GAPF-positive regions cluster in copy-number gains in cell lines Colo320DM and MCF7. Genome graph depicting locations of GAPF-positive regions (P < 0.001, log₂ signal ratio > 1.5; triangles) and copy-number gains (log₂ signal ratio > 0.3; boxes) across the genomes of colon-cancer cell line Colo320DM and breast-cancer cell line MCF7 as compared with cultured HFs. GAPF-positive regions and copy-number gains detected in Colo320DM are shown above and regions identified in MCF7 are shown below each chromosome ideogram. Clusters of at least three GAPF-positive regions that had a <5% probability of randomly occurring within a 10-Mb window are marked by asterisks (*). Chromosomes are drawn approximately to scale.

GAPF-positive regions located in copy-number gains in 16q24.1 (A) and 8q24.21 (B) in Colo320DM and MCF7. Locations of GAPF-positive regions (P < 0.001, log₂ signal ratio > 1.5) are denoted by the dark bars under the axes and the copy-number gains (log₂ signal ratio > 0.3) by the lighter boxes above the axes, with the height of the box corresponding to the log₂ signal ratio of the segment. GAPF-positive regions were determined by tiling array analysis of Colo320DM and MCF7 compared with cultured human fibroblasts. The copy-number segments were identified with SNP arrays coupled with wavelet-based statistical analysis. Regions in Colo320DM are displayed above the corresponding regions for MCF7 (as labeled). (A) The GAPF-positive regions detected in 16q24.1 in MCF7 were located in a copy-number gain, one of which was located near the boundary of the segment. In contrast, there was no amplification of 16q24.1 in Colo320DM, but one GAPF-positive region was identified. (B) The amplification in 8q24 in Colo320DM contains many GAPF-positive regions, while MCF7 has one GAPF-positive region located at a copy-number breakpoint.

Validation of a de novo DNA palindrome in chromosome 8q24.21 of breast cancer cell line MCF7. (A) GAPF analysis on tiling arrays comparing MCF7 with cultured HFs. The lower graph displays GAPF P-values (−10log₁₀). The solid bars under the graphs represent GAPF-positive regions (P-value [−10log₁₀] > 30, run > 50 bp, gap < 100 bp). The upper graph displays wavelet-derived copy-number segments (average log₂ signal ratio). Segments with a log₂ signal ratio >0.3 are designated as gained. The locations of PCR primers used in the PCR-enrichment analysis are shown. (B) PCR-based analyses to detect enrichment of genomic loci in 8q24.21 over the nonpalindromic region (ARNT) following GAPF preparations of MCF7 (MCF7 GAPF) and pooled PBL cells (Norm GAPF). Analysis of a normally occurring inverted repeat (IR) and nonpalindromic region (MYOD) are included to confirm efficient GAPF preparations. Note that the only loci that are enriched by GAPF are IR in MCF7 and pooled PBLs, and PCRB in MCF7, located in the GAPF-positive region. (C) PCR-enrichment analysis following targeted restriction-enzyme digestion preceding GAPF. Genomic DNA from MCF7 cells was predigested with SpeI, NsiI, PmeI, or EcoRV, shown on the map, and processed by GAPF. Enrichment of the GAPF-positive region in 8q24.21 over the nonpalindromic ARNT region was examined using two primer pairs, Cent and Tel. Digestion in the nonpalindromic spacer of a palindrome or IR will eliminate enrichment of the sequence by GAPF, as shown by the lack of PCR product when DNA was first digested with NsiI and enrichment of the IR was assessed (bottom gel). Note that enrichment of the 8q24.21 region was seen with the Cent primers when MCF7 DNA was digested with SpeI, but not observed when the Tel primers were used. Based on these analyses, the inferred location and orientation of palindrome places the palindromic center between SpeI and EcoRV restriction sites, shown below the map. (D–F) Southern analysis to detect rearrangements of the GAPF-positive region in 8q24.21. (D) Genomic DNA from MCF7 (M) and normal human fibroblast cell line IMR90 (I) was digested with EcoRI (R), NcoI (Nc), NheI (Nh), or EcoRV (V) shown on the map (F). Digesting genomic DNA from MCF7 with NcoI and NheI and hybridizing with a probe in the GAPF-positive region in 8q24.21 yielded abnormally sized fragments of 5 and 7 kb, respectively (white arrowheads), which differed from the expected 12-kb fragments based on the normal genome sequence. Note that we also detected normal-sized fragments, indicating the presence of rearranged and normally arranged alleles. (E) Snapback Southern to confirm palindromic nature of GAPF-positive region in 8q24.21. Genomic DNA from MCF7 (M) and IMR90 (I) were first digested with either NcoI or NheI. The digested samples were evenly split and half of the sample was processed through GAPF to create snapback DNA, shown in the + lanes. Note that the rearranged fragments in MCF7 (white arrowheads) were converted to half-sized fragments (black arrowheads) following GAPF. (F) Inferred restriction enzyme map of normal and palindromic loci is shown with the location of the palindromic center marked by the gray arrow. Probe location is denoted on inferred map.

Distribution of GAPF-positive regions and copy-number gains on chromosomes 8, 11, and 12 in breast cancers. Genome graphs of GAPF-positive regions (P < 0.001, log₂ signal ratio > 1.5; triangles) and copy-number gains (average log₂ signal ratio > 0.3; boxes) in primary invasive ductal carcinomas (A) and breast cancer cell lines (B). Locations of GAPF-positive regions were determined from tiling array analysis comparing breast cancer samples with pooled PBLs. Copy-number gains were detected by SNP arrays coupled with wavelet-based statistical analyses. Clusters of at least three GAPF-positive regions that had a <5% probability of randomly occurring within a 5-Mb window are marked by asterisks (*). Note the colocation of clusters and copy-number gains, especially in the primary tumors.

Highly amplified regions in an invasive ductal carcinoma have palindromes at amplicon breakpoints. GAPF analysis on tiling arrays of the invasive ductal carcinoma (IDC) ERP1 compared with the normal, pooled PBL reference. Graphs display GAPF P-values (−10log₁₀) and wavelet-derived copy-number segments (average log₂ signal ratio). The solid bars under the graphs represent GAPF-positive regions (P-value [−10log₁₀] > 30, run > 50 bp, gap < 100 bp). The dashed line marks where log₂ signal ratio = 1.5. (A) A highly amplified region (log₂ signal ratio > 1.5) in 8q21.13 in the IDC ERP1 has GAPF-positive regions located throughout the amplicon and at the breakpoints (arrows). (B,C) PCR-based enrichment analyses of GAPF-positive regions following targeted restriction-enzyme digestion prior to GAPF. Genomic DNA from ERP1 was digested with SbfI, SwaI, NcoI, or SpeI and processed by GAPF. The GAPF-positive regions at the centromeric (B) and telomeric (C) boundaries of the amplicon were assessed for enrichment over a nonpalindromic region (ARNT) using primer pairs Cent PCR and Tel PCR, respectively. A known inverted repeat (IR) was assessed to confirm enrichment of palindromic sequences by GAPF. Also assessed was ERP1 DNA not processed through GAPF (gDNA) and processed through the standard GAPF protocol (GAPF). The inferred orientations of the de novo palindromes are shown below the maps of the restriction enzymes, each placing the palindromic junction at the wavelet-derived breakpoint (P < 0.1).

Shared GAPF-positive regions in breast cancer cell lines. (A) Venn diagram depicting the number of overlapping GAPF-positive regions (P <0.001, log₂ signal ratio > 1.5) between four breast cancer cell lines MCF7, BT474, UACC893, and MDA231. (B) GAPF-positive region in 12p13 shared between UACC893 (lower graph) and MDA231 (upper graph) containing a normally occurring inverted repeat (Warburton et al. 2004). Graphs display P-values (−10log₁₀) of GAPF analyses comparing cell lines with normal PBL reference. The solid bars under the graphs mark GAPF-positive regions (P-value [−10log₁₀] > 30, run > 50 bp, gap < 100 bp). (C) A GAPF-positive region was present in UACC893 (upper graphs) and BT474 (lower graphs) at the same location and associated with copy-number gain. Graphs display GAPF P-values (−10log₁₀) and wavelet-derived copy-number segments (average log₂ signal ratio). Copy-number gains are defined by the shaded boxes (log₂ signal ratio > 0.3).