DNA copy number evolution in Drosophila cell lines

Background Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. Results Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). Conclusion Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1186/gb-2014-15-8-r70) contains supplementary material, which is available to authorized users.

Bottom panel. All cells exhibit large marker chromosome generated by an X-2 translocation (2t). This chromosome contained an almost entire 2 nd chromosome with the left arm conjoined to the left arm of an almost entire X chromosome, which lacked the centromere but retains some pericentromeric heterochromatin. The large X-2 translocation element was accompanied by various combinations of regular or rearranged metacentric autosomes, including a 3 rd chromosome carrying a deficiency of part of 3L euchromatin, a 2 nd chromosome with an abnormally short arm (it was impossible to assess which 6 arm was partially deleted), a 3 rd chromosome lacking the entire 3R arm (3r), and a 2 nd chromosome lacking the entire 2R arm (2r). The X and 4 th chromosomes looked wild-type in morphology.

Sex.
In the 10 karyograms analyzed the average X:A ratio was 0.88 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a female karyotype and expression pattern.

Right panel.
The most cells exhibited four large metacentric chromosomes. In most cases, one of the 2 nd chromosomes had abnormally long 2L arm and the other an abnormally short 2L arm (2r). These rearranged chromosomes were probably the result of a translocation. We also observed two types of X chromosomes displaying a small difference in the length of the euchromatic long arm (b). Karyograms displayed two X chromosomes from the most of cells, as well as two 4 th chromosomes.
Sex. In the 18 karyograms analyzed the average X:A ratio was 0.98 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a female karyotype and expression pattern.

D20-c5 (derived from antennal disc):
Top panel. 80% of the metaphases contained 11-14 chromosomes (four 3 rd chromosomes four 2 nd chromosomes, 2 X chromosomes and a variable number of 4 th chromosomes). 12% exhibited more than 20 chromosomes and were probably derived from polyploidization of cells with 11-14 chromosomes.

Bottom panel.
In most of the cells, the chromosomes exhibited relatively normal morphology, but the pericentric heterochromatin of one pair of 2 nd chromosomes was longer (clear in b) that has been seen in Oregon R. We believe that this peculiar pericentric region reflects a natural polymorphism rather than a rearrangement. In one karyogram, there was a 2 nd chromosome with a deletion, and a likely X-3 (or a 4-3 translocation) (d).
8 Sex. In the 20 karyograms analyzed the average X:A ratio was 0.47 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a male karyotype and expression pattern.

Bottom panel.
In most cells, we observed a translocation that appeared to involve the 2 nd and the X chromosome and attached most of XL to the terminus of 2R (2t). The large and the small element of this translocation were present in 40% and 7% of the cells examined, respectively. The translocation elements were independently distributed among the cells, and metaphases carrying both elements were rare (a). The autosomes not involved in the translocation had normal appearance, while the free X chromosome exhibited a heterochromatic block bigger than that of a wild-type X (Xr) (b, d).
Sex. In the 16 karyograms and 40 metaphases analyzed the average X:A ratio was 0.73 where we considered translocation's largest element was carrying most of XL (Table S1). Specifically, 23 cells displayed a male karyotype and 27 a female karyotype. The sequencing data indicated a male karyotype and expression pattern.
Note. Somatic pairing was maintained in most cells.

D8 (derived from larval stage 3 wing disc):
Top panel. 68% of the metaphases contained 8 chromosomes and exhibited a normal diploid karyotype.
19% exhibited 7 chromosomes. Of the 15 cells with 7 chromosomes, 13 lacked one of the fourth chromosomes and 2 lacked one of the X chromosomes. Most of the hyperploid cells had additional X or 4 th chromosomes.

Bottom panel.
In most cells, the autosomes and the X chromosomes looked wild-type in morphology but the 4 th chromosomes were abnormal (4r in b, c). In one karyogram there was a complex rearrangement involving the X chromosome and possibly the 2 nd chromosome (c).

Sex.
In the 16 karyograms analyzed the average X:A ratio was 0.98 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a female karyotype and expression pattern.
Note. The homologous chromosomes of this line were somatically paired as in wild-type brain cells.

Bottom panel.
Most metaphases exhibited 7-8 major autosomes including normal 2 nd and 3 rd chromosomes and abnormal derivatives of these autosomes. These derivatives included a 3 rd chromosome carrying a deletion of part of 3R, a 3 rd chromosome carrying a deletion of an entire arm, a 2 nd chromosome carrying a deletion of an entire arm, and two additional rearranged and shorter derivatives of either the 2 nd or the 3 rd or the chromosome. All cells contained an X chromosome longer 12 than its wild-type counterpart and variable number of potential X derivatives including a relatively large acrocentric chromosome bearing two blocks of fluorescent pericentric heterochromatin and two smaller chromosomes that could be either X chromosomes carrying a deletion of part their euchromatin, or elements of translocations involving the 4 th chromosome and either the X or the major autosomes (c, d).
The second alternative was supported by the fact that the average number of free 4 th chromosomes was quite low (0.7/cell). We observed several additional rearranged chromosomes of uncertain origin including an element that looked like a partially deleted Y chromosome (nd).

Sex.
Given the difficulty in recognizing the X chromosomes we could not determine the X:A ratio. The sequencing data indicated X:A ratio of 0.86, but with male-like gene expression pattern.

Bottom panel.
In most cells, they exhibited four 3 rd chromosomes and four 2 nd chromosomes but these chromosomes were often rearranged (2r and 3r). In addition they carried small derivatives of the major autosomes (a, b). We observed 3 rd chromosomes with a deficiency of part of 3L, 3 rd chromosomes with deficiencies of both 3L and 3R, a 3 rd chromosome lacking the entire 3L arm, and 2 nd chromosomes carrying a deficiency of 2L (3r and 2r). The X chromosomes varied in number (from 3 to 4) and morphology. One of the X chromosomes exhibited a fluorescent heterochromatic block bigger than that of a wild-type X chromosome, while some X chromosomes had heterochromatic blocks smaller than 14 those seen in wild-type (Xr). Some X chromosomes were deleted and contained the proximal heterochromatic block. The dot-like chromosomes varied in morphology and number, ranging from 2 to 5. Some of these small and brightly fluorescent chromosomes were bigger than a normal 4 th chromosome (a-c). They could result from rearrangements involving two 4 th chromosomes or could be grossly deleted X chromosomes that carry just the most florescent part of X heterochromatin.

Sex.
In the 12 karyograms analyzed the average X:A ratio was 0.99 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a female karyotype and expression pattern.

Right panel.
In most cells, all chromosomes looked wild-type in morphology. We observed cases of two 4 th chromosomes, or a single X chromosome and no 4 th chromosomes (from two, or one out of 36 mataphases, respectively) Sex. In the 36 metaphases analyzed the average X:A ratio was 0.95 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a male karyotype and expression pattern.

Bottom panel.
In most cells, we observed a pair of irregular 2 nd chromosomes (both carrying an abnormally long 2R arm), a pair of 3 rd chromosomes (one regular and the other carrying a deficiency that removed part of 3R euchromatin), an acrocentric chromosome that looked like a 2L arm (2r) based on heterochromatin banding pattern. This observation was consistent with DNA-Seq data that indicated an excess of 2L sequences compared to 2R, 3L and 3R sequences. A single regular X chromosome, a Y chromosome and two 4 th chromosomes were found from the most karyograms, yet the number of dot chromosome was variable ranging from 2 to 3.

Sex.
In the 12 karyograms analyzed the average X:A ratio was 0.51 (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a male karyotype and expression pattern.

Note.
In several metaphases the Y chromosome had an unusual appearance, as it exhibited sister chromatid separation in the short arm region that included the ribosomal genes and, less frequently, in the long arm region that contains the cry/su(Ste) sequences. This separation was never seen in the Y chromosome of larval neuroblasts. However, in wild-type primary spermatocytes, where the Y is functioning, the Y sister chromatids are well separated (like those of the X chromosome and the autosomes). Thus, it is possible that the Y regions that exhibited sister chromatid separation in S1 cells are transcriptionally active.
Bottom panel. Many of the cells exhibited four 3 rd chromosomes, some of which carried a pericentric inversion, and four 2 nd chromosomes, some of which carried a deficiency of part of 2L euchromatin. We observed one or two acrocentric chromosomes that looked like 2L and 3R arms based on heterochromatin banding pattern. One to three small acrocentric chromosomes that were probably derived from metacentric autosomes were observed. Karyograms exhibited two X chromosomes whose heterochromatic blocks were bigger than wild-type X chromosomes (Xr). Some cells displayed a short mbn2 (derived from hemocytes): Top panel. This line was characterized by extremely high karyotypic variability and by the presence of spontaneous chromosome breakage. Chromosome counts were carried out without distinguishing between broken and intact chromosomes. The result showed that most cells were polyploid and contained between 16 and 30 chromosomes.

Bottom panel.
A wide variety of chromosome types were observed. They included a 3 rd chromosome carrying a deletion of the entire 3L arm, a 3 rd chromosome carrying deletions in both arms, a 3 rd chromosome carrying a pericentric inversion, an entire 2 nd chromosome conjoined to a euchromatic arm of a major metacentric chromosome (an element of a translocation) (d), an entire 2 nd chromosome conjoined with a small piece of heterochromatin (an element of a translocation), an acrocentric chromosome generated by a deletion of the entire arm of a major autosome (this deleted chromosome could be either 2L or 3R) (e), several small acrocentric chromosome probably derived from the major autosomes (f). We observed regular X chromosomes as well as an X chromosome with the long arm conjoined with autosomal euchromatic material (an element of a translocation) (d-f). Several types of dot chromosomes were observed, yet some of them appeared to be regular 4 th chromosomes. All these chromosomes were found in various combinations in metaphases that often displayed chromosome breaks Sex. In the 16 karyograms analyzed the average X:A ratio was 0.54 where we considered the amount of autosomal material and not the number of metacentric autosome elements (Table S1). The observed karyotype agrees with the sequencing data indicating that this line has a male karyotype and expression pattern.