DOCSLIB.ORG

A Candidate Tumor Suppressor in Lung Cancer

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Candidate Tumor Suppressor in Lung Cancer Oncogene (2008) 27, 3761–3764 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc SHORT COMMUNICATION BHLHB3: a candidate tumor suppressor in lung cancer FS Falvella1, F Colombo1, M Spinola1, M Campiglio1, U Pastorino2 and TA Dragani1 1Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy and 2Unit of Thoracic Surgery, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy BHLHB3 is a basic helix-loop-helix (bHLH) domain- (Fujimoto et al., 2001). Both genes encode bHLH containing protein that acts as a transcriptional repressor. transcriptional repressors involved in the regulation of We found that BHLHB3 transcript levels were low in the mammalian molecular clock (Honma et al., 2002). three human lung cancer cell lines and downregulated Retroviral insertional mutagenesis analysis has impli- in human lung adenocarcinomas as compared to normal cated the mouse Bhlhb3 gene in the development of lung tissue. BHLHB3 gene overexpression inhibited mouse leukemia/lymphoma (Suzuki et al., 2002). In- colony formation of A549, NCI-H520 and NCI-H596 deed, in two cases of leukemia/lymphoma, the retrovirus lung cancer cells. The reduced colony growth was likely integration disrupted the gene coding sequence (Suzuki due to inhibition of cell proliferation as suggested by the et al., 2002) (http://genome2.ncifcrf.gov/RTCGD), sug- downregulation of cyclin D1 (CCND1) expression in gesting that Bhlhb3 loss of function is required for NCI-H520 cells transfected to overexpress the BHLHB3 leukemia/lymphoma development. No information is gene; no evidence of apoptosis was observed. These results currently available on the role of Bhlhb3 in other tumor point to the potential role of the BHLHB3 protein as a types in mice. However, on the basis of our previous tumor suppressor for lung cancer. report suggesting an association between the BHLHB3- Oncogene (2008) 27, 3761–3764; doi:10.1038/sj.onc.1211038; Ala298Val polymorphism and survival of lung adeno- published online 28 January 2008 carcinoma (ADCA) patients (Falvella et al., 2006), we tested BHLHB3for its putative tumor suppressor Keywords: BHLHB2; DEC1; DEC2; SHARP-1; activity in human lung cancer. STRA13; lung cancer BHLHB3transcript levels in human lung cancer cell lines were B100- (A549, NCI-H596) to B1000-fold (NCI-H520) lower than those in a pool of 20 RNA samples of human normal lung tissue, as determined The basic helix-loop-helix (bHLH) proteins form by kinetic reverse transcriptase–PCR. Analysis of heterodimers or homodimers via their HLH domains BHLHB3gene expression in 18 pairs of matched and bind to DNA through a region of basic amino acids normal tumor lung tissue obtained from lung ADCA immediately N terminal to the HLH dimerization patients revealed downregulation of the gene in nine domain (Davis and Turner, 2001). The bHLH transcrip- tumors (range: 2- to 11-folds) and upregulation in five tion factors are involved in coordinated regulation tumors (range: 2- to 3.5-folds); four pairs were excluded of gene expression and cell differentiation in most from the analysis since BHLHB3expression was mammalian tissues (Honma et al., 2002), affecting undetectable in either normal or tumor tissue. Overall, neurogenesis, vasculogenesis, mesoderm segmentation, there was a slight downregulation of BHLHB3expres- myogenesis and T lymphocyte development (Davis and sion in lung ADCA as compared to normal lung tissue, Turner, 2001). bHLH are targets of the Notch signaling but the difference was not statistically significant pathway (Davis and Turner, 2001; Iso et al., 2003), (Figure 1, top panel). We also measured BHLHB2 which can be dysregulated in a variety of human mRNA levels in our matched samples; all 14 pairs had neoplasms (Davis and Turner, 2001; Allenspach et al., detectable levels of BHLHB2 mRNA and the gene was 2002). Proteins encoded by the Class B bHLH protein 3 overexpressed in 2 tumor samples, downregulated in 11 (BHLHB3: also known as DEC2, SHARP-1) gene and tumors and unchanged in 1 pair (Figure 1, bottom its homolog Class B bHLH protein 2 (BHLHB2: also panel; P ¼ 0.01, Kruskal–Wallis test). The finding of known as DEC1, STRA13, SHARP-2) share 42% total downregulated BHLHB2 expression in lung cancer sequence identity and 97% identity in the bHLH region contrasts with that of (Li et al., 2003), who reported a slight upregulation of BHLHB2 (DEC1) expression in Correspondence: Dr TA Dragani, Department of Experimental lung cancer in a small series (n ¼ 4) of lung cancer/ Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale normal lung tissue pairs (Li et al., 2003). In addition, we Tumori, Via G Venezian 1, Milan 20133, Italy. found a direct correlation between BHLHB3and E-mail: [email protected] GenBank acc. number: AY665467. BHLHB2 transcript levels (Spearman’s r ¼ 0.508; Received 22 August 2007; revised 6 December 2007; accepted 11 P ¼ 0.006), whereas (Li et al., 2003) described an December 2007; published online 28 January 2008 inverse relationship between BHLHB3and BHLHB2 BHLHB3 in lung cancer FS Falvella et al 3762 cancer cells stably transfected to overexpress BHLHB3(see Figure 1 legend for details of kinetic reverse transcriptase) revealed only a slight decrease (0.56±0.10-fold, mean±s.e., n ¼ 12) as compared to vector-transfected cells, arguing against any significant, if any, direct modulation of BHLHB2 expression by BHLHB3and instead, suggesting that the observed correlation between BHLHB2 and BHLHB3expression in the normal lung and lung ADCA tissues rests in common transcription regulation. The discrepancy between the experiments carried out in mice and our findings may rest in the differences in the cell lines or species analysed. To analyse the potential functional role of BHLHB3 in human lung cancer, we assessed the clonogenicity of human lung cancer cell lines A549, NCI-H520 and NCI- H596, which expressed low levels of BHLHB3mRNA compared to normal lung tissue, after transfection with mammalian expression vectors containing full-length BHLHB3cDNAs corresponding to either the Ala298 or Val298 variant (Figure 2a). We also transfected a non- lung cancer cell line (HepG2) expressing B1000-fold lower levels of BHLHB3transcript than in normal lung tissue. Clonogenic activity decreased B3- (A549) to B15-fold (NCI-H520, NCI-H596) in the BHLHB3- overexpressing lung cancer cells (Po0.0001), with no significant difference between the two variants (Figures 2b and c). HepG2 transfectants showed no Figure 1 mRNA expression levels in normal and tumor lung difference from controls in clonogenic activity, which tissue of lung adenocarcinoma (ADCA) patients. Lung ADCA surgical specimens and corresponding normal lung tissue were was uniformly high in both cases (not shown). obtained from pathologically documented patients enrolled at To determine whether the decreased clonogenic Istituto Nazionale Tumori, Milan, Italy. Total RNAs were activity of BHLHB3-transfected NCI-H520 cells might extracted with RNeasy Midi kit (Qiagen, Valencia, CA, USA). reflect induction of apoptosis, NCI-H520 cells were cDNAs were reverse transcribed using oligo(dT) primers and transiently transfected and analysed after 48 and 72 h by ThermoScript RT–PCR System (Invitrogen, Carlsbad, CA, USA). mRNA levels were assayed by kinetic reverse transcriptase–PCR indirect immunofluorescence assay in live cells using the using Hs00229146_m1 (BHLHB3) and Hs00186419_m1 Annexin V-FITC kit (Bender MedSystems, Vienna, (BHLHB2) TaqMan Gene Expression Assays (Applied Biosys- Austria); no evidence of apoptosis was obtained at tems). The real-time PCR amplification mixture contained cDNA either time point (data not shown). As an additional template diluted in RNase-free water, 10 ml 2X TaqMan Universal PCR Master Mix No AmpErase UNG and 1 ml of 20X TaqMan assay of apoptosis, we used an anti-PARP antibody Gene Expression Assay Mix (Applied Biosystems). Reactions were (Cell Signaling Tech Inc., Danvers, MA, USA) by run in duplicate on the 7900HT System (Applied Biosystems) for western blot in stable transfected cells; again, we found 40 cycles (95 1C for 15 s, 60 1C for 1 min) after enzyme activation no evidence of PARP protein cleavage (not shown). (95 1C for 10 min). The human hypoxanthine phosphoribosyltrans- Instead, quantitative PCR of NCI-H520 cells stably ferase 1 (HPRT1) gene (Hs99999909_m1 TaqMan Gene Expres- sion Assays, Applied Biosystems) was used as control for possible transfected to overexpress BHLHB3(Figure 3)and differences in cDNA amounts. Values were calculated using the using cyclin D1 as a marker of proliferation (Berthet comparative Ct method, and relative expression levels are directly and Kaldis, 2007) revealed a decrease (0.25±0.04-fold, proportional to the ÀDCt values. kRT–PCRs for BHLHB3(top) n ¼ 8) of cyclin D1 mRNA expression (measured using or BHLHB2 (bottom) were carried out in 14 paired normal and tumor samples. The line within each box represents the median Hs00277039_m1 (CCND1) TaqMan Gene Expression ÀDCt value; upper and lower edges of each box represent the Assays; Applied Biosystems, Foster City, CA, USA) in seventy-fifth and twenty-fifth percentile, respectively; upper and BHLHB3-transfected versus vector-transfected cells, lower bars indicate the highest and lowest values determined, consistent with western blot analysis (Figure 3) and respectively. with a previous study (Azmi et al., 2004) showing that BHLHB3overexpression in mouse myoblast cell line C2C12 promotes cell cycle
Load more

Recommended publications
  • 2017.08.28 Anne Barry-Reidy Thesis Final.Pdf
    REGULATION OF BOVINE β-DEFENSIN EXPRESSION THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF DUBLIN FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2017 ANNE BARRY-REIDY SCHOOL OF BIOCHEMISTRY & IMMUNOLOGY TRINITY COLLEGE DUBLIN SUPERVISORS: PROF. CLIONA O’FARRELLY & DR. KIERAN MEADE TABLE OF CONTENTS DECLARATION ................................................................................................................................. vii ACKNOWLEDGEMENTS ................................................................................................................... viii ABBREVIATIONS ................................................................................................................................ix LIST OF FIGURES............................................................................................................................. xiii LIST OF TABLES .............................................................................................................................. xvii ABSTRACT ........................................................................................................................................xix Chapter 1 Introduction ........................................................................................................ 1 1.1 Antimicrobial/Host-defence peptides ..................................................................... 1 1.2 Defensins................................................................................................................. 1 1.3 β-defensins .............................................................................................................
    [Show full text]
  • Farnesol-Induced Apoptosis in Human Lung Carcinoma Cells Is Coupled to the Endoplasmic Reticulum Stress Response
    Research Article Farnesol-Induced Apoptosis in Human Lung Carcinoma Cells Is Coupled to the Endoplasmic Reticulum Stress Response Joung Hyuck Joo,1 Grace Liao,1 Jennifer B. Collins,2 Sherry F. Grissom,2 and Anton M. Jetten1 1Cell Biology Section, LRB, and 2Microarray Group, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina Abstract range of fruits and vegetables (9, 10). Each isoprenoid has been Farnesol (FOH) and other isoprenoid alcohols induce apopto- shown to inhibit proliferation and induce apoptosis in a number of sis in various carcinoma cells and inhibit tumorigenesis in neoplastic cell lines from different origins (4, 11–14). In addition, in vivo these isoprenoids have been reported to be effective in chemo- several models. However, the mechanisms by which in vivo they mediate their effects are not yet fully understood. In this prevention and chemotherapy in various cancer models study, we show that FOH is an effective inducer of apoptosis in (10, 12, 15, 16). FOH has been reported to exhibit chemopreventive several lung carcinoma cells, including H460. This induction is effects in colon and pancreas carcinogenesis in rats (9, 17) whereas associated with activation of several caspases and cleavage of phase I and II clinical trials have indicated therapeutic potential poly(ADP-ribose) polymerase (PARP). To obtain insight into for POH (16, 18). The mechanisms by which these isoprenoids induce these effects are not yet fully understood. Isoprenoids have the mechanism involved in FOH-induced apoptosis, we compared the gene expression profiles of FOH-treated and been reported to inhibit posttranslational protein prenylation (19) control H460 cells by microarray analysis.
    [Show full text]
  • Temporal Chip-On-Chip of RNA-Polymerase-II to Detect Novel Gene Activation Events During Photoreceptor Maturation
    Molecular Vision 2010; 16:252-271 <http://www.molvis.org/molvis/v16/a32> © 2010 Molecular Vision Received 12 July 2009 | Accepted 10 February 2010 | Published 17 February 2010 Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation Padmaja Tummala, Raghuveer S. Mali, Eduardo Guzman, Xiao Zhang, Kenneth P. Mitton (The first two authors contributed equally to this work.) Eye Research Institute, Oakland University, Rochester, MI Purpose: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites. Methods: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10–12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples.
    [Show full text]
  • Expression Signatures of the Lipid-Based Akt Inhibitors Phosphatidylinositol Ether Lipid Analogues in NSCLC Cells
    Published OnlineFirst May 6, 2011; DOI: 10.1158/1535-7163.MCT-10-1028 Molecular Cancer Therapeutic Discovery Therapeutics Expression Signatures of the Lipid-Based Akt Inhibitors Phosphatidylinositol Ether Lipid Analogues in NSCLC Cells Chunyu Zhang1, Abdel G. Elkahloun2, Hongling Liao3, Shannon Delaney1, Barbara Saber1, Betsy Morrow1, George C. Prendergast4, M. Christine Hollander1, Joell J. Gills1, and Phillip A. Dennis1 Abstract Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIA) are analogues of the products of phosphoinositide-3-kinase (PI3K) that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of five active PIAs and the PI3K inhibitor LY294002 (LY) was conducted in non–small cell lung carcinoma cells using high-density oligonucleotide arrays. Gene ontology analysis revealed that genes involved in apoptosis, wounding response, and angiogen- esis were upregulated by PIAs, whereas genes involved in DNA replication, repair, and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into three groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, and PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB, and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3, and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB), and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity.
    [Show full text]
  • The Transcriptional Repressor STRA13 Regulates a Subset of Peripheral Circadian Outputs*
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 2, Issue of January 9, pp. 1141–1150, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Transcriptional Repressor STRA13 Regulates a Subset of Peripheral Circadian Outputs* Received for publication, May 22, 2003, and in revised form, September 22, 2003 Published, JBC Papers in Press, October 27, 2003, DOI 10.1074/jbc.M305369200 Aline Gre´chez-Cassiau‡, Satchidananda Panda§¶, Samuel Lacoche‡, Miche`le Teboul‡, Sameena Azmiʈ, Vincent Laudet**, John B. Hogenesch§, Reshma Tanejaʈ‡‡, and Franck Delaunay‡§§ From the ‡Universite´ de Nice-Sophia Antipolis, CNRS UMR 6078, La Darse, 06230 Villefranche/mer, France, the §Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, the ¶Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, the ʈBrookdale Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, New York 10029, and the **Ecole Normale Supe´rieure de Lyon, CNRS UMR5665, 46 alle´e d’Italie, 69364 Lyon, France Central and peripheral mammalian circadian clocks identified a molecular oscillator generated by transcriptional/ regulate a variety of behavioral and physiological pro- translational feedback loops. In mammals, the main loop in- cesses through the rhythmic transcription of hundreds volves the E box-mediated transcriptional activation of the of clock-controlled genes. The circadian expression of Per1, Per2, Per3, Cry1, and Cry2 clock genes by the CLOCK- many transcriptional regulators suggests that a major BMAL1 heterodimer. Then PER and CRY proteins form com- part of this circadian gene network is indirectly regu- plexes that enter into the nucleus in a phosphorylation-depend- lated by clock genes.
    [Show full text]
  • Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-Like Growth Arrest in MCF-7 Breast Carcinoma Cells
    Research Article Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-like Growth Arrest in MCF-7 Breast Carcinoma Cells Yuhong Chen,1 Milos Dokmanovic,1 Wilfred D. Stein,1,2 Robert J. Ardecky,3 and Igor B. Roninson1 1Cancer Center, Ordway Research Institute, Albany, New York; 2Institute of Life Sciences, Hebrew University, Jerusalem, Israel; and 3Ligand Pharmaceuticals, Inc., San Diego, California Abstract retinoids is most often attributed to the induction of differentia- Biological effects of retinoids are mediated via retinoic acid tion, but these compounds were also shown to stop the growth of (RA) receptors (RAR) and retinoid X receptors (RXR). The tumor cells by inducing apoptosis or accelerated senescence (1, 2). best-characterized mechanism of retinoid action is stimula- In particular, treatment of two human breast carcinoma cell lines tion of transcription from promoters containing RA response with all-trans retinoic acid (RA) or fenretinide, in vitro or in vivo, elements (RARE). Retinoids induce senescence-like growth induces a senescence-like phenotype characterized by increased h arrest in MCF-7 breast carcinoma cells; this effect is cell size and expression of senescence-associated -galactosidase h associated with the induction of several growth-inhibitory (SA- -gal; refs. 3, 4). This phenotype, as investigated in MCF-7 cells, genes. We have nowfound that these genes are induced by is associated with irreversible growth arrest and up-regulation of RAR-specific but not by RXR-specific ligands. Genome-scale several intracellular and secreted proteins with known growth- microarray analysis of gene expression was used to compare inhibitory activities.
    [Show full text]
  • Supplementary Table 2
    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
    [Show full text]
  • Vertebrate Hairy and Enhancer of Split Related Proteins: Transcriptional Repressors Regulating Cellular Di€Erentiation and Embryonic Patterning
    Oncogene (2001) 20, 8342 ± 8357 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular dierentiation and embryonic patterning Robert L Davis1 and David L Turner*,2 1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, MA 02115, USA; 2Mental Health Research Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, MI 48104-1687, USA The basic-helix-loop-helix (bHLH) proteins are a super- frogs, and zebra®sh. As we discuss below, the family of DNA-binding transcription factors that vertebrate hairy and E(spl) related proteins can be regulate numerous biological processes in both inverte- grouped into distinct subfamilies based on their brates and vertebrates. One family of bHLH transcrip- primary structures. However, all proteins in these tional repressors is related to the Drosophila hairy and subfamilies contain a conserved amino acid sequence Enhancer-of-split proteins. These repressors contain a known as the Orange domain located just C-terminal tandem arrangement of the bHLH domain and an to the bHLH domain. The tandem arrangement of the adjacent sequence known as the Orange domain, so we bHLH and Orange domains is the major structural refer to these proteins as bHLH-Orange or bHLH-O feature shared among these proteins, so for conve- proteins. Phylogenetic analysis reveals the existence of nience we refer to all hairy and E(spl) related proteins four bHLH-O subfamilies, with distinct, evolutionarily collectively as bHLH-Orange (bHLH-O) proteins. conserved features.
    [Show full text]
  • System-Level Identification of Transcriptional Circuits Underlying
    LETTERS System-level identification of transcriptional circuits underlying mammalian circadian clocks Hiroki R Ueda1–3, Satoko Hayashi1, Wenbin Chen1, Motoaki Sano4, Masayuki Machida4, Yasufumi Shigeyoshi5, Masamitsu Iino3 & Seiichi Hashimoto1 Mammalian circadian clocks consist of complexly integrated (Bhlhb2 and Bhlhb3,alsocalledDec1 or Stra13 and Dec2, respectively), regulatory loops1–5, making it difficult to elucidate them one period-related gene20 (Per3), one clock-related gene21 (Npas2) without both the accurate measurement of system dynamics and three genes4,22 related to Nr1d1 and Rora (Nr1d2, Rorb and and the comprehensive identification of network circuits6. Rorc, also called RevErbAb, Rorb and Rorg, respectively), have been http://www.nature.com/naturegenetics Toward a system-level understanding of this transcriptional cloned as well. circuitry, we identified clock-controlled elements on 16 clock Gene-expression analyses have shown that the mRNA levels of 16 of and clock-controlled genes in a comprehensive surveillance of these 18 clock and clock-related genes have high-amplitude circadian evolutionarily conserved cis elements and measurement of oscillations in the central (suprachiasmatic nucleus; SCN) or periph- their transcriptional dynamics. Here we report the roles of eral (liver, etc.) clock tissues2,4,17–27 (Supplementary Fig. 1 online). E/E¢ boxes, DBP/E4BP4 binding elements7 and RevErbA/ROR The organization of the circadian regulation of the transcription of binding elements8 in nine, seven and six genes, respectively. clock and clock-related genes is suggestive of an integrated network of Our results indicate that circadian transcriptional circuits regulatory loops of great complexity, as regulators of circadian clocks are governed by two design principles: regulation of E/E¢ are themselves regulated by circadian clocks.
    [Show full text]
  • Host Cell Factors Necessary for Influenza a Infection: Meta-Analysis of Genome Wide Studies
    Host Cell Factors Necessary for Influenza A Infection: Meta-Analysis of Genome Wide Studies Juliana S. Capitanio and Richard W. Wozniak Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta Abstract: The Influenza A virus belongs to the Orthomyxoviridae family. Influenza virus infection occurs yearly in all countries of the world. It usually kills between 250,000 and 500,000 people and causes severe illness in millions more. Over the last century alone we have seen 3 global influenza pandemics. The great human and financial cost of this disease has made it the second most studied virus today, behind HIV. Recently, several genome-wide RNA interference studies have focused on identifying host molecules that participate in Influen- za infection. We used nine of these studies for this meta-analysis. Even though the overlap among genes identified in multiple screens was small, network analysis indicates that similar protein complexes and biological functions of the host were present. As a result, several host gene complexes important for the Influenza virus life cycle were identified. The biological function and the relevance of each identified protein complex in the Influenza virus life cycle is further detailed in this paper. Background and PA bound to the viral genome via nucleoprotein (NP). The viral core is enveloped by a lipid membrane derived from Influenza virus the host cell. The viral protein M1 underlies the membrane and anchors NEP/NS2. Hemagglutinin (HA), neuraminidase Viruses are the simplest life form on earth. They parasite host (NA), and M2 proteins are inserted into the envelope, facing organisms and subvert the host cellular machinery for differ- the viral exterior.
    [Show full text]
  • Stem Cell-Based Reporter Assays for the Identification of Carcinogenic Properties of (Non)-Genotoxic Chemicals
    UNIVERSIDADE DE LISBOA Faculdade de Ciências Departamento de Biologia Animal Stem cell-based reporter assays for the identification of carcinogenic properties of (non)-genotoxic chemicals Cátia Sofia Alão Gonçalves DISSERTAÇÃO MESTRADO EM BIOLOGIA HUMANA E AMBIENTE 2013 UNIVERSIDADE DE LISBOA Faculdade de Ciências Departamento de Biologia Animal Stem cell-based reporter assays for the identification of carcinogenic properties of (non)-genotoxic chemicals Cátia Sofia Alão Gonçalves Dissertação orientada por: Dr. Harry Vrieling (Toxicogenetic/LUMC) Professora Dra. Deodália Dias (DBA/FCUL) DISSERTAÇÃO MESTRADO EM BIOLOGIA HUMANA E AMBIENTE 2013 Acknowledgements First of all I would like to express my deep and sincere gratitude to Harry Vrieling, for having welcomed me in your workgroup and give me the opportunity to develop my dissertation. I also have to thank Giel Hendriks for the useful discussions and uplifting spirits that allowed me to develop my dissertation and allowed me to develop, not only the professional level, but also the personal one. A special thanks to the entire lab, in particular to Brunno for helping me with the BAC recombennering and the great talks, to Fabienne for the tips on how to culture the mES cell lines and for her constant assistance in the Lab and to Binni for helping me understand a little better the Dutch culture. I wish to thank all of my colleagues at the Department of Toxicogenetics, at the Leiden University Medical Centre, for all the support. I thank Professor Deodália Dias for all the support provided under this thesis and also in my training. I want to thank to all the friends that I made during my time in Leiden, without them, probably this dissertation would not be completed.
    [Show full text]
  • A Module of Negative Feedback Regulators Defines Growth Factor
    ARTICLES A module of negative feedback regulators defines growth factor signaling Ido Amit1,9, Ami Citri1,8,9, Tal Shay2, Yiling Lu3, Menachem Katz1, Fan Zhang3, Gabi Tarcic1, Doris Siwak3, John Lahad3, Jasmine Jacob-Hirsch4, Ninette Amariglio4, Nora Vaisman5, Eran Segal6, Gideon Rechavi4, Uri Alon7, Gordon B Mills3, Eytan Domany2 & Yosef Yarden1 Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within http://www.nature.com/naturegenetics this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks. Cells respond in a stereotypic and highly reproducible manner to phosphorylation and gene expression, with the aim of uncovering external stimuli. A major focus of current research is elucidation of the mechanisms of transcription-dependent signal attenuation. Conse- mechanisms underlying the regulation of this response. In the past, quently, we can now identify a cluster of coexpressed genes that considerable effort was invested in characterizing ‘forward-signaling’ function in feedback attenuation of growth factor signaling at specific components activated by external stimuli, with successful identifica- nodes within the network (for an overview, see Supplementary Fig.
    [Show full text]