Nucleosome repositioning in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is an intrusive malignant tumour that accounts for 60% of brain tumours in adults. GBM is aggressive and has a poor prognosis. Astrocytomas are derived from a type of brain cell called astrocyte, a star-shaped cell. Genetic or epigenetic changes in astrocytes cause abnormalities in gene expression that lead to the development of GBM (grade 4 astrocytomas). One of the important epigenetic regulators is nucleosome positioning along the genome because it determines the accessibility of DNA to regulatory proteins and can be changed in cancer. Apart from fundamental interest, nucleosome positioning is important from a practical point of view for the development of diagnostic methods based on cell-free DNA (cfDNA), which usually enters body fluids originating from apoptotic cells whose DNA fragments are protected from digestion by nucleosomes. In this project, I investigate differences in nucleosome positioning between paired cancer and healthy tissues from patients with GBM and compare these with cfDNA from blood plasma and cerebrospinal fluid. Micrococcal nuclease sequencing (MNase-seq) experiments were used for all internal dataset samples. MNase-seq involves the digestion of chromatin with micrococcal nuclease to cleaver linker DNA between nucleosomes. The results of my analysis show cancer-specific changes in nucleosome positioning and can support the use of cfDNA as a non-invasive method for the diagnosis of GBM. My analyses include the investigation of the effects of GC content and DNA fragment size distributions. The GC content of DNA fragments resulting from MNaseseq increases with the increase of the DNA fragment size. The calculation of the nucleosome repeat length (NRL) showed no significant NRL differences between normal and tumour tissues. I also investigated nucleosome occupancy profiles around binding sites of transcription factors CTCF, ASCL1, and KLF9. These profiles had similar shapes for normal and tumour samples, suggesting caution for the TF-based cfDNA analysis.