rx1 as new targets of PTF1a in pancreas progenitors.

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Prostate Apoptosis Response 4 (Par-4), a Novel Substrate of Caspase-3 during Apoptosis Activation.

Publication Date: 2012 Feb PMID: 22184067
Authors: Chaudhry, P. - Singh, M. - Parent, S. - Asselin, E.
Journal: Mol Cell Biol

Prostate apoptosis response 4 (Par-4) is a ubiquitously expressed proapoptotic tumor suppressor protein. Here, we show for the first time, that Par-4 is a novel substrate of caspase-3 during apoptosis. We found that Par-4 is cleaved during cisplatin-induced apoptosis in human normal and cancer cell lines. Par-4 cleavage generates a C-terminal fragment of approximately 25 kDa, and the cleavage of Par-4 is completely inhibited by a caspase-3 inhibitor, suggesting that caspase-3 is directly involved in the cleavage of Par-4. Caspase-3-deficient MCF-7 cells do not show Par-4 cleavage in response to cisplatin treatment, and restoration of caspase-3 in MCF-7 cells produces a decrease in Par-4 levels, with the appearance of a cleaved fragment. Additionally, knockdown of Par-4 reduces caspase-3 activation and apoptosis induction. Site-directed mutagenesis reveals that Par-4 cleavage by caspase-3 occurs at an unconventional site, EEPD(131) downward arrowG. Interestingly, overexpression of wild-type Par-4 but not the Par-4 D131A mutant sensitizes cells to cisplatin-induced apoptosis. Upon caspase-3 cleavage, the cleaved fragment of Par-4 accumulates in the nucleus and displays increased apoptotic activity. Overexpression of the cleaved fragment of Par-4 inhibits IkappaBalpha phosphorylation and blocks NF-kappaB nuclear translocation. We have identified a novel specific caspase-3 cleavage site in Par-4, and the cleaved fragment of Par-4 retains proapoptotic activity.

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Phosphorylation of the Transcription Factor YY1 by CK2alpha Prevents Cleavage by Caspase 7 during Apoptosis.

Publication Date: 2012 Feb PMID: 22184066
Authors: Riman, S. - Rizkallah, R. - Kassardjian, A. - Alexander, K. E. - Luscher, B. - Hurt, M. M.
Journal: Mol Cell Biol

In this report, we describe the phosphorylation of Yin Yang 1 (YY1) in vitro and in vivo by CK2alpha (casein kinase II), a multifunctional serine/threonine protein kinase. YY1 is a ubiquitously expressed multifunctional zinc finger transcription factor implicated in regulation of many cellular and viral genes. The products of these genes are associated with cell growth, the cell cycle, development, and differentiation. Numerous studies have linked YY1 to tumorigenesis and apoptosis. YY1 is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during apoptosis. Here, we identify serine 118 in the transactivation domain of YY1 as the site of CK2alpha phosphorylation, proximal to a caspase 7 cleavage site. CK2alpha inhibitors, as well as knockdown of CK2alpha by small interfering RNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increased CK2alpha activity by overexpression in vivo elevates S118 phosphorylation. A serine-to-alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death.

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Myc Regulates the Transcription of the PRC2 Gene To Control the Expression of Developmental Genes in Embryonic Stem Cells.

Publication Date: 2012 Feb PMID: 22184065
Authors: Neri, F. - Zippo, A. - Krepelova, A. - Cherubini, A. - Rocchigiani, M. - Oliviero, S.
Journal: Mol Cell Biol

Myc family members are critical to maintain embryonic stem cells (ESC) in the undifferentiated state. However, the mechanism by which they perform this task has not yet been elucidated. Here we show that Myc directly upregulates the transcription of all core components of the Polycomb repressive complex 2 (PRC2) as well as the ESC-specific PRC2-associated factors. By expressing Myc protein fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc, binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the acetylation of histones H3 and H4 and the recruitment of elongating RNA polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC results in reduced expression of PRC2 and H3K27me3 at Polycomb target developmental regulators and upregulation of genes involved in primitive endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor (LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with bivalent histone modifications and keep repressed the genes involved in ESC differentiation. Thus, Myc proteins control the expression of developmental regulators via the upregulation of the Polycomb PRC2 complex.

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Cyclin-Dependent Kinase 16/PCTAIRE Kinase 1 Is Activated by Cyclin Y and Is Essential for Spermatogenesis.

Publication Date: 2012 Feb PMID: 22184064
Authors: Mikolcevic, P. - Sigl, R. - Rauch, V. - Hess, M. W. - Pfaller, K. - Barisic, M. - Pelliniemi, L. J. - Boesl, M. - Geley, S.
Journal: Mol Cell Biol

Cyclin-dependent kinase 16 (CDK16, PCTK1) is a poorly characterized protein kinase, highly expressed in the testis and the brain. Here, we report that CDK16 is activated by membrane-associated cyclin Y (CCNY). Treatment of transfected human cells with the protein kinase A (PKA) activator forskolin blocked, while kinase inhibition promoted, CCNY-dependent targeting of CDK16-green fluorescent protein (GFP) to the cell membrane. CCNY binding to CDK16 required a region upstream of the kinase domain and was found to be inhibited by phosphorylation of serine 153, a potential PKA phosphorylation site. Thus, in contrast to other CDKs, CDK16 is regulated by phosphorylation-controlled cyclin binding. CDK16 isolated from murine testis was unphosphorylated, interacted with CCNY, and exhibited kinase activity. To investigate the function of CDK16 in vivo, we established a conditional knockout allele. Mice lacking CDK16 developed normally, but male mice were infertile. Spermatozoa isolated from their epididymis displayed thinning and elongation of the annulus region, adopted a bent shape, and showed impaired motility. Moreover, CDK16-deficient spermatozoa had malformed heads and excess residual cytoplasm, suggesting a role of CDK16 in spermiation. Thus, CDK16 is a membrane-targeted CDK essential for spermatogenesis.

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Vpr-Binding Protein Antagonizes p53-Mediated Transcription via Direct Interaction with H3 Tail.

Publication Date: 2012 Feb PMID: 22184063
Authors: Kim, K. - Heo, K. - Choi, J. - Jackson, S. - Kim, H. - Xiong, Y. - An, W.
Journal: Mol Cell Biol

HIV-1 Vpr-binding protein (VprBP) has been implicated in the regulation of both DNA replication and cell cycle progression, but its precise role remains unclear. Here we report that VprBP regulates the p53-induced transcription and apoptotic pathway. VprBP is recruited to p53-responsive promoters and suppresses p53 transactivation in the absence of stress stimuli. To maintain target promoters in an inactive state, VprBP stably binds to nucleosomes by recognizing unacetylated H3 tails. Promoter-localized deacetylation of H3 tails is a prerequisite for VprBP to tether and act as a bona fide inhibitor at p53 target genes. VprBP knockdown leads to activation of p53 target genes and causes an increase in DNA damage-induced apoptosis. Moreover, phosphorylation of VprBP at serine 895 impairs the ability of VprBP to bind H3 tails and to repress p53 transactivation. Our results thus reveal a new role for VprBP in regulation of the p53 signaling pathway, as well as molecular mechanisms of cancer development related to VprBP misregulation.

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Dynamic, Sex-Differential STAT5 and BCL6 Binding to Sex-Biased, Growth Hormone-Regulated Genes in Adult Mouse Liver.

Publication Date: 2012 Feb PMID: 22158971
Authors: Zhang, Y. - Laz, E. V. - Waxman, D. J.
Journal: Mol Cell Biol

Sex-dependent pituitary growth hormone (GH) secretory patterns determine the sex-biased expression of >1,000 genes in mouse and rat liver, affecting lipid and drug metabolism, inflammation, and disease. A fundamental biological question is how robust differential expression can be achieved for hundreds of sex-biased genes simply based on the GH input signal pattern: pulsatile GH stimulation in males versus near-continuous GH exposure in females. STAT5 is an essential transcriptional mediator of the sex-dependent effects of GH in the liver, but the mechanisms that underlie its sex-dependent actions are obscure. Here we elucidate the dynamic, sex-dependent binding of STAT5 and the GH/STAT5-regulated repressor BCL6 to mouse liver chromatin genome wide, revealing a counteractive interplay between these two regulators of sex differences in liver gene expression. Our findings establish a close correlation between sex-dependent STAT5 binding and sex-biased target gene expression. Moreover, sex-dependent STAT5 binding correlated positively with sex-biased DNase hypersensitivity and H3-K4me1 and H3-K4me3 (activating) marks, correlated negatively with sex-biased H3-K27me3 (repressive) marks, and was associated with sex-differentially enriched motifs for HNF6/CDP factors. Importantly, BCL6 binding was preferentially associated with repression of female-biased STAT5 targets in male liver. Furthermore, BCL6 and STAT5 common targets but not BCL6 unique targets showed strong enrichment for lipid and drug metabolism. These findings provide a comprehensive, genome-wide view of the mechanisms whereby these two GH-regulated transcription factors establish and maintain sex differences affecting liver physiology and disease. The approaches used here to characterize sex-dependent STAT5 and BCL6 binding can be applied to other condition-specific regulatory factors and binding sites and their interplay with cooperative chromatin binding factors.

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Glucocorticoid-Dependent Phosphorylation of the Transcriptional Coregulator GRIP1.

Publication Date: 2012 Feb PMID: 22158970
Authors: Dobrovolna, J. - Chinenov, Y. - Kennedy, M. A. - Liu, B. - Rogatsky, I.
Journal: Mol Cell Biol

Much of the regulatory diversity in eukaryotic transcription is provided by coregulators, which are recruited by DNA-binding factors to propagate signaling to basal machinery or chromatin. p160 family members, including the glucocorticoid receptor (GR)-interacting protein 1 (GRIP1), function as coactivators for GR, a ligand-dependent transcription factor of the nuclear receptor superfamily. Unlike other p160s, GRIP1 also potentiates GR-mediated repression of AP1 and NF-kappaB targets and, surprisingly, transcriptional activation by interferon regulatory factors. What enables GRIP1 activating or repressing properties or discrimination between physiologically antagonistic pathways is unknown. We found that endogenous GRIP1 in mammalian cells undergoes glucocorticoid-induced, GR interaction-dependent phosphorylation and identified one constitutive and six inducible phosphorylation sites and two putative GRIP1 kinases, casein kinase 2 and cyclin-dependent kinase 9. We raised phosphospecific antibodies to the four closely spaced sites in a previously uncharacterized part of GRIP1 which, combined with mutagenesis, revealed the conservation of GRIP1 phosphorylation across several cell types and species and its functional relevance to GR-activated transcription and to response element-specific recruitment of phospho-GRIP1 to native GR targets. We propose that cofactor engagement by GR is neither passive nor stochastic; rather, GR actively imparts modifications that dictate GRIP1 function in a subset of complexes, adding a layer of specificity to GR transcriptional control.

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Myocardial ATGL Overexpression Decreases the Reliance on Fatty Acid Oxidation and Protects against Pressure Overload-Induced Cardiac Dysfunction.

Publication Date: 2012 Feb PMID: 22158969
Authors: Kienesberger, P. C. - Pulinilkunnil, T. - Sung, M. M. - Nagendran, J. - Haemmerle, G. - Kershaw, E. E. - Young, M. E. - Light, P. E. - Oudit, G. Y. - Zechner, R. - Dyck, J. R.
Journal: Mol Cell Biol

Alterations in myocardial triacylglycerol content have been associated with poor left ventricular function, suggesting that enzymes involved in myocardial triacylglycerol metabolism play an important role in regulating contractile function. Myocardial triacylglycerol catabolism is mediated by adipose triglyceride lipase (ATGL), which is rate limiting for triacylglycerol hydrolysis. To address the influence of triacylglycerol hydrolysis on myocardial energy metabolism and function, we utilized mice with cardiomyocyte-specific ATGL overexpression (MHC-ATGL). Biochemical examination of MHC-ATGL hearts revealed chronically reduced myocardial triacylglycerol content but unchanged levels of long-chain acyl coenzyme A esters, ceramides, and diacylglycerols. Surprisingly, fatty acid oxidation rates were decreased in ex vivo perfused working hearts from MHC-ATGL mice, which was compensated by increased rates of glucose oxidation. Interestingly, reduced myocardial triacylglycerol content was associated with moderately enhanced in vivo systolic function in MHC-ATGL mice and increased isoproterenol-induced cell shortening of isolated primary cardiomyocytes. Most importantly, MHC-ATGL mice were protected from pressure overload-induced systolic dysfunction and detrimental structural remodeling following transverse aortic constriction. Overall, this study shows that ATGL overexpression is sufficient to alter myocardial energy metabolism and improve cardiac function.

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Intersectin 1 Enhances Cbl Ubiquitylation of Epidermal Growth Factor Receptor through Regulation of Sprouty2-Cbl Interaction.

Publication Date: 2012 Feb PMID: 22158968
Authors: Okur, M. N. - Ooi, J. - Fong, C. W. - Martinez, N. - Garcia-Dominguez, C. - Rojas, J. M. - Guy, G. - O'Bryan, J. P.
Journal: Mol Cell Biol

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.

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Embryonic lethality and fetal liver apoptosis in mice lacking all three small maf proteins.

Publication Date: 2012 Feb PMID: 22158967
Authors: Yamazaki, H. - Katsuoka, F. - Motohashi, H. - Engel, J. D. - Yamamoto, M.
Journal: Mol Cell Biol

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.

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TCERG1 Regulates Alternative Splicing of the Bcl-x Gene by Modulating the Rate of RNA Polymerase II Transcription.

Publication Date: 2012 Feb PMID: 22158966
Authors: Montes, M. - Cloutier, A. - Sanchez-Hernandez, N. - Michelle, L. - Lemieux, B. - Blanchette, M. - Hernandez-Munain, C. - Chabot, B. - Sune, C.
Journal: Mol Cell Biol

Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-x(s)) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-x(S) 5' splice site.

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Mannose 6 dephosphorylation of lysosomal proteins mediated by Acid phosphatases acp2 and acp5.

Publication Date: 2012 Feb PMID: 22158965
Authors: Makrypidi, G. - Damme, M. - Muller-Loennies, S. - Trusch, M. - Schmidt, B. - Schluter, H. - Heeren, J. - Lubke, T. - Saftig, P. - Braulke, T.
Journal: Mol Cell Biol

Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products.

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Integration of Elf-4 into Stem/Progenitor and Erythroid Regulatory Networks through Locus-Wide Chromatin Studies Coupled with In Vivo Functional Validation.

Publication Date: 2012 Feb PMID: 22158964
Authors: Smith, A. M. - Calero-Nieto, F. J. - Schutte, J. - Kinston, S. - Timms, R. T. - Wilson, N. K. - Hannah, R. L. - Landry, J. R. - Gottgens, B.
Journal: Mol Cell Biol

The ETS transcription factor Elf-4 is an important regulator of hematopoietic stem cell (HSC) and T cell homeostasis. To gain insights into the transcriptional circuitry within which Elf-4 operates, we used comparative sequence analysis coupled with chromatin immunoprecipitation (ChIP) with microarray technology (ChIP-chip) assays for specific chromatin marks to identify three promoters and two enhancers active in hematopoietic and endothelial cell lines. Comprehensive functional validation of each of these regulatory regions in transgenic mouse embryos identified a tissue-specific enhancer (-10E) that displayed activity in fetal liver, dorsal aorta, vitelline vessels, yolk sac, and heart. Integration of a ChIP-sequencing (ChIP-Seq) data set for 10 key stem cell transcription factors showed Pu.1, Fli-1, and Erg were bound to the -10E element, and mutation of three highly conserved ETS sites within the enhancer abolished its activity. Finally, the transcriptional repressor Gfi1b was found to bind to and repress one of the Elf-4 promoters (-30P), and we show that this repression of Elf-4 is important for the maturation of primary fetal liver erythroid cells. Taken together, our results provide a comprehensive overview of the transcriptional control of Elf-4 within the hematopoietic system and, thus, integrate Elf-4 into the wider transcriptional regulatory networks that govern hematopoietic development.

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Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor alpha in Mouse Liver Reveals Extensive Sharing of Binding Sites.

Publication Date: 2012 Feb PMID: 22158963
Authors: Boergesen, M. - Pedersen, T. A. - Gross, B. - van Heeringen, S. J. - Hagenbeek, D. - Bindesboll, C. - Caron, S. - Lalloyer, F. - Steffensen, K. R. - Nebb, H. I. - Gustafsson, J. A. - Stunnenberg, H. G. - Staels, B. - Mandrup, S.
Journal: Mol Cell Biol

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARalpha binding demonstrated binding of PPARalpha to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARalpha-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARalpha at the level of binding to shared genomic sites.

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MicroRNA-200c Represses Migration and Invasion of Breast Cancer Cells by Targeting Actin-Regulatory Proteins FHOD1 and PPM1F.

Publication Date: 2012 Feb PMID: 22144583
Authors: Jurmeister, S. - Baumann, M. - Balwierz, A. - Keklikoglou, I. - Ward, A. - Uhlmann, S. - Zhang, J. D. - Wiemann, S. - Sahin, O.
Journal: Mol Cell Biol

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor beta (TGF-beta)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.

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Conserved Molecular Interactions within the HBO1 Acetyltransferase Complexes Regulate Cell Proliferation.

Publication Date: 2012 Feb PMID: 22144582
Authors: Avvakumov, N. - Lalonde, M. E. - Saksouk, N. - Paquet, E. - Glass, K. C. - Landry, A. J. - Doyon, Y. - Cayrou, C. - Robitaille, G. A. - Richard, D. E. - Yang, X. J. - Kutateladze, T. G. - Cote, J.
Journal: Mol Cell Biol

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.

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Dopamine-Regulated MicroRNA MiR-181a Controls GluA2 Surface Expression in Hippocampal Neurons.

Publication Date: 2012 Feb PMID: 22144581
Authors: Saba, R. - Storchel, P. H. - Aksoy-Aksel, A. - Kepura, F. - Lippi, G. - Plant, T. D. - Schratt, G. M.
Journal: Mol Cell Biol

The dynamic expression of AMPA-type glutamate receptors (AMPA-R) at synapses is a key determinant of synaptic plasticity, including neuroadaptations to drugs of abuse. Recently, microRNAs (miRNAs) have emerged as important posttranscriptional regulators of synaptic plasticity, but whether they target glutamate receptors to mediate this effect is not known. Here we used microarray screening to identify miRNAs that regulate synaptic plasticity within the nucleus accumbens, a brain region critical to forming drug-seeking habits. One of the miRNAs that showed a robust enrichment at medium spiny neuron synapses was miR-181a. Using bioinformatics tools, we detected a highly conserved miR-181a binding site within the mRNA encoding the GluA2 subunit of AMPA-Rs. Overexpression and knockdown of miR-181a in primary neurons demonstrated that this miRNA is a negative posttranscriptional regulator of GluA2 expression. Additionally, miR-181a overexpression reduced GluA2 surface expression, spine formation, and miniature excitatory postsynaptic current (mEPSC) frequency in hippocampal neurons, suggesting that miR-181a could regulate synaptic function. Moreover, miR-181a expression was induced by dopamine signaling in primary neurons, as well as by cocaine and amphetamines, in a mouse model of chronic drug treatment. Taken together, our results identify miR-181a as a key regulator of mammalian AMPA-type glutamate receptors, with potential implications for the regulation of drug-induced synaptic plasticity.

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Cdk8 Regulates Stability of the Transcription Factor Phd1 To Control Pseudohyphal Differentiation of Saccharomyces cerevisiae.

Publication Date: 2012 Feb PMID: 22124158
Authors: Raithatha, S. - Su, T. C. - Lourenco, P. - Goto, S. - Sadowski, I.
Journal: Mol Cell Biol

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting Sigma1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

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Remodelers Organize Cellular Chromatin by Counteracting Intrinsic Histone-DNA Sequence Preferences in a Class-Specific Manner.

Publication Date: 2012 Feb PMID: 22124157
Authors: Moshkin, Y. M. - Chalkley, G. E. - Kan, T. W. - Reddy, B. A. - Ozgur, Z. - van Ijcken, W. F. - Dekkers, D. H. - Demmers, J. A. - Travers, A. A. - Verrijzer, C. P.
Journal: Mol Cell Biol

The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery.

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A Deacetylase-Deficient SIRT1 Variant Opposes Full-Length SIRT1 in Regulating Tumor Suppressor p53 and Governs Expression of Cancer-Related Genes.

Publication Date: 2012 Feb PMID: 22124156
Authors: Shah, Z. H. - Ahmed, S. U. - Ford, J. R. - Allison, S. J. - Knight, J. R. - Milner, J.
Journal: Mol Cell Biol

SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Delta2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Delta2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Delta2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Delta2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Delta2/9 binds p53 protein. SIRT1-Delta2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Delta2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Delta2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Delta2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Delta2/9 in a range of human cancers. Exogenous SIRT1-Delta2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Delta2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.

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Rhythmic Interaction between Period1 mRNA and hnRNP Q Leads to Circadian Time-Dependent Translation.

Publication Date: 2012 Feb PMID: 22124155
Authors: Lee, K. H. - Woo, K. C. - Kim, D. Y. - Kim, T. D. - Shin, J. - Park, S. M. - Jang, S. K. - Kim, K. T.
Journal: Mol Cell Biol

The mouse PERIOD1 (mPER1) protein, along with other clock proteins, plays a crucial role in the maintenance of circadian rhythms. mPER1 also provides an important link between the circadian system and the cell cycle system. Here we show that the circadian expression of mPER1 is regulated by rhythmic translational control of mPer1 mRNA together with transcriptional modulation. This time-dependent translation was controlled by an internal ribosomal entry site (IRES) element in the 5' untranslated region (5'-UTR) of mPer1 mRNA along with the trans-acting factor mouse heterogeneous nuclear ribonucleoprotein Q (mhnRNP Q). Knockdown of mhnRNP Q caused a decrease in mPER1 levels and a slight delay in mPER1 expression without changing mRNA levels. The rate of IRES-mediated translation exhibits phase-dependent characteristics through rhythmic interactions between mPer1 mRNA and mhnRNP Q. Here, we demonstrate 5'-UTR-mediated rhythmic mPer1 translation and provide evidence for posttranscriptional regulation of the circadian rhythmicity of core clock genes.

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A Novel Function of p38-Regulated/Activated Kinase in Endothelial Cell Migration and Tumor Angiogenesis.

Publication Date: 2012 Feb PMID: 22124154
Authors: Yoshizuka, N. - Chen, R. M. - Xu, Z. - Liao, R. - Hong, L. - Hu, W. Y. - Yu, G. - Han, J. - Chen, L. - Sun, P.
Journal: Mol Cell Biol

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.

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The Laforin-Malin Complex Negatively Regulates Glycogen Synthesis by Modulating Cellular Glucose Uptake via Glucose Transporters.

Publication Date: 2012 Feb PMID: 22124153
Authors: Singh, P. K. - Singh, S. - Ganesh, S.
Journal: Mol Cell Biol

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

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Genetic reevaluation of the role of f-box proteins in cyclin d1 degradation.

Publication Date: 2012 Feb PMID: 22124152
Authors: Kanie, T. - Onoyama, I. - Matsumoto, A. - Yamada, M. - Nakatsumi, H. - Tateishi, Y. - Yamamura, S. - Tsunematsu, R. - Matsumoto, M. - Nakayama, K. I.
Journal: Mol Cell Biol

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only approximately 30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.

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