DOCSLIB.ORG

A Cell Line P53 Mutation Type UM

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Cell Line P53 Mutation Type UM A Cell line p53 mutation Type UM-SCC 1 wt UM-SCC5 Exon 5, 157 GTC --> TTC Missense mutation by transversion (Valine --> Phenylalanine UM-SCC6 wt UM-SCC9 wt UM-SCC11A wt UM-SCC11B Exon 7, 242 TGC --> TCC Missense mutation by transversion (Cysteine --> Serine) UM-SCC22A Exon 6, 220 TAT --> TGT Missense mutation by transition (Tyrosine --> Cysteine) UM-SCC22B Exon 6, 220 TAT --> TGT Missense mutation by transition (Tyrosine --> Cysteine) UM-SCC38 Exon 5, 132 AAG --> AAT Missense mutation by transversion (Lysine --> Asparagine) UM-SCC46 Exon 8, 278 CCT --> CGT Missense mutation by transversion (Proline --> Alanine) B 1 Supplementary Methods Cell Lines and Cell Culture A panel of ten established HNSCC cell lines from the University of Michigan series (UM-SCC) was obtained from Dr. T. E. Carey at the University of Michigan, Ann Arbor, MI. The UM-SCC cell lines were derived from eight patients with SCC of the upper aerodigestive tract (supplemental Table 1). Patient age at tumor diagnosis ranged from 37 to 72 years. The cell lines selected were obtained from patients with stage I-IV tumors, distributed among oral, pharyngeal and laryngeal sites. All the patients had aggressive disease, with early recurrence and death within two years of therapy. Cell lines established from single isolates of a patient specimen are designated by a numeric designation, and where isolates from two time points or anatomical sites were obtained, the designation includes an alphabetical suffix (i.e., "A" or "B"). The cell lines were maintained in Eagle's minimal essential media supplemented with 10% fetal bovine serum and penicillin/streptomycin. Human normal keratinocytes (HKC) were obtained from four individuals (Cascade Biologics Inc., Portland, OR), and were maintained in keratinocyte serum-free medium 154CF containing 0.08mM of calcium chloride and supplemented with human keratinocyte growth supplements (HKGS). The final concentration of HKGS in the complete medium are: 0.2% (v/v) of bovine pituitary extract (BPE), 5 μg/ml of bovine insulin, 0.18 μg/ml of hydrocortisone and 5 μg/ml of transferrin, and 0.2ng/ml of human recombinant EGF. All HKC were used within five passages. The cultures were incubated o in a humidified cell culture incubator at 37 C and 5 % of CO2. 1 RNA isolation, Labeling of cDNA, Microarray Hybridization and Image Collection Total RNA was isolated from cultured UM-SCC cells and primary keratinocytes at their log growth phase using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and stored at -80°C. To make fluorescence-labeled target cDNA by reverse transcription, 50-100 µg total RNA was incubated in a cocktail containing Cy3 or Cy5-dUTP (Amersham Pharmacia Biotech Inc, Piscataway, NJ) and SuperScript II RT (Gibco BRL Technologies, Inc, Gaithersburg, MD). Labeled targets were purified using a Microcon column (Millipore, Bedford, MA). The appropriate Cy3 and Cy5 targets were combined, along with 2 µl (20 µg) mouse COT-1 DNA, 1 µl (8-10 µg) polyA, 2.6 µl 20X SSC and 0.45 µl 10% SDS in a final volume of 15 µl. After denaturation, labeled targets were added to processed 24K human array chips developed by National Human Genome Research Institute (NHGRI, Bethesda, MD), which were then placed in moisturized chambers and incubated overnight (10-16 hours) @ 65oC. The next day, slides were washed for 1 minute in 1X SSC, 1 minute in 0.2X SSC, 10 seconds in 0.05X SSC, then spin dried. Each hybridization condition was repeated at least three times. Fluorescence images were captured by GenePix 4000 microarray scanner (Axon Instruments, Union City, CA) with GenePix Pro software from the same company. The PMT voltage for both channels was adjusted between 750V to 890V range to give same overall intensity between both channels and the raw image was saved in TIFF format. Data Collection, Filtering and Quality Control The 24K cDNA microarray chips containing a total of 23220 spots, which includes known genes, ESTs, hypothetical genes and control spots were obtained from National 2 Human Genome Research Institute (Bethesda, MD). The study was focused on the 12270 known gene set in the array. Raw images were analyzed using the ArraySuite 2.1 extensions (Chen et al., 1997) in the IPLab program (Scanalytics, Inc., Fairfax, VA) to calibrate relative ratios and develop confidence intervals for their significance (Chen et al., 1997). Fluorescence intensities for both dyes (Cy3 and Cy5) and local background subtracted values for individual spots were obtained. Spots were filtered based on RQuality score provided in the software and visually checked. The software extracts information regarding spot quality and assigns an RQuality score to each ratio measurement, with 0 as the lowest measurement quality and 1 as the highest measurement quality. Any spots with less than 0.5 were excluded. For each spot, the calibrated ratio (the median of the pixel-by-pixel ratios of pixel intensities that have the median background intensity subtracted) was used in subsequent analysis. Expression outliners were determined using +3.0 SD and a 99% confidence interval as cutoff. The resulting gene list was further filtered to exclude EST and hypothetical clones and contain expression value from at least three human normal keratinocyte and seven tumor cell lines, resulting in a total of 9273 genes. Data Normalization, Calculation and Statistical Analysis PCA analysis was performed on normalized data from Partek Pro 5.1 software (Partek Inc., St. Louis, MO). Hierarchical clustering was carried out on gene list where the average ratio difference of ten tumor cell lines to normal keratinocytes were more than 2- fold and had a t-test score at P < 0.01. The resulting 969 genes were analyzed by cluster software based on clustering algorithm of Eisen et al (Eisen et al., 1998), and the 3 expression maps of clustered genes were visualized using Java Treeview software (Saldanha et al., 2004). Hierarchical agglomerative clustering was performed by BRB- ArrayTools developed by Dr. Richard Simon and Amy Peng (available at http://linus.nci.nih.gov/BRB-ArrayTools.html). It was applied to the normalized log ratios by using both compact linkage and average linkage and both Euclidean and one minus Pearson correlation distance metrics. Normalized log ratios were median-centered within each gene for all of the cluster analyses. The clustering results obtained by using compact linkage with one minus Pearson correlation distance applied to the 969 probe elements appeared by visual inspection to yield the most distinctive clusters, where attention is focused on the genes responsible for segregating members in sub_A and sub_B by PCA analysis in Figure 1. The presence of significant clustering was also assessed by applying the global test of clustering proposed by McShane et al. to confirm the findings (McShane et al., 2002). Gene Ontology analysis was carried out by Database for Annotation, Visualization, and Integrated Discovery (DAVID); a web-based, client/server application that allows users to access a relational database of functional annotation (NIAID, NIH, Bethesda, MD). Functional annotations are derived primarily from LocusLink at the National Center for Biotechnology Information (NCBI). Annotation pedigrees are provided via direct links to the primary sources of annotation, which also provide additional gene specific information. To analyze the differential expression between the tumor and normal group uniquely, statistical analysis of the data from the hierarchical clustering was performed using BRB-Array Tools, where a class comparison tool was used. Univariate F-tests (BRB-Array Tools 2.0) were performed on these in the two 4 classes. This tool also computes a global permutation test that excludes genes that differ significantly due to chance alone. Genes that showed significant differences (P <0.001) after 2000 permutations were designated as differentially expressed genes. A mixed-model-based F test was used to analyze differential gene expression among three groups of cells, HKC, the sub-group 1 or 2 of UM-SCC cells (Tempelman, 2005; Wolfinger et al., 2001). Restricted maximum likelihood (REML) was used as the estimation method. The F test was performed in mixed model using SAS 9.1 program (SAS Institute Inc. Cary, NC). P < 0.05 was designated as significant difference between two groups. Real Time RT-PCR Gene expression profiles generated from microarray were confirmed by real time quantitative RT-PCR. cDNA synthesis using total RNA was performed by using the High-Capacity cDNA Archive Kit, and Real time quantitative RT-PCR was performed using the Assays-on-Demand™ Gene Expression kits for specific genes and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocols. PCR was performed together with endogenous control using eukaryotic 18S ribosomal RNA (18S). The amplification was carried out by addition of 30 ng of cDNA in 30 μl of PCR reaction mix (1.5 μl of 20X assay mix and 15 μl of 2X TaqMan Universal Master Mix). Amplification conditions were as follows: activation of enzymes for 2 min at 50oC and 10 min at 95oC, followed by 40 cycles at 15 sec at 95oC and 1 min at 60oC. Relative quantitation of the expression was calculated by normalizing the target gene signals with the 18S endogenous control. An arbitrary unit was calculated after setting CT equaling to 40 as undetectable 5 expression level and used for normalization. In Situ Hybridization Human larynx tumors and normal mucosa were obtained from the Cooperative Human Tissue Network. Specimens were sectioned at 10 µm, mounted onto Poly-L-Lysine microscope slides (Polysciences, Inc, Warrington, PA), and the UM-SCC 11A xenograft tumors were used as a positive control (Ricker et al., 2004). The cRNA probes were synthesized from the following cDNA clones: BAG2 (659 base pairs, Invitrogen); CCNB2 (1315 base pairs, Open Biosystems, Huntsville, AL); PCNA (154 base pairs); and CCND1 (560 base pairs, from Dr.
Load more

Recommended publications
  • Table S1. 49 Histone Variants Were Identified with High Sequence Coverage Through LC-MS/MS Analysis Electronic Supplementary
    Electronic Supplementary Material (ESI) for Analytical Methods. This journal is © The Royal Society of Chemistry 2020 Table S1. 49 histone variants were identified with high sequence coverage through LC-MS/MS analysis Sequence Uniprot IDs Protein Name Protein Description Coverage Ratio E2+/E2- RSD P07305 H10_HUMAN 67.5% Histone H1.0 OS=Homo sapiens GN=H1F0 PE=1 SV=3 4.85 23.3% Histone H1.1 OS=Homo sapiens GN=HIST1H1A PE=1 Q02539 H11_HUMAN 74.4% SV=3 0.35 92.6% Histone H1.2 OS=Homo sapiens GN=HIST1H1C PE=1 P16403 H12_HUMAN 67.1% SV=2 0.73 80.6% Histone H1.3 OS=Homo sapiens GN=HIST1H1D PE=1 P16402 H13_HUMAN 63.8% SV=2 0.75 77.7% Histone H1.4 OS=Homo sapiens GN=HIST1H1E PE=1 P10412 H14_HUMAN 69.0% SV=2 0.70 80.3% Histone H1.5 OS=Homo sapiens GN=HIST1H1B PE=1 P16401 H15_HUMAN 79.6% SV=3 0.29 98.3% Testis-specific H1 histone OS=Homo sapiens GN=H1FNT Q75WM6 H1FNT_HUMAN 7.8% \ \ PE=2 SV=3 Histone H1oo OS=Homo sapiens GN=H1FOO PE=2 Q8IZA3 H1FOO_HUMAN 5.2% \ \ SV=1 Histone H1t OS=Homo sapiens GN=HIST1H1T PE=2 P22492 H1T_HUMAN 31.4% SV=4 1.42 65.0% Q92522 H1X_HUMAN 82.6% Histone H1x OS=Homo sapiens GN=H1FX PE=1 SV=1 1.15 33.2% Histone H2A type 1 OS=Homo sapiens GN=HIST1H2AG P0C0S8 H2A1_HUMAN 99.2% PE=1 SV=2 0.57 26.8% Q96QV6 H2A1A_HUMAN 58.0% Histone H2A type 1-A OS=Homo sapiens 0.90 11.2% GN=HIST1H2AA PE=1 SV=3 Histone H2A type 1-B/E OS=Homo sapiens P04908 H2A1B_HUMAN 99.2% GN=HIST1H2AB PE=1 SV=2 0.92 30.2% Histone H2A type 1-C OS=Homo sapiens Q93077 H2A1C_HUMAN 100.0% GN=HIST1H2AC PE=1 SV=3 0.76 27.6% Histone H2A type 1-D OS=Homo sapiens P20671
    [Show full text]
  • Propranolol-Mediated Attenuation of MMP-9 Excretion in Infants with Hemangiomas
    Supplementary Online Content Thaivalappil S, Bauman N, Saieg A, Movius E, Brown KJ, Preciado D. Propranolol-mediated attenuation of MMP-9 excretion in infants with hemangiomas. JAMA Otolaryngol Head Neck Surg. doi:10.1001/jamaoto.2013.4773 eTable. List of All of the Proteins Identified by Proteomics This supplementary material has been provided by the authors to give readers additional information about their work. © 2013 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 eTable. List of All of the Proteins Identified by Proteomics Protein Name Prop 12 mo/4 Pred 12 mo/4 Δ Prop to Pred mo mo Myeloperoxidase OS=Homo sapiens GN=MPO 26.00 143.00 ‐117.00 Lactotransferrin OS=Homo sapiens GN=LTF 114.00 205.50 ‐91.50 Matrix metalloproteinase‐9 OS=Homo sapiens GN=MMP9 5.00 36.00 ‐31.00 Neutrophil elastase OS=Homo sapiens GN=ELANE 24.00 48.00 ‐24.00 Bleomycin hydrolase OS=Homo sapiens GN=BLMH 3.00 25.00 ‐22.00 CAP7_HUMAN Azurocidin OS=Homo sapiens GN=AZU1 PE=1 SV=3 4.00 26.00 ‐22.00 S10A8_HUMAN Protein S100‐A8 OS=Homo sapiens GN=S100A8 PE=1 14.67 30.50 ‐15.83 SV=1 IL1F9_HUMAN Interleukin‐1 family member 9 OS=Homo sapiens 1.00 15.00 ‐14.00 GN=IL1F9 PE=1 SV=1 MUC5B_HUMAN Mucin‐5B OS=Homo sapiens GN=MUC5B PE=1 SV=3 2.00 14.00 ‐12.00 MUC4_HUMAN Mucin‐4 OS=Homo sapiens GN=MUC4 PE=1 SV=3 1.00 12.00 ‐11.00 HRG_HUMAN Histidine‐rich glycoprotein OS=Homo sapiens GN=HRG 1.00 12.00 ‐11.00 PE=1 SV=1 TKT_HUMAN Transketolase OS=Homo sapiens GN=TKT PE=1 SV=3 17.00 28.00 ‐11.00 CATG_HUMAN Cathepsin G OS=Homo
    [Show full text]
  • Herpes Simplex Virus Blocks Host Transcription Termination Via the Bimodal Activities of ICP27
    ARTICLE https://doi.org/10.1038/s41467-019-14109-x OPEN Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27 Xiuye Wang 1, Thomas Hennig2, Adam W. Whisnant 2, Florian Erhard 2, Bhupesh K. Prusty 2, Caroline C. Friedel 3, Elmira Forouzmand4,5, William Hu1, Luke Erber 6, Yue Chen6, Rozanne M. Sandri-Goldin 1*, Lars Dölken 2,7* & Yongsheng Shi1* Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause 1234567890():,; widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3’ processing factor CPSF. It thereby induces the assembly of a dead-end 3’ processing complex, blocking mRNA 3’ cleavage. Remarkably, ICP27 also acts as a sequence- dependent activator of mRNA 3’ processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer. 1 Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA. 2 Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany. 3 Institute of Informatics, Ludwig-Maximilians-Universität München, München, Germany. 4 Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, CA 92697, USA.
    [Show full text]
  • Environmental Influences on Endothelial Gene Expression
    ENDOTHELIAL CELL GENE EXPRESSION John Matthew Jeff Herbert Supervisors: Prof. Roy Bicknell and Dr. Victoria Heath PhD thesis University of Birmingham August 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti -angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community.
    [Show full text]
  • HIST1H2BN (H2BC15) (NM 003520) Human Tagged ORF Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC224109L3 HIST1H2BN (H2BC15) (NM_003520) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: HIST1H2BN (H2BC15) (NM_003520) Human Tagged ORF Clone Tag: Myc-DDK Symbol: H2BC15 Synonyms: H2B/d; H2BFD; HIST1H2BN Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092) E. coli Selection: Chloramphenicol (34 ug/mL) Cell Selection: Puromycin ORF Nucleotide The ORF insert of this clone is exactly the same as(RC224109). Sequence: Restriction Sites: SgfI-MluI Cloning Scheme: ACCN: NM_003520 ORF Size: 378 bp This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 HIST1H2BN (H2BC15) (NM_003520) Human Tagged ORF Clone – RC224109L3 OTI Disclaimer: The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. More info OTI Annotation: This clone was engineered to express the complete ORF with an expression tag. Expression varies depending on the nature of the gene. RefSeq: NM_003520.3 RefSeq Size: 449 bp RefSeq ORF: 381 bp Locus ID: 8341 UniProt ID: Q99877, A0A024RCJ9 Protein Pathways: Systemic lupus erythematosus MW: 13.9 kDa Gene Summary: Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes.
    [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]
  • Steroid-Dependent Regulation of the Oviduct: a Cross-Species Transcriptomal Analysis
    University of Kentucky UKnowledge Theses and Dissertations--Animal and Food Sciences Animal and Food Sciences 2015 Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis Katheryn L. Cerny University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Cerny, Katheryn L., "Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis" (2015). Theses and Dissertations--Animal and Food Sciences. 49. https://uknowledge.uky.edu/animalsci_etds/49 This Doctoral Dissertation is brought to you for free and open access by the Animal and Food Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Animal and Food Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known.
    [Show full text]
  • DNA Methylation Changes in Down Syndrome Derived Neural Ipscs Uncover Co-Dysregulation of ZNF and HOX3 Families of Transcription
    Laan et al. Clinical Epigenetics (2020) 12:9 https://doi.org/10.1186/s13148-019-0803-1 RESEARCH Open Access DNA methylation changes in Down syndrome derived neural iPSCs uncover co- dysregulation of ZNF and HOX3 families of transcription factors Loora Laan1†, Joakim Klar1†, Maria Sobol1, Jan Hoeber1, Mansoureh Shahsavani2, Malin Kele2, Ambrin Fatima1, Muhammad Zakaria1, Göran Annerén1, Anna Falk2, Jens Schuster1 and Niklas Dahl1* Abstract Background: Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages. Results: Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling. Conclusions: The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.
    [Show full text]
  • Supplement 1 Microarray Studies
    EASE Categories Significantly Enriched in vs MG vs vs MGC4-2 Pt1-C vs C4-2 Pt1-C UP-Regulated Genes MG System Gene Category EASE Global MGRWV Pt1-N RWV Pt1-N Score FDR GO Molecular Extracellular matrix cellular construction 0.0008 0 110 genes up- Function Interpro EGF-like domain 0.0009 0 regulated GO Molecular Oxidoreductase activity\ acting on single dono 0.0015 0 Function GO Molecular Calcium ion binding 0.0018 0 Function Interpro Laminin-G domain 0.0025 0 GO Biological Process Cell Adhesion 0.0045 0 Interpro Collagen Triple helix repeat 0.0047 0 KEGG pathway Complement and coagulation cascades 0.0053 0 KEGG pathway Immune System – Homo sapiens 0.0053 0 Interpro Fibrillar collagen C-terminal domain 0.0062 0 Interpro Calcium-binding EGF-like domain 0.0077 0 GO Molecular Cell adhesion molecule activity 0.0105 0 Function EASE Categories Significantly Enriched in Down-Regulated Genes System Gene Category EASE Global Score FDR GO Biological Process Copper ion homeostasis 2.5E-09 0 Interpro Metallothionein 6.1E-08 0 Interpro Vertebrate metallothionein, Family 1 6.1E-08 0 GO Biological Process Transition metal ion homeostasis 8.5E-08 0 GO Biological Process Heavy metal sensitivity/resistance 1.9E-07 0 GO Biological Process Di-, tri-valent inorganic cation homeostasis 6.3E-07 0 GO Biological Process Metal ion homeostasis 6.3E-07 0 GO Biological Process Cation homeostasis 2.1E-06 0 GO Biological Process Cell ion homeostasis 2.1E-06 0 GO Biological Process Ion homeostasis 2.1E-06 0 GO Molecular Helicase activity 2.3E-06 0 Function GO Biological
    [Show full text]
  • Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights Into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle
    animals Article Integrating Single-Step GWAS and Bipartite Networks Reconstruction Provides Novel Insights into Yearling Weight and Carcass Traits in Hanwoo Beef Cattle Masoumeh Naserkheil 1 , Abolfazl Bahrami 1 , Deukhwan Lee 2,* and Hossein Mehrban 3 1 Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj 77871-31587, Iran; [email protected] (M.N.); [email protected] (A.B.) 2 Department of Animal Life and Environment Sciences, Hankyong National University, Jungang-ro 327, Anseong-si, Gyeonggi-do 17579, Korea 3 Department of Animal Science, Shahrekord University, Shahrekord 88186-34141, Iran; [email protected] * Correspondence: [email protected]; Tel.: +82-31-670-5091 Received: 25 August 2020; Accepted: 6 October 2020; Published: 9 October 2020 Simple Summary: Hanwoo is an indigenous cattle breed in Korea and popular for meat production owing to its rapid growth and high-quality meat. Its yearling weight and carcass traits (backfat thickness, carcass weight, eye muscle area, and marbling score) are economically important for the selection of young and proven bulls. In recent decades, the advent of high throughput genotyping technologies has made it possible to perform genome-wide association studies (GWAS) for the detection of genomic regions associated with traits of economic interest in different species. In this study, we conducted a weighted single-step genome-wide association study which combines all genotypes, phenotypes and pedigree data in one step (ssGBLUP). It allows for the use of all SNPs simultaneously along with all phenotypes from genotyped and ungenotyped animals. Our results revealed 33 relevant genomic regions related to the traits of interest.
    [Show full text]
  • Analysis of Mouse Keratin 6A Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Speci®C Expression by a Keratin 6A Minigene
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Speci®c Expression by a Keratin 6a Minigene Donna Mahony, Seetha Karunaratne, Graham Cam,* and Joseph A. Rothnagel Department of Biochemistry and the Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, and *Division of Animal Production, CSIRO, Blacktown, New South Wales, Australia The analysis of keratin 6 expression is complicated atin 6 expressing tissues, including the hair follicle, by the presence of multiple isoforms that are tongue, footpad, and nail bed, showing that both expressed constitutively in a number of internal stra- transgenes retained keratinocyte-speci®c expression. ti®ed epithelia, in palmoplantar epidermis, and in Quantitative analysis of b-galactosidase activity veri- the companion cell layer of the hair follicle. In addi- ®ed that both the 1.3 and 0.12 kb keratin 6a promo- tion, keratin 6 expression is inducible in interfollicu- ter constructs produced similar levels of the reporter. lar epidermis and the outer root sheath of the Notably, bothconstructs were constitutively follicle, in response to wounding stimuli, phorbol expressed in the outer root sheath and interfollicular esters, or retinoic acid. In order to establishthecriti- epidermis in the absence of any activating stimulus, cal regions involved in the regulation of keratin 6a suggesting that they lack the regulatory elements (the dominant isoform in mice), we generated trans- that normally silence transcription in these cells. This genic mice withtwo different-sized mouse keratin 6a study has revealed that a keratin 6a minigene con- constructs containing either 1.3 kb or 0.12 kb of 5¢ tains critical cis elements that mediate tissue-speci®c ¯anking sequence linked to the lacZ reporter gene.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]