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. 2012;7(5):e37422.
doi: 10.1371/journal.pone.0037422. Epub 2012 May 14.

Genome-wide gene amplification during differentiation of neural progenitor cells in vitro

Ulrike Fischer  1 , Andreas Keller, Meike Voss, Christina Backes, Cornelius Welter, Eckart Meese
Affiliations

Genome-wide gene amplification during differentiation of neural progenitor cells in vitro

Ulrike Fischer et al. PLoS One. 2012.

Abstract

DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Whole chromosome plots.
A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.
Figure 2
Figure 2. FISH analysis of amplified loci.
For each FISH analysis, a BAC or cosmid clone containing the indicated gene was Cy3-labelled (pink) and hybridized against fixed NHNP cells that were differentiated for either 2, 5 or 7 days. Amplifications are shown for C1QL1 RP11-113A24 (5 days), SOX13 RP11876H8 (5 days), HDGF RP11-66D17 (5 days), CYP27B1 cosmid (2 days), GINS2 RP11-118F19 (2 days), CDK4 RP11-571M6 (2 days), TP53 RP11-1081A10 (7days), DIABLO RP11-568C23 (7 days). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm. Notably, the degree of amplification various within each analysis due to the high heterogeneity of the amplifications in each cell population.
Figure 3
Figure 3. Detailed gene amplification analysis on human chromosome 16 and 12.
Representative sections of log2 ratio profiles for undifferentiated (0 d) NHNP cells and cells that were differentiated for 2 and 5 days. Base count is given on the x-axis and log2 ratio on the y-axis for chromosome 16q24.1 (A) and 12q14.1 (C). Chromosomal localization of BAC probes used for FISH were indicated at the bottom of figures C and D. A GINS2 specific BAC probe that was labeled in pink and a CDH13 specific BAC probe that was labeled in green were hybridized simultaneously against fixed NHNP cells that were differentiated for 2 days. GINS2 amplification is indicated as pink speckled fluorescence signals whereas the neighboring CDH13 gene shows only single copy fluorescence signals (Bi). Neighboring cells with and without GINS2 amplification are shown in Figure Bii. A CDK4 specific BAC probe that was labeled in pink and a XRCC6BP1/KUB3 specific BAC that was labeled in green were hybridized simultaneously against NHNP cells that were differentiated for 2 days. CDK4 and KUB3 amplifications were detectable as cluster of pink and green speckled fluorescence signals. CDK4 specific signals spread over a more extended area than the KUB3 specific signals (D). Nuclei were counterstained with DAPI (B). Size calibration bar = 5 µm.
Figure 4
Figure 4. FISH and immunofluorescence analysis.
FISH with GINS2 specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed GINS2 amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with GFAP revealed CDK4 amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed CDK4 amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.
Figure 5
Figure 5. Correlation of gene content to chromosome region.
The correlation is shown for human chromosome 16 with several amplified and one deleted region in NHNP cells after 5 d differentiation. The log2 ratio profile of the 10× window averaged data is presented at 25 kb resolution (A). Fused lasso analysis is performed using the same data points (B). GC repeats, gene content and banding pattern is shown as indicated by Ensembl genome browser (C). Both the log2 ratio profile and the fused lasso analysis indicate an overlap between amplifications and regions with high GC and gene content and an overlap between deletion and regions with low GC and gene content.
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References

    1. Bostock CJ. Mechanisms of DNA sequence amplification and their evolutionary consequences. Philos Trans R Soc Lond B Biol Sci. 1986;312:261–273. - PubMed
    1. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A. 1969;63:378–383. - PMC - PubMed
    1. Spradling AC, Mahowald AP. Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1980;77:1096–1100. - PMC - PubMed
    1. Tower J. Developmental gene amplification and origin regulation. Annu Rev Genet. 2004;38:273–304. - PubMed
    1. Graves JA. Gene amplification in a mouse embryo? Double minutes in cell lines independently derived from a Mus musculus X M. caroli fetus. Chromosoma. 1984;89:138–142. - PubMed

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