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.

Figure 1. TOP1 inhibition reduces expression of long genes in neurons

a, Mouse cortical neurons treated with vehicle (v) or 300 nM topotecan (drug; d) for 3 days (n=5 biological replicates). RNA-seq gene expression versus gene length. b, Mean expression change in bins of 200 genes by length. c, Percentage of genes that were reduced in expression by topotecan; plotted as a sliding window of 100 genes by length, RNA-seq data (log scale). Inset, same data on linear scale. d, iPSC-derived human neurons treated with 1 μM topotecan for 6 days relative to vehicle, RNA-seq data in bins of 200 genes by length (n=2 biological replicates).

Figure 2. Lentiviral shRNA knockdown of Top1 or Top2b in mouse cortical neurons reduces expression of long genes

a, b, Representative western blots and quantification of TOP1 and TOP2B seven days post infection. Normalized to β-actin in arbitrary units (A.U.). **P<0.01 relative to scrambled (Scr) control, Student’s t-test. Error bars, s.e.m. n=3 biological replicates. c, d, Gene expression from Affymetrix microarrays, relative to scrambled control shRNA. Plotted as mean expression change in bins of 200 genes by length.

Figure 3. Topotecan impairs transcription elongation of long genes

a, Mouse cortical neurons treated with vehicle or topotecan (300 nM for 3 days). Pol II density in gene bodies and promoter regions, averaged for bins of 200 genes by length. b, c, Change in Pol II density across genes and (c) across the first 10 kb, averaged for groups of 200 genes by length. Gene bins aligned relative to the transcription start site (TSS). d, Mouse cortical neurons treated with 100 μM DRB for 3 days. Affymetrix microarray expression compared to controls in bins of 200 genes by length.