Single Chromosome Aneuploidy Induces Genome-Wide Perturbation of Nuclear Organization and Gene Expression

Abstract

Chromosomal aneuploidy is a defining feature of carcinomas and results in tumor-entity specific genomic imbalances. For instance, most sporadic colorectal carcinomas carry extra copies of chromosome 7, an aneuploidy that emerges already in premalignant adenomas, and is maintained throughout tumor progression and in derived cell lines. A comprehensive understanding on how chromosomal aneuploidy affects nuclear organization and gene expression, i.e., the nucleome, remains elusive. We now analyzed a cell line established from healthy colon mucosa with a normal karyotype (46,XY) and its isogenic derived cell line that acquired an extra copy of chromosome 7 as its sole anomaly (47,XY,+7). We studied structure/function relationships consequent to aneuploidization using genome-wide chromosome conformation capture (Hi-C), RNA sequencing and protein profiling. The gain of chromosome 7 resulted in an increase of transcript levels of resident genes as well as genome-wide gene and protein expression changes. The Hi-C analysis showed that the extra copy of chromosome 7 is reflected in more interchromosomal contacts between the triploid chromosomes. Chromatin organization changes are observed genome-wide, as determined by changes in A/B compartmentalization and topologically associating domain (TAD) boundaries. Most notably, chromosome 4 shows a profound loss of chromatin organization, and chromosome 14 contains a large A/B compartment switch region, concurrent with resident gene expression changes. No changes to the nuclear position of the additional chromosome 7 territory were observed when measuring distances of chromosome painting probes by interphase FISH. Genome and protein data showed enrichment in signaling pathways crucial for malignant transformation, such as the HGF/MET-axis. We conclude that a specific chromosomal aneuploidy has profound impact on nuclear structure and function, both locally and genome-wide. Our study provides a benchmark for the analysis of cancer nucleomes with complex karyotypes.

Figure 1

(A) SKY analysis of the HCEC+7 cells. The trisomy of chromosome 7 is the sole cytogenetic anomaly. (B) Differential, global, chromosome-wide gene expression between HCEC and HCEC+7 cells. Upregulation (green) is most profound on chromosome 7, however, changes were not restricted to the aneuploid chromosome.

Figure 2

Hi-C maps show an increase in chromosome 7 contacts genome-wide. (A) Genome-wide Hi-C contact maps for HCEC (left), HCEC+7 (right), and the difference between the two samples (center). Matrices are shown at 1 Mb resolution and log-scale. Red regions in the middle matrix are enriched in HCEC+7, blue regions in the middle matrix are enriched in HCEC. A clear increase in HCEC+7 contacts is observed in regions involving chromosome 7 (green arrow). (B) Chromosome 7 Hi-C contact maps for HCEC (left), HCEC+7 (right), and the difference between the 2 samples (center). Matrices are shown at 1 Mb resolution and log-scale. Red regions in the middle matrix are enriched in HCEC+7, blue regions in the middle matrix are enriched in HCEC. A clear increase in HCEC+7 contacts is observed for the majority of intra-chromosomal contacts in chromosome 7.

Figure 3

HCEC+7 results in genome-wide structural changes. (A) A change in chromatin partitioning is observed in chromosome 14. The left and right side show HCEC and HCEC+7, respectively. The top row shows the average gene expression for each 100 kb bin. The middle row shows the Fiedler vector partitioning. A clear change is observed between 425 and 450 (blue square). The bottom row is the Hi-C contact map for chromosome 14, shown at 100 kb resolution, log-scale. (B) HCEC+7 shows a clear change in chromosome 4 patterning. The left and right side show HCEC and HCEC+7, respectively. The top row shows the Fiedler vector partitioning. The middle row is the Hi-C contact map for chromosome 4, shown at 100 kb resolution, log-scale. Squares (green and blue) along the diagonal of the Hi-C matrix depict the TAD structure. A direct comparison of the TAD structure between samples is shown below.

Figure 4

3D-FISH image analysis of chromosome territories (CT). (A) Cell nuclei were automatically selected from DAPI images. (B) Fluorescent channels corresponding to chromosome 7 and 19 were analyzed, and CTs were automatically selected. (C) CT size, distance from CT to nucleus edge (d_1), and distance between CTs (d_2) was extracted.

Figure 5

Identification of differentially regulated pathways. (A) GSAA analysis. The top four signaling networks that were enriched in the HCEC+7 cells are shown; P53-pathway (0.003), MYC-targets (0.003), TNF-α via NF-κB (0.126), KRAS signaling up (0.160). (B) IPA network analysis of differentially expressed proteins identified by 2-DIGE and MS. Red indicates over-expression, green indicates under-expression.