Scientific Understanding of Consciousness
Autism Linked to Transcription of Long Genes
Nature 501, 58–62 (05 September 2013)
Topoisomerases facilitate transcription of long genes linked to autism
Ian F. King, et.al.
Carolina Institute for Developmental Disabilities, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, USA
Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.
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.
Autism is a neurodevelopmental disorder with symptoms that include repetitive behaviours and deficits in social interactions. Hundreds of genes are now associated with ASD, suggesting that there are diverse genetic risk factors for autism. Environmental factors, including chemicals that are ingested during critical periods of brain development, can also increase autism risk. Many ASD candidate genes regulate synapse function; however, whether there are additional mechanisms that unite ASD patients or expression of ASD genes is unclear.
Recently, we found that topoisomerase inhibitors can transcriptionally un-silence the paternal allele of Ube3a in mouse cortical neurons. Ube3a is located adjacent to a cluster of imprinted genes, is normally expressed only from the maternal allele in neurons, and regulates synaptic function. In humans, UBE3A is associated with two distinct neurodevelopmental disorders. Specifically, deletion or mutation of maternal UBE3A causes Angelman syndrome, whereas duplication of the chromosomal region containing maternal UBE3A is frequently detected in individuals with autism.
Intriguingly, mutations in topoisomerases were recently identified in some individuals with ASD. However, precisely how topoisomerases regulate the expression of Ube3a and possibly other genes associated with autism is unknown. Topoisomerases, including Top1 and Top2b, are expressed throughout the developing and adult mouse brain. Topoisomerases are integral to gene expression, as they resolve DNA supercoiling that is generated during transcription. Here we sought to determine whether topoisomerases preferentially regulate the expression of additional imprinted genes in neurons, or whether they have broader effects on gene expression. Using genome-wide approaches, we unexpectedly found that topoisomerases facilitate the expression of long genes, including numerous long genes associated with synaptic function and ASD. In addition, our study uncovers a transcriptional mechanism that is particularly important for maintaining the expression of numerous ASD genes at normal levels.
Topoisomerases interact directly with RNA polymerase II (Pol II) and are required for transcription elongation.
To investigate further the biological consequences of TOP1 inhibition in neurons, we defined a list of genes that were differentially expressed with high confidence. From our RNA-seq expression data, we found that topotecan significantly downregulated 155 genes and significantly upregulated 28 genes (Benjamini–Hochberg method, 5% false discovery rate). The topotecan-downregulated genes were significantly longer (mean 591 kb, median 548 kb) than all expressed genes in cortical neurons (mean 59.3 kb, median 23.5 kb) and were significantly longer than topotecan-upregulated genes (mean 29.3 kb, median 16.4 kb) (one-way analysis of variance versus all expressed genes P = 2.2 × 10−16, versus upregulated genes P = 3.7 × 10−14), further indicating that topotecan has pronounced effects on long genes.
On the basis of Gene Ontology and functional annotation terms, we found that many topotecan-downregulated genes were involved in neuronal development and synaptic function. Because ASD is thought to be a neurodevelopmental disorder that affects synapses, we cross-referenced our list of downregulated genes with known ASD candidate genes, combining genes in the SFARI Gene database with candidates identified in recent exome sequencing studies. Notably, 27% (n = 49) of the 183 differentially expressed genes are known ASD candidate genes, a proportion that is highly significant compared to chance (P = 4.4 × 10−8, Fisher’s exact test). Independent microarray experiments showed that these ASD genes were dose-dependently downregulated by topotecan. Notably, ASD candidate genes are exceptionally long as a group; genes in the SFARI Gene database are 3.7-fold longer on average than all genes expressed in cortical neurons (means of 217.3 kb versus 59.3 kb). Thus, mutations that alter topoisomerase activity might reduce expression of numerous long ASD genes and might contribute significantly to ASD. Consistent with this hypothesis, recent sequencing studies of autism patient cohorts uncovered rare de novo missense mutations in TOP1, TOP3B, TOPORS (a TOP1-SUMO ligase) and several other genes that directly connect to TOP1.
Our study shows that topoisomerases facilitate the expression of a large number of ASD candidate genes, including many that are long and that are thought to have large effects on ASD pathology in isolation. Pharmacological inhibition of topoisomerases also reduced expression of long genes in other cell types, suggesting that this length-dependent transcriptional effect is fundamental to all mammalian cells. Our data rule out numerous possibilities as to why topoisomerase inhibitors reduce the expression of long genes (for example, cell death, DNA damage, formation of covalent complexes), and instead implicate a gene-length-dependent block in transcription elongation. Pol II and topoisomerases dynamically form and remodel large supercoiling domains, and the effects of topoisomerases on gene expression are strongly influenced by genomic structure and context. Thus, we speculate that higher order structure differentially constrains shorter and longer genes, and that this creates distinct length-dependent requirements for topoisomerases in transcription elongation.
Intriguingly, numerous genes associated with transcription are mutated in autism patients, although how these diverse transcriptional regulators contribute to autism is unclear. Our study highlights a mechanistic link between a critical step in transcription elongation and expression of numerous long ASD candidate genes. Our data suggest that chemicals or genetic mutations that impair topoisomerases, and possibly other components of the transcription elongation machinery that interface with topoisomerases, have the potential to profoundly affect the expression of long ASD candidate genes. Length-dependent impairment of gene transcription, particularly in neurons and during critical periods of brain development, may thus represent a unifying cause of pathology in many individuals with ASD and other neurodevelopmental disorders.
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