Pan-cancer analysis of somatic mutations and epigenetic alterations in insulated neighbourhood boundaries

Abstract

Recent evidence shows that the disruption of constitutive insulated neighbourhoods might lead to oncogene dysregulation. We present here a systematic pan-cancer characterisation of the associations between constitutive boundaries and genome alterations in cancer. Specifically, we investigate the enrichment of somatic mutation, abnormal methylation, and copy number alteration events in the proximity of CTCF bindings overlapping with topological boundaries (junctions) in 26 cancer types. Focusing on CTCF motifs that are both in-boundary (overlapping with junctions) and active (overlapping with peaks of CTCF expression), we find a significant enrichment of somatic mutations in several cancer types. Furthermore, mutated junctions are significantly conserved across cancer types, and we also observe a positive selection of transversions rather than transitions in many cancer types. We also analyzed the mutational signature found on the different classes of CTCF motifs, finding some signatures (such as SBS26) to have a higher weight within in-boundary than off-bounday motifs. Regarding methylation, we find a significant number of over-methylated active in-boundary CTCF motifs in several cancer types; similarly to somatic-mutated junctions, they also have a significant conservation across cancer types. Finally, in several cancer types we observe that copy number alterations tend to overlap with active junctions more often than in matched normal samples. While several articles have recently reported a mutational enrichment at CTCF binding sites for specific cancer types, our analysis is pan-cancer and investigates abnormal methylation and copy number alterations in addition to somatic mutations. Our method is fully replicable and suggests several follow-up tumour-specific analyses.

Fig 1. Classification of CTCF motifs, within a short portion of chromosome 11.

Motifs are classified as active (confirmed by a CTCF ChIP-seq peak) and inactive (not confirmed). Active motifs are further divided into in-boundary and off-boundary according to whether they overlap a boundary, as defined by a ChIA-PET experiment.

Fig 2. Somatic mutation frequency in a range of (-1kbp, +1kbp) surrounding (a) an active in-boundary and (b) an active off-boundary CTCF motifs in eEsophageal adenocarcinoma (ESAD).

A central peak overlapping the 19bp CTCF motif is evident in (a).

Fig 3. Distribution of the number of mutations on active in-boundary CTCF motifs within and outside promoters and exons in Esophageal Adenocarcinoma using the hESC cell line.

A higher frequency of mutations is found outside promoters and exons, ruling out the possibility that the enrichment of somatic mutations in active CTCF motifs is due to their higher probability of having open chromatin.

Fig 4. Genes close to mutated CTCF in-boundary motifs in melanoma.

The height of the bars represents the number of samples in which the gene is close to a mutated in-boundary active CTCF motif. We considered only genes with top mutations whose TSS is within 180 kbp from the center of the mutated motif.

Fig 5. Mutations in active in-boundary CTCF motifs and flanking regions (19 bp ±50 bp).

Clear peaks are observed within CTCF motifs in all cancers types. Additional peaks are observed in the flanking regions of the CTCF motifs in Liver Cancer, Esophageal Adenocarcinoma and Breast Cancer tumour types (sub-plots A,B,C).

Fig 6. The average contributions or weights (i.e., exposures) of mutational signatures for the Esophageal Adenocarcinoma dataset.

Signature refitting was done based on mutations falling a) in-boundaries motifs, b)in the whole genome, and and then c) on the difference between the two.

Fig 7. A representative distribution of delta methylation values on CTFC in-boundary motifs for Breast Cancer.

Hypo-methylated refers to probes with a delta beta value lower than -0.2, hyper-methylated to probes with a delta beta value greater than 0.2 and normal-methylated to all the others. The x-axis reports the CTCF motif. Two distinguishable methylation peaks are observed at positions 4 and 15, corresponding to the presence of 2 pairs of CpG dinucleotides.