Skip to main content
Advertisement
  • Other Publications
    • EMBO Press
    • EMBO Molecular Medicine (Home)
    • The EMBO Journal
    • EMBO reports
    • Molecular Systems Biology
    • Life Science Alliance
Login

   

Search

Advanced Search

Journal

  • Home
  • Latest Online
  • Current Issue
  • Archive
  • Article Collections
  • Subject Collections

Authors & Referees

  • Submit
  • Author Guidelines
  • Aims & Scope
  • Editors & Board
  • Transparent Process
  • Referee Guidelines
  • Bibliometrics
  • Open Access

Info

  • E-Mail Editorial Office
  • Alerts
  • RSS Feeds
  • Reprints & Permissions
  • Advertise & Sponsor
  • Media Partners
  • News & Press
  • Customer Service
  • Home
  • Latest Online

Open Access

Transparent Process

Report

A complex genomic locus drives mtDNA replicase POLG expression to its disease‐related nervous system regions

Joni Nikkanen, Juan Cruz Landoni, Diego Balboa, Maarja Haugas, Juha Partanen, Anders Paetau, Pirjo Isohanni, Virginia Brilhante, View ORCID ProfileAnu Suomalainen
DOI 10.15252/emmm.201707993 | Published online 06.11.2017
EMBO Molecular Medicine (2017) e201707993
Joni Nikkanen
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Juan Cruz Landoni
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Diego Balboa
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maarja Haugas
Department of Biosciences, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Juha Partanen
Department of Biosciences, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anders Paetau
HUSLAB and Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Pirjo Isohanni
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland Department of Pediatric Neurology, Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Virginia Brilhante
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anu Suomalainen
Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland Department of Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Neuroscience Center, University of Helsinki, Helsinki, Finland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site

Author Affiliations

  1. Joni Nikkanen1,
  2. Juan Cruz Landoni1,
  3. Diego Balboa1,2,
  4. Maarja Haugas3,
  5. Juha Partanen3,
  6. Anders Paetau4,
  7. Pirjo Isohanni1,5,
  8. Virginia Brilhante1 and
  9. Anu Suomalainen (anu.wartiovaara{at}helsinki.fi)*,1,6,7
  1. 1Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland
  2. 2Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland
  3. 3Department of Biosciences, University of Helsinki, Helsinki, Finland
  4. 4HUSLAB and Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  5. 5Department of Pediatric Neurology, Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  6. 6Department of Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  7. 7Neuroscience Center, University of Helsinki, Helsinki, Finland
  1. ↵*Corresponding author. Tel: +358 9 4717 1965; E‐mail: anu.wartiovaara{at}helsinki.fi
View Abstract
  • Article
  • Figures & Data
  • Transparent Process
Loading

Article Figures & Data

Figures

  • Supplementary Materials
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1. Three distant enhancers drive POLG expression in the central nervous system

    • A. (Left) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter. (Right) Luciferase expression with mutated CAAT boxes in POLG promoter. The error bars indicate standard deviation in three biological replicates. AU; arbitrary units.

    • B. POLG promoter sequence showing predicted CAAT boxes. Red shows disrupted nucleotides by site‐directed mutagenesis in (A).

    • C. Expression pattern driven by 500‐bp Polg proximal promoter in E12.5 mouse embryo. LacZ‐positive cell populations in the developing midbrain (black arrows), dorsal root ganglia (gray arrows), and motoneuron progenitors (arrowhead) of the neural tube. Somites show some expression (white arrow). Sectioning planes indicated by red lines. Scale bars 100 μm.

    • D. Prediction of enhancers in the genomic loci of mtDNA maintenance genes, 100 kb upstream of the analyzed gene, found in human–mouse and human–rat comparisons. EEL score for individual elements: red bars. Protein‐coding genes upstream from mtDNA maintenance genes are shown with black lines under each locus (picture not in scale). POLG shows three highly conserved elements in a gene‐poor region. TWNK shows one distant element, with several genes between the element and the gene, suggesting the element not be a specific regulator for TWNK. TWNK, Twinkle mtDNA helicase; POLG, DNA polymerase gamma, catalytic subunit; POLG2, DNA polymerase gamma, accessory subunit; SSBP1, single‐stranded DNA‐binding protein 1; TFAM, mitochondrial transcription factor A.

    • E–G POLG enhancer elements are functional in vivo and drive expression in E12.5 transgenic mice. Sectioning planes indicated by red lines. Black line in (E) marks the expression in the dorsal neural tube, whereas rostral and caudal regions lack dorsal expression (black arrows).

    • H–J Neural tube lacZ expression driven by (H) EE1: immature neuronal precursors (gray arrow), (I) EE2: dorsal neural tube (gray arrow) and dorsal root ganglia (black arrow), and (J) EE3: dorsal neural tube (gray arrow). Scale bars 100 μm.

    • K–M Midbrain lacZ expression, driven by (K) EE1, (L) EE2, and (M) EE3. Black arrow indicates neuronal population from EE1 embryo stained in (N). Scale bars 100 μm.

    • N. EE1 drives expression in oculomotor complex; immunofluorescent costaining with antibodies against ISL1/2 (motoneurons of oculomotor complex; red) and β‐Gal (green). LacZ staining of the region in (K); black arrow. Scale bars 50 μm.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2. Polg enhancer EE2 drives expression in adult brain, and EE2 and 3 in sensory interneurons of the adult spinal cord

    • A. Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus.

    • B. EE2‐driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 μm.

    • C–G Adult mouse spinal cord, (C) EE2 and (D, left panel in E) EE3 expression pattern. Dorsal horns, laminae I‐III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicate the region of dorsal horn shown in (D), (F), and (G). Insets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (gray arrow), respectively. Scale bars: 100 μm unless indicated otherwise. Calbindin (red) and β‐Gal (green) expression in interneurons of the dorsal horn laminae I‐III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 μm.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3. LINC00925 regulates POLG expression

    1. The number of distal DNase I hypersensitive sites (DHSs) of mtDNA maintenance genes and genes in the genomic POLG locus correlating > 0.85 with target promoter DHS across 125 cell lines. TWNK, Twinkle protein; POLG, DNA polymerase gamma, catalytic subunit; POLG2, DNA polymerase gamma, accessory subunit; SSBP1, single‐stranded DNA‐binding protein 1; TFAM, mitochondrial transcription factor A; FANCI, Fanconi anemia group I protein; RHCG, ammonium transporter Rh type C; TICRR, Treslin.

    2. Distribution histogram of DHSs for mtDNA maintenance genes.

    3. Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.

    4. Genes surrounding LINC00925.

    5. Conservation of regulatory elements of POLG genomic locus in species: LINC00925, POLG EEs, MIR9‐3. PanTro, Pan troglodytes; Mm, Mus musculus; MonDom, Monodelphis domestica; Xen, Xenopus levis. Adapted from https://rvista.dcode.org.

    6. Expression of LINC00925 and POLG in different human tissues and cell types. Quantitative PCR amplification of cDNA. iPS, induced pluripotent stem cell; SH5Y, neuroblastoma line; HepG2, liver hepatocellular carcinoma line; U2OS, bone osteosarcoma line. Error bars indicate standard deviation in three technical replicates. AU; arbitrary units.

    7. Polg and long non‐coding RNA Ai854517 (mouse homolog of human LINC00925) correlate tightly in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).

    8. Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; M, cerebellar molecular layer; PC, Purkinje cell layer; GC, granular cell layer. Scale bars: hippocampus 760 μm, cerebellum 300 μm.

    9. Expression levels of MIR9‐3 and Ai854517 correlate in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).

    10. Predicted targets of MIR9. Six targets were predicted by all prediction programs, TargetScan, PicTar, miRDB, and PITA: low‐density lipoprotein receptor adaptor protein‐1, LDLRAP1; methylene tetrahydrofolate dehydrogenase‐2, MTHFD2; follistatin‐like 1, FSTL1; capping actin protein of muscle Z‐line alpha‐subunit 1, CAPZA1; PR/SET domain 1, PRDM1; paired related homeobox 1, PRRX1. One cut homeobox 1 and 2 (ONECUT1 and ONECUT2) and nuclear receptor subfamily 2 group E member 1 (NR2E1) are recently discovered MIR9 targets.

    11. RNA expressions of MIR9, NR2E2, LDLDRAP1, and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre‐MIR9 or scrambled RNA. Shown is mean with standard error of the mean of 5 analyzed replicates. Statistical testing was performed using one‐way ANOVA with Dunnett's correction for multiple comparisons. AU; arbitrary units.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4. POLG defects cause degeneration of enhancer‐active regions of CNS

    1. Spinal cord (C8 level) of MIRAS patient, neurofilament staining. Dorsal columns, especially the gracile, show pallor (dashed line with star); motoneurons in the anterior horns are preserved (dashed line with black arrow). Scale bar 1 mm.

    2. Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin–eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 μm.

Supplementary Materials

  • Figures
  • Appendix [emmm201707993-sup-0001-Appendix.pdf]

Next Article in this Issue
Back to top

  • PDF
  • Share
  • Export
  • Print
Loading

PDF

Review Process

In this Issue
Volume 10, Issue 4
01 April 2018 | pp -
EMBO Molecular Medicine: 10 (4)
About the cover
Alert me when this article is cited
Alert me if a correction is posted

Article

  • Article
    • Abstract
    • Synopsis
    • Introduction
    • Results and Discussion
    • Materials and Methods
    • Author contributions
    • Conflict of interest
    • Supplementary Information
    • Acknowledgements
    • References
  • Figures & Data
  • Transparent Process

Related Content

More Reports

  • G‐quadruplex‐binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo
    Roberto Simone, Rubika Balendra, Thomas G Moens, Elisavet Preza, Katherine M Wilson, Amanda Heslegrave, Nathan S Woodling, Teresa Niccoli, Javier Gilbert‐Jaramillo, Samir Abdelkarim, Emma L Clayton, Mica Clarke, Marie‐Therese Konrad, Andrew J Nicoll, Jamie S Mitchell, Andrea Calvo, Adriano Chio, Henry Houlden, James M Polke, Mohamed A Ismail, Chad E Stephens, Tam Vo, Abdelbasset A Farahat, W David Wilson, David W Boykin, Henrik Zetterberg, Linda Partridge, Selina Wray, Gary Parkinson, Stephen Neidle, Rickie Patani, Pietro Fratta, Adrian M Isaacs
    EMBO Molecular Medicine 10: 22-31
  • Peroxisome proliferator‐activated receptor gamma (PPARγ) regulates lactase expression and activity in the gut
    Mathurin Fumery, Silvia Speca, Audrey Langlois, Anne‐Marie Davila, Caroline Dubuquoy, Marta Grauso, Anthony Martin Mena, Martin Figeac, Daniel Metzger, Christel Rousseaux, Jean‐Frederic Colombel, Laurent Dubuquoy, Pierre Desreumaux, Benjamin Bertin
    EMBO Molecular Medicine 9: 1471-1481
More Report

Related Articles

Cited By...

Request Permissions

Subject Areas

  • Chromatin, Epigenetics, Genomics & Functional Genomics
  • Genetics, Gene Therapy & Genetic Disease
  • Neuroscience

Journal

  • Latest Online
  • Current Issue
  • Archive
  • Focus Pages
  • Bibliometrics
  • E-Mail Editorial Office

Authors & References

  • Aims & Scope
  • Editors & Board
  • Transparent Process
  • Author Guidelines
  • Referee Guidelines
  • Open Access
  • Submit

Info

  • Alerts
  • RSS Feeds
  • Reprints & Permissions
  • Advertise & Sponsor
  • News & Press
  • Customer Service

EMBO

  • Funding & Awards
  • Events
  • Science Policy
  • Members
  • About EMBO

Online ISSN  1757-4684

Copyright© 2018 EMBO

This website is best viewed using the latest versions of all modern web browsers. Older browsers may not display correctly.