Nucleosome repositioning in chronic lymphocytic leukemia

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral-blood B cells from patients with chronic lymphocytic leukaemia (CLL) and healthy individuals at single base pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes – the nucleosome repeat length (NRL) – was shortened in CLL. This effect was stronger in the more aggressive IGHV-unmutated than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites were linked to active chromatin remodelling and reduced DNA methylation. Nucleosomes lost or gained in CLL marked differential binding of 3D chromatin organisers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to monitor disease progression. We have performed genome-wide comparison of nucleosome positioning in B-cells from peripheral blood of CLL patients and healthy donors at a sequencing depth of more than 4 billion DNA reads. The median age of the CLL patients was 60 years (range, 47 to 75). Non-malignant control samples were enriched from peripheral blood of in total 36 age-matched donors (median, 58 years, range, 51 to 70) that were collected in 9 sample pools. All samples, from CLL patients and from non-malignant donors, were enriched for CD19-positive B cells. The purity of the CD19-positive fraction was 98 ± 2% in non-malignant donor samples and 99 ± 1% in CLL patient samples. 79% of CLL patients had favorable genetic aberrations [del(13q) with deletions of SF3B1 and ATM] as sole abnormality associated with increased survival times, 21% had normal karyotype. 71% of the patients were diagnosed with hypermutated IGHV associated with good prognosis. Due to the high numbers of B-cells required for multiple sequencing, we selected asymptomatic CLL patients who had been approached with the watch-and-wait strategy. None of the patients analyzed had previous treatment of the CLL disease, thereby high numbers of CLL cells unaffected by the potential impact of prior therapy had accumulated in the peripheral blood with a median leukocyte count of 105 x 109/L (range, 82 to 187 x 109/L). Patient derived cells were crosslinked with 1 % methanol-free formaldehyde for 10 min. After quenching with glycine, cells were washed three times with PBS and the cell pellet was frozen in liquid nitrogen. For analysis, the cell pellet was thawn and treated with 4U MNase per 1x106 cells for 15 min. MNase was stopped with 10x covaris buffer and the chromatin was sheared for an additional 15 min with the S2 covaris device and the soluble chromatin was then recovered and subjected to a background-minimizing pre-clearing step with an unspecific IgG antibody. For each ChIP-assay an equivalent of 3x106 cells were used with the following antibody: H3 (Abcam, ab1791). After the IP, chromatin was digested with RNaseA and proteinase K. The purified DNA was cloned into illumina sequencing libraries with the NEBNext Ultra library preparation kit (NEB). Original sequencing data for all samples are available at the European Genome-phenome Archive (http://www.ebi.ac.uk/ega/), hosted by the European Bioinformatics Institute under the accession number EGAS00001002518.