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Topoisomerases facilitate transcription of long genes linked to autism

Abstract

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

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Figure 1: TOP1 inhibition reduces expression of long genes in neurons.
Figure 2: Lentiviral shRNA knockdown of Top1 or Top2b in mouse cortical neurons reduces the expression of long genes.
Figure 3: Topotecan impairs transcription elongation of long genes.

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Gene Expression Omnibus

Data deposits

Data from Microarray, RNA-seq and ChIPseq experiments have been deposited in the Gene Expression Omnibus under accession number GSE43900.

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Acknowledgements

We thank M. Vernon at the UNC Expression Profiling Core for assistance with microarray experiments and with data analysis, H. Kelkar for pilot bioinformatics support, P. Mieczkowski and A. Brandt at the UNC High Throughput Sequencing Facility for advice and assistance with Illumina library preparation and sequencing and T. Kafri and P. Zhang at the UNC Lenti-shRNA Core for assistance with preparation of lentiviral vectors. This work was supported by grants to M.J.Z. and B.D.P. from The Angelman Syndrome Foundation, The Simons Foundation (SFARI 10-3625) and The National Institute of Mental Health (R01MH093372). I.F.K. and A.M.M. were supported by Joseph E. Wagstaff Postdoctoral Research Fellowships from the Angelman Syndrome Foundation. J.M.C. was supported by a grant from the American Cancer Society (117571-PF-09-124-01-DDC). J.M.C., J.S. and T.M. were supported by a grant from the National Institutes of Health (NIH) (R01GM101974). B.L.P. was supported by a NIH postdoctoral training grant (T32HD040127). S.J.C. was supported by a grant from National Institute of Child Health and Human Development (NICHD) (R01HD068730). The expression profiling and bioinformatics cores were funded by grants from the National Institute of Neurological Disorders and Stroke (P30NS045892) and NICHD (P30HD03110).

Author information

Authors and Affiliations

Authors

Contributions

I.F.K., H.-S.H., A.M.M., J.S.H., S.J.C., B.D.P. and M.J.Z. conceived and designed experiments. I.F.K. performed RNA-seq and ChIP-seq experiments with mouse neurons. I.F.K., C.N.Y., J.M.C., J.S. and J.S.P. analysed data from genome-wide experiments and from published datasets. J.M.C. performed SNP filtering of RNA-seq data and J.S. performed statistical analysis of RNA-seq data. A.M.M. performed lentiviral shRNA knockdown experiments. J.S.H. and S.J.C. performed all experiments with iPSC-derived human neurons. B.L.P. assessed propensity of compounds to kill neurons and damage DNA. T.M. provided bioinformatics support. I.F.K., H.-S.H. and A.M.M. performed microarray experiments. I.F.K. and M.J.Z. wrote the manuscript.

Corresponding authors

Correspondence to Benjamin D. Philpot or Mark J. Zylka.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Topotecan affects allelic expression of Ube3a and Ube3a-ATS but not expression of nearby genes.

a, Parent-of-origin-specific RNA-seq reads for Ube3a and Ube3a-ATS in vehicle- and topotecan-treated (300 nM for 3 days) mouse cortical neurons (n = 5 biological replicates). Shown are SNP-filtered read counts per million mapped reads (RPM) from the maternally (mat) and paternally (pat) inherited chromosomes. Reads from exons and 3′-untranslated region of Ube3a are indicated by arrows. b, Expression of Ube3a and imprinted genes near Ube3a in mouse cortical neurons ± 300 nM topotecan for 3 days. *P < 0.05, Student’s t-test. Error bars represent s.e.m. c, Expression of UBE3A and UBE3A-ATS in iPSC-derived neurons from an Angelman syndrome patient carrying a maternal deletion of the 15q11–q13 region. Differentiated neuronal cultures were treated with 10 nM–10 μM topotecan or vehicle for 6 days. Expression quantified by qPCR. **P < 0.01, one-way analysis of variance with Dunnet’s post-hoc test. n = 4. Error bars represent s.e.m.

Extended Data Figure 2 Topotecan dose–response.

Mouse cortical neurons were treated with 3 nM, 30 nΜ, 150 nM, 300 nM and 1,000 nM topotecan for 3 days (n = 3 for 300 nM topotecan, all other doses n = 1). Gene expression was analysed by Affymetrix microarrays, plotted as mean expression change in bins of 200 genes.

Extended Data Figure 3 DNA damage does not inhibit gene expression in a length-dependent manner.

a, Cultured mouse cortical neuron viability, assayed by Sytox Green staining after 72 h treatment with 300 nM topotecan, and after 24 h with 100 μM H2O2 or 10 μM paraquat, compared to vehicle-treated controls. Error bars represent s.e.m. n = 4. b, Average number of γ-H2AX foci per nucleus for cultured cortical neurons treated with 300 nM topotecan for 72 h, 100 μM H2O2 for 24 h and 10 μM paraquat for 24 h, compared to vehicle-treated controls. **P < 0.01, Student’s t-test. Number of cells counted is indicated for each sample. c, Gene expression compared to vehicle controls in bins of 200 genes by length, for cultured cortical neurons treated with 100 μM H2O2 for 24 h. d, Gene expression compared to vehicle controls in bins of 200 genes by length, for cultured cortical neurons treated with 10 μM paraquat for 24 h. e, Gene expression in cultured cortical neurons treated with 300 nM topotecan for 24 h, or treated for 24 h followed by 48 h without drug (washout). Average change in expression for bins of 200 genes by length.

Extended Data Figure 4 Topotecan and irinotecan have highly similar effects on gene expression.

a, Affymetrix microarray analysis of gene expression in cultured mouse cortical neurons treated with vehicle or 10 μM irinotecan (n = 3 biological replicates), an inhibitor of TOP1, for 3 days. Mean expression fold change in bins of 200 genes, plotted by average gene length. b, Scatterplot of fold change with 1 μM topotecan (n = 6 biological replicates) versus fold change with 10 μM irinotecan for all expressed genes, measured by Affymetrix microarray. Pearson’s R = 0.860. c, Overlap between genes showing positive or negative fold change of log2 = 0.5 or greater with topotecan and irinotecan treatment. d, Overlap between genes reduced or increased in expression. e, Overlap between differentially expressed genes that are greater or less than 67 kb.

Extended Data Figure 5 TOP1 and TOP2 inhibitors reduce expression of long genes in human cell lines.

Re-analysis of microarray gene expression data sets from other laboratories. All plots are mean fold change in expression compared to vehicle controls in bins of 200 genes, plotted by average gene length. a, MCF7 cells treated with 10 μM irinotecan for 24 h, from the CMAP2 project44. b, MCF7 cells treated with 165 nM SN38, the active metabolite of irinotecan, for 6 h45. ce, Gene expression in three human cell lines treated with camptothecin. c, MCF7 cells treated for 24 h with 10 μM camptothecin, from CMAP2. d, 293T cells treated with 2 μM camptothecin for 48 h46. e, HeLa cells treated with 10 μM camptothecin for 8 h47. f, g, Re-analysis of microarray data from ref. 26, comparing gene expression in TOP2 inhibitor- and vehicle-treated ME16C cells. f, ME16C cells treated with 0.5 μM doxorubicin for 36 h. g, ME16C cells treated with 50 μM etoposide for 36 h.

Extended Data Figure 6 Topotecan and the TOP2 inhibitor ICRF-193 have similar effects on gene expression.

a, Affymetrix microarray analysis of gene expression in cultured mouse cortical neurons treated with vehicle or 3 μM ICRF-193 (n = 3 biological replicates), an inhibitor of TOP2 enzymes, for 3 days. Mean expression fold change in bins of 200 genes, plotted by average gene length. b, Scatterplot of fold change with 300 nM topotecan (n = 3 biological replicates) versus fold change with 3 μM ICRF-193 for all expressed genes, measured by Affymetrix microarray. Pearson’s R = 0.588. c, Overlap between genes showing positive or negative fold change of log2 = 0.5 or greater with topotecan and ICRF-193 treatment. d, Overlap between genes reduced or increased in expression. e, Overlap between differentially expressed genes that are greater or less than 67 kb in length.

Extended Data Figure 7 Pharmacological inhibition of TOP2 or genetic deletion of Top2b reduces expression of long genes in ES-cell-derived neurons.

a, b, Re-analysis of microarray expression data from ref. 25. Mean fold change in expression in bins of 200 genes, plotted by average gene length. a, Gene expression in ES-cell-derived neurons treated with vehicle or 50 μM ICRF-193 for 3 days. b, Gene expression in ES cells treated with vehicle or 50 μM ICRF-193 for 3 days. c, Re-analysis of microarray expression data from ref. 14, comparing gene expression in whole brain from wild-type (WT) and Top2b−/− embryonic mice. Expression data from three developmental time points (E16.5, E17.5, E18.5) were averaged for each gene then plotted as mean fold change in expression between wild-type and Top2b−/− mice (in bins of 100 genes, by average gene length). dg, Re-analysis of microarray expression data from ref. 25. Mean fold change in gene expression between wild-type and Top2b−/− cells, for bins of 200 genes, plotted by average gene length. Expression data from ES cells (d), neuronal progenitors (e), ES-cell-derived neurons, 2 days after plating of neuronal progenitors (f) and ES-cell-derived neurons, 6 days after plating of neuronal progenitors (g). ES cells and neuronal progenitors express Top2a and Top2b (indicated within parentheses) and do not show reduced expression of long genes when Top2b is knocked out, suggesting that Top2a and Top2b redundantly regulate the expression of long genes.

Extended Data Figure 8 Topotecan increases travelling ratio in genes larger than 67 kb.

a, b, Travelling ratio was calculated for all genes bound by Pol II in vehicle- and topotecan-treated (300 nM for 3 days) cultured cortical neurons. 92.1% of all genes had travelling ratio values greater than 2, consistent with a previous report using mouse ES cells30. Frequency of travelling ratios for genes less than 67 kb in length (a) and for genes greater than 67 kb in length (b). Mean travelling ratio of vehicle- and topotecan-treated samples was significantly different for genes greater than 67 kb (P = 2.4 × 10−21, Student’s t-test) but not for genes less than 67 kb (P = 0.648, Student’s t-test).

Extended Data Figure 9 Topotecan dose-dependently reduces expression of ASD candidate genes in cortical neurons.

a, Topotecan dose–response for high-confidence autism candidate genes (n = 3 for 300 nM topotecan, all other doses n = 1). bg, Topotecan dose–response for other ASD candidate genes organized by length from longest to shortest. Mouse cortical neurons were treated with vehicle or the indicated doses of topotecan for 3 days. Gene expression was quantified using Affymetrix microarrays. Dose–responses are from all topotecan-downregulated ASD candidate genes that were identified by RNA-seq (Table 1) and that were present on the Affymetrix microarrays.

Extended Data Table 1 Topotecan does not alter parent-of-origin bias of imprinted genes.

Supplementary information

Supplementary Data 1

This file contains a Table of all genes differentially expressed with topotecan treatment of cultured mouse cortical neurons. (XLSX 24 kb)

Supplementary Data 2

This file contains the functional annotation clustering data for topotecan upregulated and downregulated genes. (XLSX 55 kb)

Supplementary Data 3

This file contains the expression information for topotecan treated neurons for all ASD candidate genes considered in this study. (XLSX 59 kb)

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King, I., Yandava, C., Mabb, A. et al. Topoisomerases facilitate transcription of long genes linked to autism. Nature 501, 58–62 (2013). https://doi.org/10.1038/nature12504

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