A Dissertation

Total Page:16

File Type:pdf, Size:1020Kb

A Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2014-03-28 Identification and Characteristics of Factors Regulating Hepatocellular Carcinoma Progression and Metastasis: A Dissertation Leanne G. Ahronian University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cancer Biology Commons, Digestive System Diseases Commons, Molecular Genetics Commons, and the Neoplasms Commons Repository Citation Ahronian LG. (2014). Identification and Characteristics of Factors Regulating Hepatocellular Carcinoma Progression and Metastasis: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/ M2002W. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/705 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. IDENTIFICATION AND CHARACTERISTICS OF FACTORS REGULATING HEPATOCELLULAR CARCINOMA PROGRESSION AND METASTASIS A Dissertation Presented By LEANNE G. AHRONIAN Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MARCH 28, 2014 CANCER BIOLOGY IDENTIFICATION AND CHARACTERISTICS OF FACTORS REGULATING HEPATOCELLULAR CARCINOMA PROGRESSION AND METASTASIS A Dissertation Presented By LEANNE G. AHRONIAN The signatures of the Dissertation Defense Committee signify completion and approval as to the style and content of the Dissertation _______________________________________________________________ Brian C. Lewis, Ph.D., Thesis Advisor _______________________________________________________________ JeanMarie Houghton, M.D., Ph.D., Member of Committee _______________________________________________________________ Alec Kimmelman, M.D., Ph.D., Member of Committee _______________________________________________________________ Karl Simin, Ph.D., Member of Committee _______________________________________________________________ Scot Wolfe, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _______________________________________________________________ Leslie Shaw, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. _______________________________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Cancer Biology March 28, 2014 iv Acknowledgements I would like to thank Brian Lewis for his mentorship during my graduate studies. His encouragement and insights during this journey were indispensable, and I value all of the time he invested in my training. I also appreciate the members of the Lewis Lab, past and present, for their technical support and insights. Particularly, I would like to thank Jiufeng Cai and Victor Adelanwa for their technical assistance. Experimental contributions from YaGWen Chen were critical to the KLF6 project, and I also thank her for taking the time to share her expertise with me after I joined the lab. Lihua (Julie) Zhu provided me with excellent suggestions for experimental design, and I thank her for the bioinformatics analysis and support that she provided. Leslie Shaw, Karl Simin, and Scot Wolfe provided excellent advice and comments throughout my graduate studies, and I truly appreciate the time they dedicated to considering my work. I would also like to thank Justine Landis, Brian Quattrochi, and Kirsten Tracy for providing comments on portions of my thesis. Of course, I must thank my friends and family for the unwavering support they continue to provide. v Abstract Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancerGrelated death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of KrüppelGlike factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of KrüppelGlike factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. SingleGcopy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6Gmediated tumor and metastasis suppression, we performed gene expression profiling and ChIPGsequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 vi and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6Gmediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gainGofG function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gainGofGfunction properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformationGassociated phenotypes in these cells. However, p53R172HGexpressing mice did not have enhanced tumor formation or metastasis compared to p53Gnull mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gainGofGfunction mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gainGofGfunction effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53Gnull and p53R172HGexpressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. vii Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients. viii Table of Contents Title Page ii Signature Page iii Acknowledgments iv Abstract v Table of Contents viii List of Tables x List of Figures xi List of Abbreviations xvi CHAPTER I: Introduction 1 CHAPTER II 51 KLF6 is a repressor of HCC cell migration, tumor formation, and metastasis Introduction 53 Results 57 Materials and Methods 96 Discussion 105 CHAPTER III 111 KLF6 suppresses RHO family GTPase activity through transcriptional repression of VAV3 and CDC42EP3 Introduction 113 Results 118 Materials and Methods 140 ix Discussion 148 CHAPTER IV 152 The p53R172H mutant does not enhance hepatocellular carcinoma development and progression Introduction 154 Results 159 Materials and Methods 177 Discussion 183 CHAPTER V: Discussion 187 Appendix A 217 Appendix B 227 Bibliography 237 x List of Tables Table 2.1 64 Gene Ontology terms present in the subpopulation gene expression profiling dataset. Table 2.2 103 Antibody conditions used for Western blotting and immunohistochemistry Table 2.3 104 Primers used for amplification of target genes by qRTGPCR. Table 3.1 129 Transcriptional targets of KLF6 as detected by ChIPGsequencing and gene expression profiling of KLF6 knockdown cells Table 3.2 147 Antibodies and conditions used for Western blotting Table 4.1 166 The incidence of HCC or gross metastases is not different at any period of time in HCC development. Table 4.2 182 Antibodies and conditions used for Western blotting and immunohistochemistry Table 4.3 182 Primer sequences used for detection of transcripts by qRTGPCR xi List of Figures Figure 1.1 5 Development of hepatocellular carcinoma Figure 1.2 26 The Klf6 locus is alternatively spliced to generate four known variants Figure 1.3 41 p53 incorporates cellular stress signals and transcribes a myriad of target genes to regulate multiple cell processes Figure 2.1 59 Subpopulations of an HCC cell line display increased proliferation, transformation and migration Figure 2.2 61
Recommended publications
  • 4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation
    Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4).
    [Show full text]
  • Transcriptional Control of Tissue-Resident Memory T Cell Generation
    Transcriptional control of tissue-resident memory T cell generation Filip Cvetkovski Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Filip Cvetkovski All rights reserved ABSTRACT Transcriptional control of tissue-resident memory T cell generation Filip Cvetkovski Tissue-resident memory T cells (TRM) are a non-circulating subset of memory that are maintained at sites of pathogen entry and mediate optimal protection against reinfection. Lung TRM can be generated in response to respiratory infection or vaccination, however, the molecular pathways involved in CD4+TRM establishment have not been defined. Here, we performed transcriptional profiling of influenza-specific lung CD4+TRM following influenza infection to identify pathways implicated in CD4+TRM generation and homeostasis. Lung CD4+TRM displayed a unique transcriptional profile distinct from spleen memory, including up-regulation of a gene network induced by the transcription factor IRF4, a known regulator of effector T cell differentiation. In addition, the gene expression profile of lung CD4+TRM was enriched in gene sets previously described in tissue-resident regulatory T cells. Up-regulation of immunomodulatory molecules such as CTLA-4, PD-1, and ICOS, suggested a potential regulatory role for CD4+TRM in tissues. Using loss-of-function genetic experiments in mice, we demonstrate that IRF4 is required for the generation of lung-localized pathogen-specific effector CD4+T cells during acute influenza infection. Influenza-specific IRF4−/− T cells failed to fully express CD44, and maintained high levels of CD62L compared to wild type, suggesting a defect in complete differentiation into lung-tropic effector T cells.
    [Show full text]
  • The Correlation of Keratin Expression with In-Vitro Epithelial Cell Line Differentiation
    The correlation of keratin expression with in-vitro epithelial cell line differentiation Deeqo Aden Thesis submitted to the University of London for Degree of Master of Philosophy (MPhil) Supervisors: Professor Ian. C. Mackenzie Professor Farida Fortune Centre for Clinical and Diagnostic Oral Science Barts and The London School of Medicine and Dentistry Queen Mary, University of London 2009 Contents Content pages ……………………………………………………………………......2 Abstract………………………………………………………………………….........6 Acknowledgements and Declaration……………………………………………...…7 List of Figures…………………………………………………………………………8 List of Tables………………………………………………………………………...12 Abbreviations….………………………………………………………………..…...14 Chapter 1: Literature review 16 1.1 Structure and function of the Oral Mucosa……………..…………….…..............17 1.2 Maintenance of the oral cavity...……………………………………….................20 1.2.1 Environmental Factors which damage the Oral Mucosa………. ….…………..21 1.3 Structure and function of the Oral Mucosa ………………...….……….………...21 1.3.1 Skin Barrier Formation………………………………………………….……...22 1.4 Comparison of Oral Mucosa and Skin…………………………………….……...24 1.5 Developmental and Experimental Models used in Oral mucosa and Skin...……..28 1.6 Keratinocytes…………………………………………………….….....................29 1.6.1 Desmosomes…………………………………………….…...............................29 1.6.2 Hemidesmosomes……………………………………….…...............................30 1.6.3 Tight Junctions………………………….……………….…...............................32 1.6.4 Gap Junctions………………………….……………….….................................32
    [Show full text]
  • MALE Protein Name Accession Number Molecular Weight CP1 CP2 H1 H2 PDAC1 PDAC2 CP Mean H Mean PDAC Mean T-Test PDAC Vs. H T-Test
    MALE t-test t-test Accession Molecular H PDAC PDAC vs. PDAC vs. Protein Name Number Weight CP1 CP2 H1 H2 PDAC1 PDAC2 CP Mean Mean Mean H CP PDAC/H PDAC/CP - 22 kDa protein IPI00219910 22 kDa 7 5 4 8 1 0 6 6 1 0.1126 0.0456 0.1 0.1 - Cold agglutinin FS-1 L-chain (Fragment) IPI00827773 12 kDa 32 39 34 26 53 57 36 30 55 0.0309 0.0388 1.8 1.5 - HRV Fab 027-VL (Fragment) IPI00827643 12 kDa 4 6 0 0 0 0 5 0 0 - 0.0574 - 0.0 - REV25-2 (Fragment) IPI00816794 15 kDa 8 12 5 7 8 9 10 6 8 0.2225 0.3844 1.3 0.8 A1BG Alpha-1B-glycoprotein precursor IPI00022895 54 kDa 115 109 106 112 111 100 112 109 105 0.6497 0.4138 1.0 0.9 A2M Alpha-2-macroglobulin precursor IPI00478003 163 kDa 62 63 86 72 14 18 63 79 16 0.0120 0.0019 0.2 0.3 ABCB1 Multidrug resistance protein 1 IPI00027481 141 kDa 41 46 23 26 52 64 43 25 58 0.0355 0.1660 2.4 1.3 ABHD14B Isoform 1 of Abhydrolase domain-containing proteinIPI00063827 14B 22 kDa 19 15 19 17 15 9 17 18 12 0.2502 0.3306 0.7 0.7 ABP1 Isoform 1 of Amiloride-sensitive amine oxidase [copper-containing]IPI00020982 precursor85 kDa 1 5 8 8 0 0 3 8 0 0.0001 0.2445 0.0 0.0 ACAN aggrecan isoform 2 precursor IPI00027377 250 kDa 38 30 17 28 34 24 34 22 29 0.4877 0.5109 1.3 0.8 ACE Isoform Somatic-1 of Angiotensin-converting enzyme, somaticIPI00437751 isoform precursor150 kDa 48 34 67 56 28 38 41 61 33 0.0600 0.4301 0.5 0.8 ACE2 Isoform 1 of Angiotensin-converting enzyme 2 precursorIPI00465187 92 kDa 11 16 20 30 4 5 13 25 5 0.0557 0.0847 0.2 0.4 ACO1 Cytoplasmic aconitate hydratase IPI00008485 98 kDa 2 2 0 0 0 0 2 0 0 - 0.0081 - 0.0
    [Show full text]
  • Experimental Eye Research 129 (2014) 93E106
    Experimental Eye Research 129 (2014) 93e106 Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq S. Scott Whitmore a, b, Alex H. Wagner a, c, Adam P. DeLuca a, b, Arlene V. Drack a, b, Edwin M. Stone a, b, Budd A. Tucker a, b, Shemin Zeng a, b, Terry A. Braun a, b, c, * Robert F. Mullins a, b, Todd E. Scheetz a, b, c, a Stephen A. Wynn Institute for Vision Research, The University of Iowa, Iowa City, IA, USA b Department of Ophthalmology and Visual Sciences, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA c Department of Biomedical Engineering, College of Engineering, The University of Iowa, Iowa City, IA, USA article info abstract Article history: Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal Received 14 September 2014 diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmen- Received in revised form tosa, preferentially affect distinct topographic regions of the retina. While much is known about the 31 October 2014 distribution of cell types in the retina, the distribution of molecular components across the posterior pole Accepted in revised form 4 November 2014 of the eye has not been well-studied. To investigate regional difference in molecular composition of Available online 5 November 2014 ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq.
    [Show full text]
  • Strand Breaks for P53 Exon 6 and 8 Among Different Time Course of Folate Depletion Or Repletion in the Rectosigmoid Mucosa
    SUPPLEMENTAL FIGURE COLON p53 EXONIC STRAND BREAKS DURING FOLATE DEPLETION-REPLETION INTERVENTION Supplemental Figure Legend Strand breaks for p53 exon 6 and 8 among different time course of folate depletion or repletion in the rectosigmoid mucosa. The input of DNA was controlled by GAPDH. The data is shown as ΔCt after normalized to GAPDH. The higher ΔCt the more strand breaks. The P value is shown in the figure. SUPPLEMENT S1 Genes that were significantly UPREGULATED after folate intervention (by unadjusted paired t-test), list is sorted by P value Gene Symbol Nucleotide P VALUE Description OLFM4 NM_006418 0.0000 Homo sapiens differentially expressed in hematopoietic lineages (GW112) mRNA. FMR1NB NM_152578 0.0000 Homo sapiens hypothetical protein FLJ25736 (FLJ25736) mRNA. IFI6 NM_002038 0.0001 Homo sapiens interferon alpha-inducible protein (clone IFI-6-16) (G1P3) transcript variant 1 mRNA. Homo sapiens UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 15 GALNTL5 NM_145292 0.0001 (GALNT15) mRNA. STIM2 NM_020860 0.0001 Homo sapiens stromal interaction molecule 2 (STIM2) mRNA. ZNF645 NM_152577 0.0002 Homo sapiens hypothetical protein FLJ25735 (FLJ25735) mRNA. ATP12A NM_001676 0.0002 Homo sapiens ATPase H+/K+ transporting nongastric alpha polypeptide (ATP12A) mRNA. U1SNRNPBP NM_007020 0.0003 Homo sapiens U1-snRNP binding protein homolog (U1SNRNPBP) transcript variant 1 mRNA. RNF125 NM_017831 0.0004 Homo sapiens ring finger protein 125 (RNF125) mRNA. FMNL1 NM_005892 0.0004 Homo sapiens formin-like (FMNL) mRNA. ISG15 NM_005101 0.0005 Homo sapiens interferon alpha-inducible protein (clone IFI-15K) (G1P2) mRNA. SLC6A14 NM_007231 0.0005 Homo sapiens solute carrier family 6 (neurotransmitter transporter) member 14 (SLC6A14) mRNA.
    [Show full text]
  • Maintenance of Mammary Epithelial Phenotype by Transcription Factor Runx1 Through Mitotic Gene Bookmarking Joshua Rose University of Vermont
    University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2019 Maintenance Of Mammary Epithelial Phenotype By Transcription Factor Runx1 Through Mitotic Gene Bookmarking Joshua Rose University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biochemistry Commons, and the Genetics and Genomics Commons Recommended Citation Rose, Joshua, "Maintenance Of Mammary Epithelial Phenotype By Transcription Factor Runx1 Through Mitotic Gene Bookmarking" (2019). Graduate College Dissertations and Theses. 998. https://scholarworks.uvm.edu/graddis/998 This Thesis is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. MAINTENANCE OF MAMMARY EPITHELIAL PHENOTYPE BY TRANSCRIPTION FACTOR RUNX1 THROUGH MITOTIC GENE BOOKMARKING A Thesis Presented by Joshua Rose to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Master of Science Specializing in Cellular, Molecular, and Biomedical Sciences January, 2019 Defense Date: November 12, 2018 Thesis Examination Committee: Sayyed Kaleem Zaidi, Ph.D., Advisor Gary Stein, Ph.D., Advisor Seth Frietze, Ph.D., Chairperson Janet Stein, Ph.D. Jonathan Gordon, Ph.D. Cynthia J. Forehand, Ph.D. Dean of the Graduate College ABSTRACT Breast cancer arises from a series of acquired mutations that disrupt normal mammary epithelial homeostasis and create multi-potent cancer stem cells that can differentiate into clinically distinct breast cancer subtypes. Despite improved therapies and advances in early detection, breast cancer remains the leading diagnosed cancer in women.
    [Show full text]
  • 3. Inflammasomes
    UNIVERSIDAD DE MURCIA ESCUELA INTERNACIONAL DE DOCTORADO Characterization of Caiap and Wdr90 as Novel Inflammasome Components Involved in the Resistance to Salmonella enterica serovar Typhimurium. Caracterización de Caiap y Wdr90 como Nuevos Componentes del Inflamasoma Implicados en la Resistencia a Salmonella enterica serovar Typhimurium Dña. Ana Valera Pérez 2018 TABLE OF CONTENTS ABBREVIATIONS ................................................................................................................ 9 SUMMARY....................................................................................................................... 19 INTRODUCTION .............................................................................................................. 23 1. The immune system .............................................................................. 25 1.1. The innate immune system in fish ....................................................... 26 1.2. Adaptive immune system in fish .......................................................... 29 2. Immune system and inflammasomes ................................................... 30 2.1. PRRs ................................................................................................... 31 2.2. Toll like receptors .............................................................................. 32 2.3. NOD-like receptors ............................................................................ 32 3. Inflammasomes ....................................................................................
    [Show full text]
  • The Genetic Program of Pancreatic Beta-Cell Replication in Vivo
    Page 1 of 65 Diabetes The genetic program of pancreatic beta-cell replication in vivo Agnes Klochendler1, Inbal Caspi2, Noa Corem1, Maya Moran3, Oriel Friedlich1, Sharona Elgavish4, Yuval Nevo4, Aharon Helman1, Benjamin Glaser5, Amir Eden3, Shalev Itzkovitz2, Yuval Dor1,* 1Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 3Department of Cell and Developmental Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 4Info-CORE, Bioinformatics Unit of the I-CORE Computation Center, The Hebrew University and Hadassah, The Institute for Medical Research Israel- Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 5Endocrinology and Metabolism Service, Department of Internal Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel *Correspondence: [email protected] Running title: The genetic program of pancreatic β-cell replication 1 Diabetes Publish Ahead of Print, published online March 18, 2016 Diabetes Page 2 of 65 Abstract The molecular program underlying infrequent replication of pancreatic beta- cells remains largely inaccessible. Using transgenic mice expressing GFP in cycling cells we sorted live, replicating beta-cells and determined their transcriptome. Replicating beta-cells upregulate hundreds of proliferation- related genes, along with many novel putative cell cycle components. Strikingly, genes involved in beta-cell functions, namely glucose sensing and insulin secretion were repressed. Further studies using single molecule RNA in situ hybridization revealed that in fact, replicating beta-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in beta-cell function.
    [Show full text]
  • Analyses of Histological and Transcriptome Differences in the Skin
    Ding et al. BMC Genomics (2019) 20:140 https://doi.org/10.1186/s12864-019-5503-x RESEARCH ARTICLE Open Access Analyses of histological and transcriptome differences in the skin of short-hair and long-hair rabbits Haisheng Ding, Huiling Zhao, Guanglong Cheng, Yongxin Yang, Xiaofei Wang, Xiaowei Zhao, Yunxia Qi and Dongwei Huang* Abstract Background: Hair fibre length is an important economic trait of rabbits in fur production. However, molecular mechanisms regulating rabbit hair growth have remained elusive. Results: Here we aimed to characterise the skin traits and gene expression profiles of short-hair and long-hair rabbits by histological and transcriptome analyses. Haematoxylin-eosin staining was performed to observe the histological structure of the skin of short-hair and long-hair rabbits. Compared to that in short-hair rabbits, a significantly longer anagen phase was observed in long-hair rabbits. In addition, by RNA sequencing, we identified 951 genes that were expressed at significantly different levels in the skin of short-hair and long-hair rabbits. Nine significantly differentially expressed genes were validated by quantitative real-time polymerase chain reaction. A gene ontology analysis revealed that epidermis development, hair follicle development, and lipid metabolic process were significantly enriched. Further, we identified potential functional genes regulating follicle development, lipid metabolic, and apoptosis as well as important pathways including extracellular matrix-receptor interaction and basal cell carcinoma pathway. Conclusions: The present study provides transcriptome evidence for the differences in hair growth between short- hair and long-hair rabbits and reveals that lipid metabolism and apoptosis might constitute major factors contributing to hair length.
    [Show full text]
  • Types I and II Keratin Intermediate Filaments
    Downloaded from http://cshperspectives.cshlp.org/ on October 10, 2021 - Published by Cold Spring Harbor Laboratory Press Types I and II Keratin Intermediate Filaments Justin T. Jacob,1 Pierre A. Coulombe,1,2 Raymond Kwan,3 and M. Bishr Omary3,4 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 2Departments of Biological Chemistry, Dermatology, and Oncology, School of Medicine, and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21205 3Departments of Molecular & Integrative Physiologyand Medicine, Universityof Michigan, Ann Arbor, Michigan 48109 4VA Ann Arbor Health Care System, Ann Arbor, Michigan 48105 Correspondence: [email protected] SUMMARY Keratins—types I and II—are the intermediate-filament-forming proteins expressed in epithe- lial cells. They are encoded by 54 evolutionarily conserved genes (28 type I, 26 type II) and regulated in a pairwise and tissue type–, differentiation-, and context-dependent manner. Here, we review how keratins serve multiple homeostatic and stress-triggered mechanical and nonmechanical functions, including maintenance of cellular integrity, regulation of cell growth and migration, and protection from apoptosis. These functions are tightly regulated by posttranslational modifications and keratin-associated proteins. Genetically determined alterations in keratin-coding sequences underlie highly penetrant and rare disorders whose pathophysiology reflects cell fragility or altered
    [Show full text]
  • Oryctolagus Cuniculus)
    Genome Gene Expression Profiling Analysis Reveals Fur Development in Rex Rabbits (Oryctolagus cuniculus) Journal: Genome Manuscript ID gen-2017-0003.R2 Manuscript Type: Article Date Submitted by the Author: 31-Jul-2017 Complete List of Authors: Zhao, Bohao; Yangzhou University Chen, Yang; Yangzhou University Yan, Xiaorong ; Yangzhou University Hao, Ye; YangzhouDraft University Zhu, Jie; Yangzhou University Weng, Qiiaoqing; Zhejiang Yuyao Xinnong Rabbit Industry Co., Ltd. Wu, Xinsheng; Yangzhou University, College of Animal Science and Technology Is the invited manuscript for consideration in a Special This submission is not invited Issue? : Keyword: Chinchilla rex rabbit, fur development, key gene, transcriptome https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 138 Genome 1 Gene Expression Profiling Analysis Reveals Fur Development in Rex 2 Rabbits ( Oryctolagus cuniculus ) 3 BoHao Zhao 1, Yang Chen 1, XiaoRong Yan 1, Ye Hao 1, Jie Zhu 1, QiaoQing Weng 2, and 4 XinSheng Wu 1* 5 1 The Key Laboratory of Animal Genetics & Breeding and Molecular Design of Jiangsu Province, 6 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. 7 China. ; 8 2 Zhejiang Yuyao Xinnong Rabbit Industry Co., Ltd., Yuyao, Zhejiang 315400, China 9 *Corresponding author E-mail: [email protected] 10 Draft 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 138 11 Abstract 12 Fur is an important economic trait in rabbits. The identification of genes that 13 influence fur development and knowledge regarding the actions of these genes 14 provides useful tools for improving fur quality. However, the mechanism of fur 15 development is unclear. To obtain candidate genes related to fur development, the 16 transcriptomes of tissues from backs and bellies of Chinchilla rex rabbits were 17 compared.
    [Show full text]