Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions

Abstract

We have previously found that the borders of evolutionarily conserved chromosomal regions often coincide with tumor-associated deletion breakpoints within human 3p12-p22. Moreover, a detailed analysis of a frequently deleted region at 3p21.3 (CER1) showed associations between tumor breaks and gene duplications. We now report on the analysis of 54 chromosome 3 breaks by multipoint FISH (mpFISH) in 10 carcinoma-derived cell lines. The centromeric region was broken in five lines. In lines with highly complex karyotypes, breaks were clustered near known fragile sites, FRA3B, FRA3C, and FRA3D (three lines), and in two other regions: 3p12.3-p13 ( approximately 75 Mb position) and 3q21.3-q22.1 ( approximately 130 Mb position) (six lines). All locations are shown based on NCBI Build 36.1 human genome sequence. The last two regions participated in three of four chromosome 3 inversions during primate evolution. Regions at 75, 127, and 131 Mb positions carry a large ( approximately 250 kb) segmental duplication (tumor break-prone segmental duplication [TBSD]). TBSD homologous sequences were found at 15 sites on different chromosomes. They were located within bands frequently involved in carcinoma-associated breaks. Thirteen of them have been involved in inversions during primate evolution; 10 were reused by breaks during mammalian evolution; 14 showed copy number polymorphism in man. TBSD sites showed an increase in satellite repeats, retrotransposed sequences, and other segmental duplications. We propose that the instability of these sites stems from specific organization of the chromosomal region, associated with location at a boundary between different CG-content isochores and with the presence of TBSDs and "instability elements," including satellite repeats and retroviral sequences.

Figure 1.

Distribution of chr 3 features from 3-pter (top, 0 Mb) to 3-qter (bottom, 200 Mb). Megabase position is shown on vertical scale. (A) Approximate positions of known instability regions, including fragile sites FRA3B, FRA3C, and FRA3D (Schwartz et al. 2006) and pericentromeric region (cen). (B) Cumulative profile for 54 break regions detected by mpFISH in 10 carcinoma cell lines (for explanations, see Supplemental Fig. 6). Red arrows show newly identified tumor break-prone regions (TBRs). (C) SDs of different lengths. Blue rhomb (“alignL”), length of aligned duplicated sequence; white spot (“matchB”), number of match-pairs in aligned unit. (D) Dot-plot alignment of the duplicated (>90% homology) sequences longer than 10 kb, to different human chromosomes (shown with different colors) in relation to their megabase position on a chromosome (horizontal axis). (E) Dot-plot alignment of the rhesus orthologous sequences in relation to their megabase position on chromosome (horizontal axis). The human–rhesus synteny breaks, which correspond to human branch evolution, are shown by red horizontal lines. The human–mouse synteny break, which corresponds to a chromosome rearrangement during early primate evolution, is shown by a green horizontal line. (F) Four inversions in human chr 3 evolution: three occurred after divergence from rhesus branch (yellow arrows), the fourth after divergence from mouse (green arrow). (G) SATR1 and SATR2 satellite repeats, flanking the moderate score HERVE element (HERVE M) are associated with the TBRs, long SDs, and evolutionary chr 3 breaks. HERVE L and HERVE H: low and high score HERVE elements, respectively. Horizontal axis: number of the repeat elements within a particular site.

Figure 2.

Two-color FISH of TBR1 probes on normal human chromosomes. DAPI staining (blue) based karyotype. (A) BACs RP11-71K3 and RP11-1053M22 (red in Supplemental Fig. 7E) give red and green FISH signals, respectively, at 3p12-p13, 3q21-q22, 4-pter, 7-pter, 7q21, 8-pter, 11-pter, 11q13, 12-pter, and 16-pter, corresponding to large TBSD. (B) BACs RP11-666K17 and RP11-139H7 (green in Supplemental Fig. 7E), give red and green signals, respectively, at 3p12-p13 and on the short arms of acrocentric chromosomes, suggesting that this region represents an “SD-amplicon” homologous to ribosomal-gene-cluster boundary (RBSD), which was not identified by sequence analysis. Both probes were hybridizing also to the pericentromeric regions of chromosomes 1, 4, 20, and 22, corresponding to SDs 6, 7, and 8 in Supplemental Fig. 7A–D.

Figure 3.

Schematic organization of the TBSDs from different human chromosomal sites as determined by comparison with the chr3:75 TBSD parts (see Supplemental Fig. 7G). All TBSDs, with the exception of those that are marked with a red arrow, are shown in pter–qter direction from bottom to top. Chromosomal segments, which have clearly identified conservation in rhesus genome, are shown in gray with corresponding rhesus chromosome number shown within the bar, and megabase position shown adjacent to the bar. Arrowhead on the bar indicates telomeric location of the rhesus orthologous sequence. Rounded end of the bar shows breakpoint of human–rhesus evolutionary inversions, indicated by black double-head arrows. The orange double-headed arrows show the inversions that occurred within this chromosomal region before human–rhesus, but after human–mouse divergence.

Figure 4.

Two-color FISH of TBR1 probes on orangutan chromosomes. BAC RP11-266L17 (red) identifies orangutan region, orthologous to unique human site telomeric to TBSD (see blue in Supplemental Fig. 7E). (A) Mixed BACs RP11-71K3 and RP11-1053M22 (green) identify TBSD orthologous sequences on two orangutan chromosomes, different from human chr 3 ortholog. (B) Mixed BACs, RP11-666K17 and RP11-139H7 (green), identify RBSD orthologous sequences as multiple signals on the short arms of acrocentric chromosomes. BAC RP11-266L17 signal (red) is not overlapping to green signal, showing that a chromosomal rearrangement occurred within the TBSD site during human–orangutan divergence.

Figure 5.

Frequencies of breaks within regions surrounding the TBSDs during mammalian evolution. (A) Total number of human–rhesus, rhesus–mouse, and mouse–chicken synteny breaks per megabase within regions surrounding the TBSDs (gray) and within random 500-kb sites (black) taken from each 10 Mb of the particular chromosome (chr 3, chr 4, etc) sequence. (B) Percentage of regions, which had human–rhesus (h-rh), rhesus–mouse (rh-m), and mouse–chicken (m-ch) synteny breaks, counted from total number of analyzed 500-kb regions, which surrounded either TBSDs (gray) or random sites taken at each 10 Mb of TBSD containing chromosome sequences (black). (C) Frequency of reuse for synteny breakpoint region at TBSD (gray) and at random synteny break (black). Synteny breakpoint region is defined as a 500-kb region containing a minimum of one break in either human–rhesus or in rhesus–mouse or in mouse–chicken branch. Reuse of the synteny breakpoint region is defined as the occurrence of breaks in at least two of three mentioned branches during evolution. (D) Gradual decrease in frequency of rhesus–mouse and mouse–chicken synteny breaks with increase of distance from TBSD sites. Cumulative area shows the number of breaks within the 1st, 2nd, and 3rd sequence windows (point 1, 2, and 3, respectively) from different TBSD locations. For each TBSD location, windows 1, 2, and 3 represent successive sequence windows equal in size to TBSD, which flank TBSD (1), located next to both flanks (2), and most distant windows (3).

Figure 6.

Sequence features in the region surrounding TBSD. Cumulative area shows contents (in percentages) of particular sequence features, which were identified in different TBSD sites and in three successive sequence windows of the same size departing from the TBSD sites (points 1, 2, and 3, respectively). Red arrowheads show cumulative value for the same number of random regions, calculated based on average values for the human genome. Regions are pter–qter oriented, as described in the Results section, “TBSDs are located at the transitions between CG- rich and poor areas.”

Figure 7.

Isochore surrounding of TBSDs. Average GC levels were assessed over 500-kb DNA stretches distal and proximal to each TBSD. The colored boxes show correspondence of these stretches to different class isochores (see at right). (Ovals) TBSD; (black circles) telomeres; (double-headed arrows) recent human branch-specific evolutionary inversions. In the case of chr 3, three successive inversions changed the orientation of TBSD regions. In the case of chr 7, one additional inversion changed the orientation of the chr7:97 region as shown by the green arrow.