TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

Journal name:
Nature Genetics
Volume:
46,
Pages:
516–521
Year published:
DOI:
doi:10.1038/ng.2929
Received
Accepted
Published online

Topoisomerase II (TOP2) removes torsional stress from DNA and facilitates gene transcription by introducing transient DNA double-strand breaks (DSBs). Such DSBs are normally rejoined by TOP2 but on occasion can become abortive and remain unsealed. Here we identify homozygous mutations in the TDP2 gene encoding tyrosyl DNA phosphodiesterase-2, an enzyme that repairs 'abortive' TOP2-induced DSBs, in individuals with intellectual disability, seizures and ataxia. We show that cells from affected individuals are hypersensitive to TOP2-induced DSBs and that loss of TDP2 inhibits TOP2-dependent gene transcription in cultured human cells and in mouse post-mitotic neurons following abortive TOP2 activity. Notably, TDP2 is also required for normal levels of many gene transcripts in developing mouse brain, including numerous gene transcripts associated with neurological function and/or disease, and for normal interneuron density in mouse cerebellum. Collectively, these data implicate chromosome breakage by TOP2 as an endogenous threat to gene transcription and to normal neuronal development and maintenance.

At a glance

Figures

  1. TDP2 mutation in individuals with intellectual disability, epilepsy and ataxia.
    Figure 1: TDP2 mutation in individuals with intellectual disability, epilepsy and ataxia.

    (a) Pedigree analysis. Black symbols denote probands, and gray symbols denote individuals with multiple congenital abnormalities who died soon after birth. SNP genotyping for homozygosity mapping was conducted on the individuals underlined. Segregation of the TDP2 putative splice-site mutation c.425+1G>A is indicated (+, normal allele; −, mutated allele). (b) Clinical phenotypes of the indicated probands. SGE, symptomatic generalized epilepsy; M/S, moderate to severe intellectual disability (estimated IQ of 30–40). *, an individual with compromised breathing and cyanosis during seizures; **, no additional neurodegenerative symptoms noted. Note that the probands lacked core symptoms of autism (for example, repetitive or restrictive behaviors). (c) Location of the putative donor splice-site mutation (bold) in intron 3 of TDP2. Exons 5 and 6 were omitted for simplicity.

  2. Loss of TDP2 protein and activity in individuals with intellectual disability, epilepsy and ataxia.
    Figure 2: Loss of TDP2 protein and activity in individuals with intellectual disability, epilepsy and ataxia.

    (a) 5′-TDP and 3′-TDP activities were measured in protein extracted from the blood of three affected brothers (IV-9, IV-14, IV-16), an unaffected sibling (IV-2), the unaffected mother (III-2) and a non-related additional control (NR). Reactions contained 1 μl of lysis buffer (B) or total protein extract (from 50 μl of total extract). Schematics depict the [32P]-labeled (asterisk) oligonucleotide substrates for TDP2 (top) and TDP1 (bottom), harboring a 5′- or 3′-phosphotyrosyl terminus (Y-P), respectively. (b) 5′-TDP and 3′-TDP activities in lymphoblastoid cell extract (9 μg of total protein) from the indicated individuals were measured as in a. (c) Protein blots showing TDP1, TDP2, TOP2α, TOP2β and actin amounts in lymphoblastoid cell extracts from the indicated individuals.

  3. Phenotypic impact of TDP2 disruption in lymphoblastoid cells from affected humans and neural cells from Tdp2[Delta]1-3 mice.
    Figure 3: Phenotypic impact of TDP2 disruption in lymphoblastoid cells from affected humans and neural cells from Tdp2Δ1–3 mice.

    (a) Growth of normal (IV-2) or affected (IV-9) human lymphoblastoid cells in the absence or presence of 15 nM etoposide. Data are the mean (±s.e.m.) of four independent experiments, and statistically significant differences are indicated (two-tailed t test, *P < 0.05). NS, not significant. (b) Cell death (Sytox staining) in normal (NRC, IV-2) or affected (IV-9, IV-14, IV-16) human lymphoblastoid cells in the presence or absence of etoposide or MMS. Data are the mean (±s.e.m.) of at least three independent experiments. Statisticallysignificant differences compared to the non-related control (NRC) are indicated (two-tailed t test, *P < 0.05, **P < 0.01). (c) Decreased repair of TOP2-induced DSBs in normal (IV-2) or affected (IV-9) human lymphoblastoid cells. The average number of γH2AX foci per cell was quantified before (−) and after (time 0) a 30-min incubation with 20 μM etoposide and at indicated times after drug removal. Data are the mean (±s.e.m.) of four independent experiments. Statistically significant differences are indicated (two-tailed t test, ***P < 0.005). (d) Left, 5′-TDP activity in total protein extract (9 μg) from the brain and cerebellum of wild-type and Tdp2Δ1–3 mice. Right, 5′-TDP activity in total protein extract (2 μg) from quiescent primary cortical astrocytes and granule cerebellar neurons. Reactions were conducted for the times indicated. (e) Decreased repair of TOP2-induced DSBs in primary cortical astrocytes and cerebellar granule neurons from Tdp2Δ1–3 mice. Data are the mean (±s.e.m.) of three independent experiments and are as described in b and c. (f) Reduced interneuron density in the molecular layer of the cerebellum of Tdp2Δ1–3 mice. Left, representative images of Nissl-stained sections from the cerebellum of heterozygous (Tdp2+/Δ1–3) and mutant (Tdp2Δ1–3) 10-week-old mice. Arrows denote interneurons in the molecular layer (ML). Scale bars, 200 μm. Right, quantification of interneuron density in the molecular layer of Tdp2+/Δ1–3 and Tdp2Δ1–3 cerebellum. Data are the mean (±s.e.m.) from three control and five Tdp2-mutant littermates (two-tailed t test, **P < 0.01).

  4. TDP2 is required to maintain gene transcription at sites of abortive TOP2 activity.
    Figure 4: TDP2 is required to maintain gene transcription at sites of abortive TOP2 activity.

    (a) TDP2 is required for high levels of AR-induced gene expression. mRNA levels are shown for the indicated AR-responsive (KLK2, TMPRSS2 and KLK3) and unresponsive (ACTB, TBP and MLN51) genes in TDP2-depleted (TDP2 siRNA) or mock-depleted (scrambled) LNCaP cells before and 4 h after stimulation with DHT or after 4-h induction with DHT in the presence of 20 μM etoposide. mRNA levels were quantified by qRT-PCR and are expressed as arbitrary units (AU) normalized first to ACTB levels under the same experimental condition and then to the levels of the relevant gene of interest under control conditions without DHT. Data are the mean (±s.e.m.) of four independent experiments. In all experiments, statistically significant differences are indicated (two-tailed t test; *P < 0.05, **P < 0.005, ***P < 0.001). NS, not statistically significant (P > 0.05). (b) Recruitment of RNAP II at the KLK3 promoter in the absence of gene induction, 8 h after gene induction with 100 nM DHT and after gene induction with 100 μM etoposide present for two additional hours. Data are presented as the amount of DNA precipitated relative to the recovery of a non-transcribed region of chromosome 5 and are the mean (±s.e.m.) of three independent experiments. (c) Top, representative images of EU pulse labeling in Tdp2+/+ and Tdp2Δ1–3 astrocytes and Tdp2+/Δ1–3 and Tdp2Δ1–3 granule neurons before and after 2-h treatment with 0.5 mM etoposide and after a further 90-min recovery period in drug-free medium. Bottom, transcription was quantified using SimplePCI 6.0. Data are the mean (±s.e.m.) of three independent experiments (two-tailed t test, **P < 0.005). (d) mRNA levels of the indicated developmentally regulated TOP2β-dependent genes (Cacna2d1, Kcnd2, Syt1) and controls (Actb, Tubb3, Gapdh) in E16.5 brain from wild-type and Tdp2Δ1–3 mice. mRNA levels were quantified by qRT-PCR as in a but were normalized to Gapdh levels instead of to Actb levels. Data are the mean (±s.e.m.) for three independent littermate pairs. Statistically significant differences are indicated (two-tailed t test, *P < 0.05, **P < 0.01). NS, not statistically significant (P > 0.05). (e) Physiological processes that are significantly over-represented in terms of altered gene transcription in embryonic brain from Tdp2Δ1–3 mice. The percentage of affected genes (n = 165) that fall into each category is plotted. Bars are sorted by the P-value range for the relevant genes: (top to bottom) P = 1.5 × 10−2 to 5.9 × 10−13 (53 genes); P = 1.5 × 10−2 to 3.4 × 10−7 (28 genes); P = 8.0 × 10−5 (5 genes); P = 1.5 × 10−2 to 8.2 × 10−5 (20 genes); P = 1.5 × 10−2 to 8.2 × 10−5 (20 genes). Data are from three independent littermate pairs.

  5. Regions of homozygosity (ROH) in patients with mutated TDP2.
    Supplementary Fig. 1: Regions of homozygosity (ROH) in patients with mutated TDP2.

    a, Summary of ROHs of ≥1 Mb per individual. Phe, phenotype status (U, unaffected, A, affected); NSEG, number of ROH segments; Mb, total size of ROH; MbAVG, average size of each ROH. b, Visualisation of shared ROH segments in the UCSC Genome Browser among affected (red) and unaffected (blue) individuals. A 9.08-Mb region along chromosome 6 is the sole region unique to all three affected siblings.

  6. Putative splice-site mutation in TDP2.
    Supplementary Fig. 2: Putative splice-site mutation in TDP2.

    a, Raw sequence reads from exome sequencing of inidividual IV-9 showing the homozygous c.425+1G>A mutation. The orientation of the genomic sequence is 5′ to 3′ and is the transcribed/template strand. b, Confirmation by Sanger sequencing of the homozygous mutation in individual IV-9, the heterozygous mutation in the mother, and the wild-type allele in an unrelated control individual. The orientation of the genomic sequence is 5′ to 3′ and is the transcribed/template strand. c, Predicted effect of the c.425+1G>A mutation on mRNA splicing. Scenario A involves retention of intron 3, resulting in the introduction of a premature stop codon in the p.Leu142fs* alteration. Scenario B involves skipping of exon 3, resulting in the introduction of a premature stop codon in the p.Tyr84* alteration. Scenario C involves the use of an alternative splice-donor site resulting in the frameshift p.Gly135fs*16.

  7. Evolutionary conservation of TDP2.
    Supplementary Fig. 3: Evolutionary conservation of TDP2.

    Alignment (CLUSTALW) of TDP2 orthologs (frog, Xenopus tropicalis; chick, Gallus gallus; worm, Caenorhabditis elegans; zebrafish, Danio rerio). Identical residues are indicated by blue boxes, and conserved motifs of the metal-dependent phosphodiesterase superfamily are indicated by red boxes. Catalytic residues are indicated by yellow ovals. The three consequences of the putative splice-site mutation in the Irish patients are indicated in red, and the consequence of the dinucleotide substitution in the Egyptian patient is indicated in green.

  8. Impact of the TDP2 splice-site mutation (c.425+1G>A) on TDP2 mRNA.
    Supplementary Fig. 4: Impact of the TDP2 splice-site mutation (c.425+1G>A) on TDP2 mRNA.

    a, Impact of the splice-site mutation on TDP2 mRNA size, as measured by non-quantitative RT-PCR amplification of TDP2 exons 1–6 in RNA from lymphoblastoid cells of the three affected individuals (IV-9, IV-14, IV-16) and an unrelated control individual (NR). Primer sequences are indicated in Supplementary Table 5. b, Impact of the splice-site mutation on TDP2 mRNA levels as measured by qRT-PCR. Data are the mean expression of TDP2 mRNA in all three affected individuals (A, n = 3) compared to the non-related control (NR, n = 8) in the absence (– CH) or presence (+ CH) of cycloheximide treatment to inhibit nonsense-mediated mRNA decay. Quantifications were performed in duplicate and were normalized against GUSB and PPIB levels.

  9. Absence of 5[prime]-TDP activity in Irish and Egyptian patients harbouring homozygous truncation mutations in TDP2.
    Supplementary Fig. 5: Absence of 5′-TDP activity in Irish and Egyptian patients harbouring homozygous truncation mutations in TDP2.

    5′-TDP (left) and 3′-TDP (right) activity in protein extract from total blood from an unrelated control (UC) and from affected Irish (IV-9) or Egyptian (E) patients with independent TDP2 mutations. B, negative control lacking protein extract. The radioactively labeled strand of the substrate is indicated with an asterisk.

  10. TDP2 mRNA expression in different fetal and adult human tissues.
    Supplementary Fig. 6: TDP2 mRNA expression in different fetal and adult human tissues.

    Relative expression levels are given as the fold change in comparison to the tissue with the lowest expression level. Quantifications were performed in duplicate and normalized against GUSB and PPIB levels.

  11. Repair of TOP2-induced DSBs in mouse neural cells.
    Supplementary Fig. 7: Repair of TOP2-induced DSBs in mouse neural cells.

    Decreased repair of TOP2-induced DSBs in Tdp2Δ1–3 mouse cortical astrocytes (top) and cerebellar granule neurons (bottom). Representative images are shown of etoposide-induced (20 mM) γH2AX immunofoci in Tdp2+/+ and in Tdp2Δ1–3 astrocytes and
    Tdp2+/δ1–3 and Tdp2Δ1–3 granule neurons after a 3-h recovery period in etoposide-free medium.

  12. Transcription of AR-responsive genes is inhibited by abortive TOP2 activity.
    Supplementary Fig. 8: Transcription of AR-responsive genes is inhibited by abortive TOP2 activity.

    a, mRNA levels of three AR-responsive genes (KLK2, TMPRSS2 and KLK3) and three genes unresponsive to AR (ACT, TBP and MLN51) before and at the indicated times (h) after incubation with 100 nM DHT. mRNA levels were normalized to ACTIN and then made relative to the untreated time point (– DHT). b, Inhibition of AR-induced gene transcription by etoposide-induced abortive TOP2 activity. mRNA levels of the indicated AR-responsive (KLK2, TMPRSS2 and KLK3) and unresponsive (ACT, TBP and MLN51) genes before (– DHT) and after (+ DHT) stimulation with 100 nM DHT and after induction with DHT in the presence of the indicated concentration of etoposide. mRNA levels were quantified as described in the main text. c, Recruitment of RNAP II at the KLK3 and TMPRSS2 promoters in the absence of gene induction (– DHT), 8 h after gene induction with 100 nM DHT (+ DHT) and after gene induction with 100 μM etoposide present for two additional hours (+ DHT/+ Etop.). Data are presented as percentage of DNA precipitated (left) or relative to the recovery of a non-transcribed region of chromosome 5 (right) and are the mean (± s.e.m.) of at least three independent experiments.

  13. TDP2 depletion by RNA interference.
    Supplementary Fig. 9: TDP2 depletion by RNA interference.

    TDP2 depletion in LNCaP cells by short hairpin RNA (shRNA) (left) or siRNA (right). Cells employed to measure RNAP II promoter occupancy by chromatin immunoprecipitation (left) or AR-dependent gene expression by qRT-PCR (right) were subjected to protein blotting to measure the levels of the proteins indicated.

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Author information

  1. These authors contributed equally to this work.

    • Fernando Gómez-Herreros,
    • Janneke H M Schuurs-Hoeijmakers &
    • Mark McCormack

Affiliations

  1. Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Sussex, UK.

    • Fernando Gómez-Herreros,
    • Stuart Rulten &
    • Keith W Caldecott
  2. Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

    • Janneke H M Schuurs-Hoeijmakers,
    • Arjan P M de Brouwer &
    • Bert B A de Vries
  3. Department of Cognitive Neurosciences, Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands.

    • Janneke H M Schuurs-Hoeijmakers,
    • Arjan P M de Brouwer &
    • Bert B A de Vries
  4. Molecular and Cellular Therapeutics, The Royal College of Surgeons in Ireland, Dublin, Ireland.

    • Mark McCormack,
    • Norman Delanty &
    • Gianpiero L Cavalleri
  5. National Centre for Medical Genetics, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.

    • Marie T Greally
  6. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Departamento de Genética, CSIC (Centro Superior de Investigaciones Científicas)–Universidad de Sevilla, Sevilla, Spain.

    • Rocío Romero-Granados &
    • Felipe Cortés-Ledesma
  7. Department of Neurology, University Hospital Galway, Galway, Ireland.

    • Timothy J Counihan
  8. Division of Neurology, Beaumont Hospital, Dublin, Ireland.

    • Elijah Chaila &
    • Norman Delanty
  9. School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.

    • Judith Conroy &
    • Sean Ennis
  10. Kreb's Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.

    • Sherif F El-Khamisy
  11. Center of Genomics, Helmy Institute, Zewail City of Science and Technology, Giza, Egypt.

    • Sherif F El-Khamisy

Contributions

K.W.C. devised and coordinated the project. K.W.C. and F.G.-H. designed and interpreted the biochemical, cell biology and mouse experiments and wrote the manuscript. F.G.-H. conducted all biochemical and cell biology experiments. S.R. analyzed mouse interneurons, and F.C.-L. and R.R.-G. measured TDP2 activity in mouse brain tissue. J.H.M.S.-H., A.P.M.d.B. and B.B.A.d.V. conducted and interpreted exome sequencing under the supervision of B.B.A.d.V. and identified the human splice-site mutation in Nijmegen. M.M., J.C., S.E. and G.L.C. conducted and interpreted genome-wide association study and homozygosity mapping in Ireland under the supervision of G.L.C. and identified the TDP2 splice-site mutation by exome sequencing in collaboration with the Duke Center for Human Genome Variation. E.C., N.D. and T.J.C. recruited and phenotyped patients in Ireland. M.T.G. consulted, phenotyped and liaised with patients and their families in Ireland. S.F.E.-K. identified and coordinated the analysis of the TDP2 patient in Egypt.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Regions of homozygosity (ROH) in patients with mutated TDP2. (280 KB)

    a, Summary of ROHs of ≥1 Mb per individual. Phe, phenotype status (U, unaffected, A, affected); NSEG, number of ROH segments; Mb, total size of ROH; MbAVG, average size of each ROH. b, Visualisation of shared ROH segments in the UCSC Genome Browser among affected (red) and unaffected (blue) individuals. A 9.08-Mb region along chromosome 6 is the sole region unique to all three affected siblings.

  2. Supplementary Figure 2: Putative splice-site mutation in TDP2. (345 KB)

    a, Raw sequence reads from exome sequencing of inidividual IV-9 showing the homozygous c.425+1G>A mutation. The orientation of the genomic sequence is 5′ to 3′ and is the transcribed/template strand. b, Confirmation by Sanger sequencing of the homozygous mutation in individual IV-9, the heterozygous mutation in the mother, and the wild-type allele in an unrelated control individual. The orientation of the genomic sequence is 5′ to 3′ and is the transcribed/template strand. c, Predicted effect of the c.425+1G>A mutation on mRNA splicing. Scenario A involves retention of intron 3, resulting in the introduction of a premature stop codon in the p.Leu142fs* alteration. Scenario B involves skipping of exon 3, resulting in the introduction of a premature stop codon in the p.Tyr84* alteration. Scenario C involves the use of an alternative splice-donor site resulting in the frameshift p.Gly135fs*16.

  3. Supplementary Figure 3: Evolutionary conservation of TDP2. (705 KB)

    Alignment (CLUSTALW) of TDP2 orthologs (frog, Xenopus tropicalis; chick, Gallus gallus; worm, Caenorhabditis elegans; zebrafish, Danio rerio). Identical residues are indicated by blue boxes, and conserved motifs of the metal-dependent phosphodiesterase superfamily are indicated by red boxes. Catalytic residues are indicated by yellow ovals. The three consequences of the putative splice-site mutation in the Irish patients are indicated in red, and the consequence of the dinucleotide substitution in the Egyptian patient is indicated in green.

  4. Supplementary Figure 4: Impact of the TDP2 splice-site mutation (c.425+1G>A) on TDP2 mRNA. (51 KB)

    a, Impact of the splice-site mutation on TDP2 mRNA size, as measured by non-quantitative RT-PCR amplification of TDP2 exons 1–6 in RNA from lymphoblastoid cells of the three affected individuals (IV-9, IV-14, IV-16) and an unrelated control individual (NR). Primer sequences are indicated in Supplementary Table 5. b, Impact of the splice-site mutation on TDP2 mRNA levels as measured by qRT-PCR. Data are the mean expression of TDP2 mRNA in all three affected individuals (A, n = 3) compared to the non-related control (NR, n = 8) in the absence (– CH) or presence (+ CH) of cycloheximide treatment to inhibit nonsense-mediated mRNA decay. Quantifications were performed in duplicate and were normalized against GUSB and PPIB levels.

  5. Supplementary Figure 5: Absence of 5′-TDP activity in Irish and Egyptian patients harbouring homozygous truncation mutations in TDP2. (24 KB)

    5′-TDP (left) and 3′-TDP (right) activity in protein extract from total blood from an unrelated control (UC) and from affected Irish (IV-9) or Egyptian (E) patients with independent TDP2 mutations. B, negative control lacking protein extract. The radioactively labeled strand of the substrate is indicated with an asterisk.

  6. Supplementary Figure 6: TDP2 mRNA expression in different fetal and adult human tissues. (110 KB)

    Relative expression levels are given as the fold change in comparison to the tissue with the lowest expression level. Quantifications were performed in duplicate and normalized against GUSB and PPIB levels.

  7. Supplementary Figure 7: Repair of TOP2-induced DSBs in mouse neural cells. (183 KB)

    Decreased repair of TOP2-induced DSBs in Tdp2Δ1–3 mouse cortical astrocytes (top) and cerebellar granule neurons (bottom). Representative images are shown of etoposide-induced (20 mM) γH2AX immunofoci in Tdp2+/+ and in Tdp2Δ1–3 astrocytes and
    Tdp2+/δ1–3 and Tdp2Δ1–3 granule neurons after a 3-h recovery period in etoposide-free medium.

  8. Supplementary Figure 8: Transcription of AR-responsive genes is inhibited by abortive TOP2 activity. (163 KB)

    a, mRNA levels of three AR-responsive genes (KLK2, TMPRSS2 and KLK3) and three genes unresponsive to AR (ACT, TBP and MLN51) before and at the indicated times (h) after incubation with 100 nM DHT. mRNA levels were normalized to ACTIN and then made relative to the untreated time point (– DHT). b, Inhibition of AR-induced gene transcription by etoposide-induced abortive TOP2 activity. mRNA levels of the indicated AR-responsive (KLK2, TMPRSS2 and KLK3) and unresponsive (ACT, TBP and MLN51) genes before (– DHT) and after (+ DHT) stimulation with 100 nM DHT and after induction with DHT in the presence of the indicated concentration of etoposide. mRNA levels were quantified as described in the main text. c, Recruitment of RNAP II at the KLK3 and TMPRSS2 promoters in the absence of gene induction (– DHT), 8 h after gene induction with 100 nM DHT (+ DHT) and after gene induction with 100 μM etoposide present for two additional hours (+ DHT/+ Etop.). Data are presented as percentage of DNA precipitated (left) or relative to the recovery of a non-transcribed region of chromosome 5 (right) and are the mean (± s.e.m.) of at least three independent experiments.

  9. Supplementary Figure 9: TDP2 depletion by RNA interference. (46 KB)

    TDP2 depletion in LNCaP cells by short hairpin RNA (shRNA) (left) or siRNA (right). Cells employed to measure RNAP II promoter occupancy by chromatin immunoprecipitation (left) or AR-dependent gene expression by qRT-PCR (right) were subjected to protein blotting to measure the levels of the proteins indicated.

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  1. Supplementary Text and Figures (10,174 KB)

    Supplementary Figures 1–9, Supplementary Tables 1–6 and Supplementary Note

Additional data