Article Figures & Data
Figures
- 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. 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. LINC00925 regulates POLG expression
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.
Distribution histogram of DHSs for mtDNA maintenance genes.
Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.
Genes surrounding LINC00925.
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.
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.
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).
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.
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).
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.
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. POLG defects cause degeneration of enhancer‐active regions of CNS
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.
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
