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.
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Gene Expression Omnibus
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- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Supplementary Text and Figures (10,174 KB)
Supplementary Figures 1–9, Supplementary Tables 1–6 and Supplementary Note