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Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63

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Abstract

The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of ∼6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5–7 ‘bands’ spanning a single chromosome termed the ‘IFN chromosome’. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage–fusion–bridge events.

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Abbreviations

aCGH:

array comparative genomic hybridization

BAC:

bacterial artificial chromosome

BFB:

breakage–fusion–bridge

CCD:

charge-coupled device

CCR:

complex chromosome rearrangements

CENP:

centromeric protein

FISH:

fluorescence in-situ hybridization

FRA:

fragile sites

IFN:

interferon

SKY:

spectral karyotyping

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Acknowledgements

We thank Dr William Earnshaw, Wellcome Trust Centre for Cell Biology, University of Edinburgh for providing us with centromeric protein antibodies and advice. This work was supported by a grant from the National Institute of Health (GM-072131) to R. Berezney, grants LC535, MSM0021620806, AV0Z50110509 to I. Raska and the Excellence Initiative ‘REBIRTH’, the SFB 738 and the CliniGene Network of Excellence (European Commission FP6 Research Program, contract LSHB-CT-2006-018933) to J. Bode.

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Authors and Affiliations

  1. Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, NY, 14260, USA

    Narasimharao V. Marella, Michael J. Zeitz, Kishore S. Malyavantham, Artem Pliss & Ronald Berezney

  2. SKY Core Resource Facility, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA

    Sei-ichi Matsui

  3. HZI, Helmholtz Centre for Infection Research / Epigenetic Regulation, 38124, Braunschweig, Germany

    Sandra Goetze & Juergen Bode

  4. First Faculty of Medicine, Charles University in Prague and Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Albertov 4, 128 000, Prague, Czech Republic

    Ivan Raska

  5. IMMCO Diagnostics Ltd, Buffalo, NY, 14228, USA

    Kishore S. Malyavantham

  6. Center for Model Organism Proteomes, Institute of Molecular Biology, University of Zurich, 8057, Zurich, Switzerland

    Sandra Goetze

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  1. Narasimharao V. Marella
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Corresponding author

Correspondence to Ronald Berezney.

Electronic Supplementary Material

Below is the image is a link to a high resolution version.

Supplementary Figure S1

Spectral karyotyping (SKY) of MG63 cell line. (A–E) Spectral karyotyping analyses of six representative MG63 metaphase chromosome spreads are shown. (F, G) Spectral karyotyping of normal diploid human fibroblast (NHF-1) cell line. (F) Representative image of metaphase chromosomes of NHF-1 cell line labelled with SKY probes and the corresponding (G) karyotype analysis of the above spread is shown. (GIF 247 kb, 196 kb, 270 kb)

High resolution image file (TIFF 402 kb)

High resolution image file (TIFF 305 kb)

High resolution image file (TIFF 607 kb)

Supplementary Figure S2

Interferon gene probe labelling on MG63 metaphase spreads. (A–D) Four representative images of MG63 metaphase chromosomes hybridized with cosmid probe 133D4 (green) that spans across the interferon ω gene are shown. Chromosomes showing amplification of the IFN cluster are indicated by arrows, while arrow heads point to other chromosomes in the spread that show signal for the IFN gene probe. Chromosomes are stained with DAPI (blue). (GIF 119 kb)

High resolution image file (TIFF 1.18 mb)

Supplementary Figure S3

Magnified images of IFN chromosome. (A–L) Magnified images of the IFN chromosome showing amplification for the cosmid probe 133D4 (green) are shown. Chromosomes are stained with DAPI (blue). (GIF 221 kb)

High resolution image file (TIFF 2 mb)

Supplementary Figure S4

Labelling of IFN gene probes, whole-chromosome painting and centromere labelling of 4, 9 and 8 over three other osteosarcoma cell lines. (A–C) Labelling of chromosomes with whole-chromosome 9 paint (green) and G20 BAC probe in (A) Saos-2, (B) U-2 OS and (C) SK-ES-1 cell lines. (D–F) Labelling of centromere 4 (green) in (D) Saos-2, (E) U-2 OS and (F) SK-ES-1 cell lines. (G–I) Labelling of centromere 9 (red) on metaphase chromosomes of (G) Saos-2, (H) U-2 OS and (I) SK-ES-1 cell lines. (J–L) Double labelling of whole-chromosome painting of chromosome 4 (green) and chromosome 8 (red) in (J) Saos-2, (K) U-2 OS and (L) SK-ES-1 cell lines. Chromosomes are stained with DAPI (blue). (GIF 265)

High resolution image file (TIFF 2.63 mb)

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Supplementary Figure S5

Array-based comparative genomic hybridization (aCGH) analysis of MG63 cell line. Graphs for individual chromosomes with normalized ratio for each spot in relation to their location on the chromosome are plotted. Normal genomic content is represented by a ratio of 1. Upward peaks from this ratio indicate gains in genomic material while downward peaks indicate a loss in the genome. Array results showed gains and losses at various regions in the genome. Significant gains have been observed in chromosomes 1, 3, 8, 9 and 15 while major losses are seen in chromosomes 3, 4, 7, 9 and 16. (PDF 180 kb)

Supplementary Table S1

Comparative genomic hybridization (CGH) analysis of chromosome arm 9p in MG63 cell line. The table represents the various probes that span the chromosome arm 9p and the corresponding ratios of amplification or deletion in MG63 cell line are shown. Normal genomic content is represented by a ratio of 1. A portion of this table is displayed in Figure S5. (PDF 30.9 kb)

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Marella, N.V., Zeitz, M.J., Malyavantham, K.S. et al. Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63. Chromosome Res 16, 1177–1192 (2008). https://doi.org/10.1007/s10577-008-1267-x

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  • DOI: https://doi.org/10.1007/s10577-008-1267-x

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