Ectopic CENP-A clusters at a large domain at the subtelomeric 8q24/
locus in colorectal cancer cells and tumors. (A) CENP-A hotspots cluster (indicated by gray boxes, additional examples in Figure
7) at subtelomeric regions of chromosome 8 in SW480 cells, but not in normal colon cells. (B) Chromatin immunoprecipitation (ChIP)-seq profiles (tag density peaks) and input-adjusted hotspot analysis (vertical bars below each profile) demonstrate CENP-A distribution relative to input chromatin upon the 30 MB domain spanning the cytogenetic band 8q24 in SW480, HeLa, and normal colon cells (note that input tag density peaks reflect increased copy number of this region in SW480 cells, but hotspot analysis takes into account copy number variation). Base numbers above indicate the genomic location, and horizontal lines below the genome browser profile indicate position of fluorescent in situ hybridization (FISH) probes used for FISH/co-IF in (C). (C) Upper left panel: qtPCR graph demonstrating CENP-A and CENP-C enrichment at the 8q24 locus in SW480 cells (positions of PCR primers are indicated in (A), negative control (NC) primers were selected from chromosomes 1 and 11 from regions with no CENP-A peaks based on ChIP-seq data). Upper right panel contains cytological analysis of metaphase chromosome spreads stained with FISH probe for 8q24 (in green), indicating amplification of this region in SW480 cells. One of these translocated 8q24 loci (green) associates with CENP-A (red) (middle left panel) and the inner kinetochore protein CENP-C (red) (middle right panel). Combining FISH and IF in three primary colon tumors demonstrates that CENP-A (red) co-localizes with one of the amplified regions of 8q24/Myc (green) in the tumor (right bottom panel) but not in normal tissue (left bottom panel). White insets and arrowheads point to co-localization spots detected by Image J automated co-localization algorithm. Quantification is provided in Table
7. (D) Additional controls for the FISH/IF experiments in (C). First panel depicts FISH of 8q24 to metaphase spreads of normal human lymphocytes, demonstrating specific hybridization to 2 subtelomeric locations. Second panel depicts Co-IF/FISH of native centromere 8 and CENP-A on metaphase chromosome spreads in SW480 cells demonstrating that native centromere 8 still contains CENP-A and is active. Remaining panels demonstrate that CENP-A co-localizes to one of the amplified and translocated regions of 8q24/Myc in SW480 cells (right panels), but not in HeLa (left panels) cells. Quantification of this data is presented in Table
7. Merge of DAPI (blue or gray), 8q24 or Myc (green) and CENP-A (red) is indicated for each cell line at the top of each image. Automated co-localization analysis was performed using Image J; white is indicative of co-localization shown as insets.