Thesis Final 140410

Total Page:16

File Type:pdf, Size:1020Kb

Thesis Final 140410 SUMO-1 mapping in the human genome and its implications for transcription control DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hui-wen Liu Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Jeffrey D. Parvin, M.D., Ph.D., Advisor Ching-shih Chen, Ph.D., Mark Parthun, Ph.D., Amanda Toland, Ph.D. Copyright by Hui-wen Liu 2014 Abstract SUMOylation, a post-translational modification with SUMO proteins covalently conjugated to a variety of proteins, regulates a range of cellular processes, including cell proliferation and maintenance of genome stability. In this study, we investigated how SUMO-1 functions as a chromatin mark on the human genome during cell cycle progression by ChIP-seq approach. Surprisingly, despite the known repressive role of SUMOylation on histones, we found that SUMO-1 localizes to the promoters of constitutively active genes involved in protein translation and proliferation during interphase. For example, ribosomal protein genes; and SUMO-1 marks on these promoters were absent during mitosis. In addition, SUMO-1 association on the promoters recruits RNAPII, and depletion of SUMO-1 leads to down regulation of those ribosomal protein genes, suggesting a positive role of SUMO-1 in gene activation. To further elucidate how SUMOylation regulates transcription process related to protein synthesis, we identified that SUMO-1 marks the promoters via the Scaffold Associated Factor B (SAFB) protein. The results showed that SAFB is SUMOylated, and depletion of SAFB caused the decrease of SUMO-1 marks on the promoters of those housekeeping genes transcribed by RNAPII. In addition, depletion of SAFB decreased the splicing of the mRNAs and disrupted the organization of Cajal body, which is important for snRNP and snoRNP biogenesis. All these findings suggested that SUMOylation plays an ii important role in the regulatory process for transcription initiation and splicing of mRNA of ribosomal protein genes. iii Dedication This dissertation is dedicated to my family. iv Acknowledgements I would like to thank my advisor, Dr. Jeffrey Parvin for supporting and guiding me throughout the past 5 years. Whenever I feel lost in my research, you are always willing to help. You have set a great example to be an outstanding researcher, mentor, and role model. I am truly grateful to have such an excellent mentor in my life. I also thank my thesis committee members, Dr. Mandy Toland, Dr. Mark Parthun, and Dr. Ching-shih Chen. Not only your time and patience, but also your invaluable feedback and discussion, have guided me on the path as a researcher. I am also thankful to have my lab-mates, Zeina, Mansi, Muhtadi, Grace, Cindy, Shweta, Derek, Alaina, Eliana, and Ian. You have been very helpful and fun to work with. I will easily miss the time we hanging out (and working in the lab, of course!). I thank my friends for all the support and suggestions, and it is always a great pleasure to hanging out with you guys. I am a person who gets homesick a lot, but you guys make me feel like home, and I really appreciate that. Last but not least, I would like to thank my family and my husband Aaron Chen, for unconditional love and support. There are times when I feel frustrated, and you are always there for me. This journey would not have been possible without all the support from everyone, and I am truly thankful for everything I have. v Vita 2008-Present…...…………….PhD Candidate, The Ohio State University, Columbus, OH 2005………... MS, Microbiology and Biochemistry, National Taiwan University, Taiwan 2003…………………...BS, Agricultural Chemistry, National Taiwan University, Taiwan Publications 1. Liu HW, Zhang J, Heine GF, Arora M, Ozer HG, Onti-Srinivasan R, Huang K, Parvin JD, Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes. Nucleic acid research. 40(20): 10172-86, 2012. 2. Arora M, Zhang J, Heine GF, Liu HW, Ozer G, Huang K, Parvin JD. Chromatin ubiquitination: a bookmark for transcription and DNA replication through mitosis. Nucleic acid research. 40(20): 10187-202, 2012. 3. Zhang J, Lu K, Xiang Y, Islam M, Kotian S, Kais Z, Lee C, Arora M, Liu HW, Parvin JD, Huang K. Weighted Frequent Gene Co-expression Network Mining to Identify Genes Involved in Genome Stability. PLOS Computational biology. 8(8): e1002656, 2012 4. Shen YF, Chen YH, Chu SY, Lin MI, Wu PY, Hsu HT, Wu CJ, Liu HW, Lin FY, Lin G, Hsu PH, Yang AS, Cheng SH, Wu YT, Wong CH, Tsai MD. E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc Natl Acad Sci USA. 108(40):16515-20, 2011 5. Chen SC, Liu HW, Lee KT, Yamakawa T. High-efficiency Agrobacterium rhizogenes-mediated transformation of sHSP18.2-GUS in Nicotiana tabacum. Plant Cell Reports. 26: 29-37, 2007. Fields of Study Major Field: Molecular, Cellular and Developmental Biology vi Table of Contents Abstract ...…...................................................................................................................... .ii Dedication......... ................................................................................................................. iv Acknowledgements ............................................................................................................. v Vita......................................................................................................................................vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: Introduction ..................................................................................................... 1 1.1 Chromatin functions in eukaryotes ........................................................................ 1 1.1.1 Chromatin structures are well organized in eukaryotes ................................... 1 1.1.2 Epigenetics and histone dynamics ................................................................... 2 1.1.3 Histone modifications ...................................................................................... 4 1.2 SUMO pathway ...................................................................................................... 9 1.2.1 Enzymes involved in SUMOylation ................................................................ 9 1.2.2 SUMO proteases ............................................................................................ 11 1.2.3 SUMO proteins .............................................................................................. 12 vii 1.2.4 SUMO-Interaction Motif ............................................................................... 13 1.3 The role of SUMOylation in chromatin remodeling ............................................ 14 1.3.1 SUMO localization on chromatin .................................................................. 14 1.3.2 SUMO modification of Histones and HDACs .............................................. 15 1.3.3 Crosstalk between histone methylation and SUMOylation ........................... 17 1.4 SUMOylation and transcription regulation .......................................................... 17 1.4.1 Transcription repression ................................................................................ 17 1.4.2 Transcription activation ................................................................................. 18 1.4.3 SUMO, transcription, and chromatin structure ............................................. 19 1.5 SUMO function in subnuclear structure ............................................................... 20 1.5.1 Polycomb bodies ............................................................................................ 20 1.5.2 PML bodies ................................................................................................... 21 1.5.3 Nucleolus and speckles .................................................................................. 22 1.6 Interactions between SUMO and mRNA biogenesis ........................................... 23 1.7 The role of SUMOylation in genome stability and tumorigenesis ....................... 24 1.7.1 SUMOylation regulates cell cycle progression ............................................. 24 1.7.2 SUMOylation regulates DNA damage response ........................................... 25 1.7.3 Deregulation of SUMO system causes tumorigenesis .................................. 25 Chapter 2: Rationale ........................................................................................................ 30 viii Chapter 3: Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes ............................................................................................ 32 3.1 Abstract ................................................................................................................ 33 3.2 Introduction .......................................................................................................... 34 3.3 Materials and Methods ........................................................................................
Recommended publications
  • In Silico Prediction of High-Resolution Hi-C Interaction Matrices
    ARTICLE https://doi.org/10.1038/s41467-019-13423-8 OPEN In silico prediction of high-resolution Hi-C interaction matrices Shilu Zhang1, Deborah Chasman 1, Sara Knaack1 & Sushmita Roy1,2* The three-dimensional (3D) organization of the genome plays an important role in gene regulation bringing distal sequence elements in 3D proximity to genes hundreds of kilobases away. Hi-C is a powerful genome-wide technique to study 3D genome organization. Owing to 1234567890():,; experimental costs, high resolution Hi-C datasets are limited to a few cell lines. Computa- tional prediction of Hi-C counts can offer a scalable and inexpensive approach to examine 3D genome organization across multiple cellular contexts. Here we present HiC-Reg, an approach to predict contact counts from one-dimensional regulatory signals. HiC-Reg pre- dictions identify topologically associating domains and significant interactions that are enri- ched for CCCTC-binding factor (CTCF) bidirectional motifs and interactions identified from complementary sources. CTCF and chromatin marks, especially repressive and elongation marks, are most important for HiC-Reg’s predictive performance. Taken together, HiC-Reg provides a powerful framework to generate high-resolution profiles of contact counts that can be used to study individual locus level interactions and higher-order organizational units of the genome. 1 Wisconsin Institute for Discovery, 330 North Orchard Street, Madison, WI 53715, USA. 2 Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53715, USA. *email: [email protected] NATURE COMMUNICATIONS | (2019) 10:5449 | https://doi.org/10.1038/s41467-019-13423-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13423-8 he three-dimensional (3D) organization of the genome has Results Temerged as an important component of the gene regulation HiC-Reg for predicting contact count using Random Forests.
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Gene Therapy in Cancer Treatment: Why Go Nano?
    pharmaceutics Review Gene Therapy in Cancer Treatment: Why Go Nano? Catarina Roma-Rodrigues 1 , Lorenzo Rivas-García 1,2 , Pedro V. Baptista 1,* and Alexandra R. Fernandes 1,* 1 UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Campus de Caparica, 2829-516 Caparica, Portugal; [email protected] (C.R.-R.); [email protected] (L.R.-G.) 2 Biomedical Research Centre, Institute of Nutrition and Food Technology, Department of Physiology, Faculty of Pharmacy, University of Granada, Avda. del Conocimiento s/n. 18071 Armilla, Granada, Spain * Correspondence: [email protected] (P.V.B.); [email protected] (A.R.F.); Tel.: +351-212-948-530 (P.V.B. & A.R.F.) Received: 29 January 2020; Accepted: 3 March 2020; Published: 5 March 2020 Abstract: The proposal of gene therapy to tackle cancer development has been instrumental for the development of novel approaches and strategies to fight this disease, but the efficacy of the proposed strategies has still fallen short of delivering the full potential of gene therapy in the clinic. Despite the plethora of gene modulation approaches, e.g., gene silencing, antisense therapy, RNA interference, gene and genome editing, finding a way to efficiently deliver these effectors to the desired cell and tissue has been a challenge. Nanomedicine has put forward several innovative platforms to overcome this obstacle. Most of these platforms rely on the application of nanoscale structures, with particular focus on nanoparticles. Herein, we review the current trends on the use of nanoparticles designed for cancer gene therapy, including inorganic, organic, or biological (e.g., exosomes) variants, in clinical development and their progress towards clinical applications.
    [Show full text]
  • 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]
  • A Small De Novo 16Q24.1 Duplication in a Woman with Severe Clinical Features
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by HAL-Université de Bretagne Occidentale A small de novo 16q24.1 duplication in a woman with severe clinical features. Sylvia Qu´em´ener-Redon,Caroline B´enech, S´everine Audebert-Bellanger, Ga¨elleFriocourt, Marc Planes, Philippe Parent, Claude F´erec To cite this version: Sylvia Qu´em´ener-Redon, Caroline B´enech, S´everine Audebert-Bellanger, Ga¨elle Friocourt, Marc Planes, et al.. A small de novo 16q24.1 duplication in a woman with severe clini- cal features.. European Journal of Medical Genetics, Elsevier, 2013, epub ahead of print. <10.1016/j.ejmg.2013.01.001>. <inserm-00788405> HAL Id: inserm-00788405 http://www.hal.inserm.fr/inserm-00788405 Submitted on 14 Feb 2013 HAL is a multi-disciplinary open access L'archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destin´eeau d´ep^otet `ala diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publi´esou non, lished or not. The documents may come from ´emanant des ´etablissements d'enseignement et de teaching and research institutions in France or recherche fran¸caisou ´etrangers,des laboratoires abroad, or from public or private research centers. publics ou priv´es. A small de novo 16q24.1 duplication in a woman with severe clinical features Sylvia Quéméner-Redon1,2, Caroline Bénech1,3, Séverine Audebert-Bellanger4, Gaëlle Friocourt1, Marc Planes4, Philippe Parent4 and Claude Férec1,2,3 1 Institut National de la Santé et de la Recherche Médicale (INSERM), UMR1078, Brest, France, 2 Laboratoire de Génétique Moléculaire et d’Histocompatibilité, Centre Hospitalier Universitaire (CHU), Hôpital Morvan, Brest, France, 3 Etablissement Français du Sang (EFS) – Bretagne, Brest, France, 4Service de Pédiatrie et de Génétique Médicale, Centre Hospitalier Universitaire de Brest, Brest, France.
    [Show full text]
  • FAM158A Polyclonal Antibody Catalog Number:23919-1-AP
    For Research Use Only FAM158A Polyclonal antibody www.ptgcn.com Catalog Number:23919-1-AP Catalog Number: GenBank Accession Number: Recommended Dilutions: Basic Information 23919-1-AP BC002491 WB 1:200-1:1000 Size: GeneID (NCBI): IHC 1:20-1:200 500 μg/ml 51016 IF 1:20-1:200 Source: Full Name: Rabbit family with sequence similarity 158, Isotype: member A IgG Calculated MW: Purification Method: 208 aa, 23 kDa Antigen Affinity purified Observed MW: Immunogen Catalog Number: 23 kDa AG20744 Applications Tested Applications: Positive Controls: IF, IHC, WB,ELISA WB : HEK-293 cells; Species Specificity: IHC : human pancreas tissue; human Note-IHC: suggested antigen retrieval with IF : HEK-293 cells; TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0 UPF0172 protein FAM158A, also known as c14orf122 or CGI112, is a protein that in humans is encoded by the Background Information FAM158A gene located on chromosome 14q11.2. Human FAM158A and its paralogs in other species are part of the uncharacterized protein family UPF0172 family, which is a subset of the JAB1/Mov34/MPN/PAD-1 ubiquitin protease protein family. The MPN superfamily contributes to ubiquitination and de-ubiquitination activity within the cell. The UPF0172 subset no longer has a functional ubiquitination domain and the function is uncharacterized. Fam158a is nearly ubiquitously expressed throughout the human body. The homolog in mice also shows expression throughout the entire body. Several micro-arrays demonstrate the variable expression of Fam158a in response to other factors and in various cancer types. None of this information gives any indication of a specific function but the wide expression of the gene and its high conservation indicate that Fam158a plays an important role in cellular function.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [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]
  • 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]
  • 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]
  • Loss of Fam60a, a Sin3a Subunit, Results in Embryonic Lethality and Is Associated with Aberrant Methylation at a Subset of Gene
    RESEARCH ARTICLE Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters Ryo Nabeshima1,2, Osamu Nishimura3,4, Takako Maeda1, Natsumi Shimizu2, Takahiro Ide2, Kenta Yashiro1†, Yasuo Sakai1, Chikara Meno1, Mitsutaka Kadota3,4, Hidetaka Shiratori1†, Shigehiro Kuraku3,4*, Hiroshi Hamada1,2* 1Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan; 2Laboratory for Organismal Patterning, RIKEN Center for Developmental Biology, Kobe, Japan; 3Phyloinformatics Unit, RIKEN Center for Life Science Technologies, Kobe, Japan; 4Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Abstract We have examined the role of Fam60a, a gene highly expressed in embryonic stem cells, in mouse development. Fam60a interacts with components of the Sin3a-Hdac transcriptional corepressor complex, and most Fam60a–/– embryos manifest hypoplasia of visceral organs and die in utero. Fam60a is recruited to the promoter regions of a subset of genes, with the expression of these genes being either up- or down-regulated in Fam60a–/– embryos. The DNA methylation level of the Fam60a target gene Adhfe1 is maintained at embryonic day (E) 7.5 but markedly reduced at –/– *For correspondence: E9.5 in Fam60a embryos, suggesting that DNA demethylation is enhanced in the mutant. [email protected] (SK); Examination of genome-wide DNA methylation identified several differentially methylated regions, [email protected] (HH) which were preferentially hypomethylated, in Fam60a–/– embryos. Our data suggest that Fam60a is †These authors contributed required for proper embryogenesis, at least in part as a result of its regulation of DNA methylation equally to this work at specific gene promoters.
    [Show full text]
  • Mutation in the COX4I1 Gene Is Associated with Short Stature, Poor Weight Gain and Increased Chromosomal Breaks, Simulating Fanconi Anemia
    European Journal of Human Genetics (2017) 25, 1142–1146 & 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1018-4813/17 www.nature.com/ejhg ARTICLE Mutation in the COX4I1 gene is associated with short stature, poor weight gain and increased chromosomal breaks, simulating Fanconi anemia Bassam Abu-Libdeh*,1,4, Liza Douiev2,3,4, Sarah Amro1, Maher Shahrour1, Asaf Ta-Shma2, Chaya Miller2,3, Orly Elpeleg2 and Ann Saada*,2,3 We describe a novel autosomal recessive form of mitochondrial disease in a child with short stature, poor weight gain, and mild dysmorphic features with highly suspected Fanconi anemia due to a mutation in COX4I1 gene. Whole Exome Sequencing was performed then followed by Sanger confirmation, identified a K101N mutation in COX4I1, segregating with the disease. This nuclear gene encodes the common isoform of cytochrome c oxidase (COX) subunit 4 (COX 4-1), an integral regulatory part of COX (respiratory chain complex IV) the terminal electron acceptor of the mitochondrial respiratory chain. The patient’s fibroblasts disclosed decreased COX activity, impaired ATP production, elevated ROS production, decreased expression of COX4I1 mRNA and undetectable (COX4) protein. COX activity and ATP production were restored by lentiviral transfection with the wild-type gene. Our results demonstrate the first human mutation in the COX4I1 gene linked to diseases and confirm its role in the pathogenesis. Thus COX4I1 mutations should be considered in any patient with features suggestive of this diagnosis. European Journal of Human Genetics (2017) 25, 1142–1146; doi:10.1038/ejhg.2017.112; published online 2 August 2017 INTRODUCTION normal vaginal delivery with birth weight of 2.8 kg after an uneventful Mitochondrial cytochrome c oxidase (COX, complex IV) is the terminal pregnancy.
    [Show full text]