Abstract
Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.
Highlights
Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcriptionassociated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase
Another enzyme associated with genomic stress is activationinduced cytidine deaminase (AID), which is normally responsible for the DNA cleavage in the switch (S) and variable (V) regions of the immunoglobulin (Ig) locus, which initiates class switch recombination (CSR) and somatic hypermutation (SHM), respectively[19,20,21]
To overcome the shortcomings of dithiobis-succinimidyl propionate (DSP)-IP, such as nonspecific protein trapping, we performed IP without crosslinking, proteins with labile Topoisomerase 1 (TOP1) interactions may be lost with this method (Fig. 1a–d; Supplementary Fig. 1c)
Summary
Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcriptionassociated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. It is important to stress that these genome instabilities in the nerve cells do not depend on replication, but depend on transcription in which TOP1 plays a critical role Another enzyme associated with genomic stress is activationinduced cytidine deaminase (AID), which is normally responsible for the DNA cleavage in the switch (S) and variable (V) regions of the immunoglobulin (Ig) locus, which initiates class switch recombination (CSR) and somatic hypermutation (SHM), respectively[19,20,21]. The reduced expression of TOP1 in Top[1] heterozygous knockout mice leads to a dramatic increase in the frequency of SHM in Peyer’s patch B cells[24] These findings indicate that reduction in the TOP1 protein level enhances the DNA cleavage that augments SHM or CSR. Non-canonical AID targets such as MYC, MALAT1 and SNHG3, identified in a genome-wide study, are highly transcribed, flanked by repetitive sequences, and enriched with H3K4me[3] as well as FACT25,26,32,33
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