Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues

Abstract

We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200 to 400 base pairs long, are derived from unique nonrepetitive sequence, and are enriched in the 5'-untranslated regions of genes, exons, and CpG islands. Chromosomal loci that are enriched sources of microDNA in the adult brain are somatically mosaic for microdeletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a previously unknown DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.

Fig. 1. Tiny circular DNA are detected in the extrachromosomal DNA fraction

a. Outward-directed PCR primers (Out) amplified DNA fragments from extrachromosomal DNA (E), but not from genomic DNA (G). DNA was amplified by inward-directed PCR primers (In) from both (E) and (G). b. Sequencing of fragments amplified by Out primers on extrachromosomal fraction. Underlined sequences indicate primers. Junctions between red and blue sequences were the same as that observed in clones in Fig. S2. c. Length distribution of microDNAs from various tissues and cell lines. The library abbreviations are explained in SOM. d. EM of double-stranded microDNA examined by the cytochrome c drop spreading method (16) (50 nm = 150 bp). e. EM of single-stranded microDNA after binding with the T4 gene 32 single stranded DNA binding protein (17).

Fig. 2. Properties of the loci that give rise to microDNAs

a. Enrichment of microDNAs observed in the indicated genomic region relative to the expected percentage based on random distribution. b. Distribution of GC composition in microDNAs in the EMB1 library and their up- and down-stream regions (of same length as microDNA). Vertical line: the genomic average GC content. c. Presence of micro homology near the start and end of a microDNA. "MicroDNA island (blue curve)" is a contiguous stretch of the genome to which the PE-tags map uniquely and correctly. Direct repeats of 2–15 bp (red letters) were observed at the junction of the circle (Upper case) with flanking genomic DNA (Lower case). d. Direct repeats are enriched in different microDNA libraries compared to the random model (RM), generated from the EMB1 sequences. e. Intersection of microDNAs from EMB1 with positioned nucleosome-occupied regions in the mouse liver (13). Obs: observed overlap with nucleosome-occupied DNA. Exp: expected overlap of 1000 randomizations of each microDNA in the library (p<0.0001). A similar enrichment is seen with other microDNA libraries (Fig. S10).

Fig. 3. Microdeletions in genomic loci known to yield microDNAs

a. Algorithm for finding microdeletions in genomic DNA. Details in SOM. b. Micro-deletions found in the KCNK3 locus. DNA spanning the indicated locus was amplified from 200,000 copies of 6 month old mouse brain genomic DNA, and paired-end-sequenced. White square is KCNK3 exon1 and solid line is KCNK3 intron1. Blue squares are positions of microDNAs identified in three independent embryonic brain libraries, and red squares are microdeletions found in the genome in this study. c. Direct repeats observed near the junctions of microdeletions. d. GC composition of the microdeletions identified in the two loci. The deleted sequences were rich in GC content compared to the genomic average of 46%.

Fig. 4. Germline deletions of <1000 bp in the Thousand Genomes Project have properties similar to microDNAs

a. Length distribution peaks at 100 bp and 350 bp. b. Deletions in genic areas are enriched in 5'UTRs, exons, CpG islands and regions 200bp upstream from genes. c. GC content of deletion and up-stream and down-stream regions is greater than genomic average. The up-stream and down-stream sequence was of same length as the deletions. d. 70% of the microdeletions had flanking direct repeats. Length distribution of the direct repeats is shown. Direct repeats ≥15 bp are shown at 15 bp.