Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration [Mate-Pair]
Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's Disease (AD) with the potential to disrupt genome integrity. We show increased mosaic structural variations and gene fusions in neurons burdened with DSBs in the CK-p25 inducible mouse model of neurodegeneration. Next, we used full-transcript single-nucleus RNA-seq across 47 human post-mortem prefrontal cortex and find that excitatory neurons in AD are enriched for mosaic gene fusions. In addition, gene fusions are particularly enriched in excitatory neurons with senescence and DNA repair gene signatures. Neurons enriched for DSBs also have elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic and neuronal development genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases. Overall design: Mate-Pair library preparation was performed using the Nextera® Mate Pair Library Preparation (FC-132-1001). Briefly, formaldehyde-fixed FANS sorted nuclei were reverse crosslinked and genomic DNA was purified. Genomic DNA was tagmented to a size range of 1-5kb using mate pair transposome containing biotinylated adaptors followed by strand displacement to repair single-stranded gaps. Tagmented genomic DNA was circularized and then fragmented using sonication. The junctions were purified using streptavidin beads. Sequencing libraries were prepared using TruSeq DNA LT Sample Prep (15027084) provided with the mate pair kit or NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S) and NEBNext Multiplex Oligos for Illumina (E6440S). Illumina libraries were sequenced in 75bp or 40bp paired-end mode using Nextseq500. FASTQ files were trimmed to remove Illumina adaptors and paired-end reads were mapped separately using Bowtie2. Mapped sam files were converted to bam files using samtools view. Bam files were sorted and PCR duplicates were removed using samtools rmdup. Bam files were converted to bed files using bedtools bamtobed. BED files from corresponding paired reads were merged based on FASTQ ID to create the matepair.txt files for each sample.