Abstract
Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.
Highlights
Topoisomerases are enzymes that resolve DNA supercoils by creating transient single (Type I topoisomerases) or double (Type II topoisomerases) strand breaks [1,2]
The Topoisomerase 1 (TOP1) inhibitor topotecan suppresses expression of long genes and unsilences the paternal copy of Ube3a in neurons [13,22]. To determine if these transcriptional effects could be recapitulated by deletion of Top1, we generated a Top1 conditional knockout mouse, as homozygous deletion of Top1 is embryonic lethal with failure occurring between the 4 and 16-cell stages [31]
We found that deletion of Top1 results in down- and upregulation of only a fraction of genes compared to treatment with the TOP1 inhibitor, topotecan (Fig 2C and 2D)
Summary
Topoisomerases are enzymes that resolve DNA supercoils by creating transient single (Type I topoisomerases) or double (Type II topoisomerases) strand breaks [1,2]. These enzymes facilitate DNA replication, chromosomal segregation, DNA repair, and gene transcription [3]. Topoisomerases predominantly regulate gene transcription and DNA repair [4]. Topoisomerase I (TOP1) relieves DNA supercoiling ahead of RNA polymerase to facilitate transcription elongation [5,6,7]. Long noncoding RNAs (lncRNA) can act as transcriptional activators or repressors in postmitotic neurons and other cell types [8,9].
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