Unraveling the internal structure of neocentromeres in ring chromosomes harboring genomic amplification in soft tissue tumors

Neocentromeres have been reported as frequently arising on ring/rod-shaped chromosomes, particularly in sarcomas. It has been reported that the CENP-A centromeric protein is rapidly recruited at doublestrand break (DSB) by the DNA repair machinery. This finding suggested a potential link between the process of DSB repair and neocentromere seeding. We studied four cancer cell lines (three well-differentiated liposarcomas and a lung-sarcomatoid carcinoma) carrying neocentromeres on ring/rod-shaped chromosomes, to investigate this phenomenon. SNP array, FISH, and anti-CENP-A ChIP-seq experiments were performed to characterize the marker chromosomes and the neocentromeric domains. Whole genome nextgeneration sequencingwas also carried out to finely define the inner structure of the marker chromosomes. Our results revealed that neocentromeres were seeded on a “patchwork” of short-sized (<100 Kb) amplified fragments, some from different chromosomes, underlining the epigenetic nature of this phenomenon and suggesting that the massive recruitment of CENPA to repair DSB might trigger the neocentromere formation. We are currently evaluating the chromatin status of the neocentromeric regions by performing targeted bisulphite sequencing and H3K4me1-3/H3K4me27 ChIP-seq assays. Furthermore, we are performing RNA-Seq experiments to identify transcripts likely involved in the neocentromeres seeding and maintenance. Moreover, two of our liposarcoma cell lines derive from the same primary tumour; this will allow us to investigate the evolution of marker chromosomes and neocentromere from the original tumour. Finally, several cases of soft tissue tumours carrying neocentromeres will be analysed to gain a broader view of this phenomenon in primary malignant sarcomas. The expected results will unveil the existing link between DSB repair and neocentromere seeding, and add new insight into the mechanisms behind the genesis of ring/rod-shaped marker chromosomes.