Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer

Nature Genetics volume 37, pages391–400(2005)Cite this article

Abstract

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

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Figure 1: Quantification of global histone H4 post-translational modifications.
Figure 2: Loss of monoacetylation and trimethylation of histone H4 occurs in cancer.
Figure 3: Loss of monoacetylation of histone H4 in cancer cells occurs mainly at Lys16.
Figure 4: Loss of trimethylation of histone H4 in cancer cells occurs mainly at Lys20.
Figure 5: Loss of monoacetylation at H4-Lys16 and trimethylation at H4-Lys20 in cancer cells is not associated with a shift in the histone modification pattern of epigenetically silenced genes.
Figure 6: Loss of monoacetylation at H4-Lys16 and trimethylation at H4-Lys20 in cancer cells is not associated with promoter-associated CpG islands.
Figure 7: Loss of monoacetylation at H4-Lys16 and trimethylation at H4-Lys20 in cancer cells is associated with DNA hypomethylation of repetitive sequences.
Figure 8: Loss of methylation induced by treatment with demethylating drugs in repetitive Sat2 and D4Z4 sequences is associated with a loss of monoacetylation at H4-Lys16 and trimethylation at H4-Lys20.

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Acknowledgements

We thank H. Lindner for advice regarding the optimization of the capillary electrophoresis method for quantification of histone H4 modifications and the Tumor Bank of the Spanish National Cancer Center for providing human primary tumor samples. This work was supported by the Health and Science Departments of the Spanish Government. M.F.F. is funded by the Spanish Association Against Cancer.

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Affiliations

  1. Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center, Melchor Fernández Almagro 3, Madrid, 28029, Spain

    Mario F Fraga, Esteban Ballestar, Ana Villar-Garea, Manuel Boix-Chornet, Jesus Espada, Santiago Ropero, Kevin Petrie & Manel Esteller

  2. Research Institute of Molecular Pathology, The Vienna Biocenter, Vienna, A-1030, Austria

    Gunnar Schotta & Thomas Jenuwein

  3. Histone Modifications Group, Ludwig-Maximillians Universität München, Munich, Germany

    Tiziana Bonaldi & Axel Imhof

  4. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA

    Claire Haydon & Natalie Ahn

  5. Department of Oncology, Cancer Genomics Program, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 2XZ, UK

    N Gopalakrishna Iyer & Carlos Caldas

  6. Lymphoma Laboratory, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain

    Alberto Pérez-Rosado & Miguel Ángel Piris

  7. Proteomics Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

    Enrique Calvo & Juan A Lopez

  8. Biochemistry Department, School of Medicine, Universidad Autónoma de Madrid, Instituto de Investigaciones Biomédicas, Madrid, Spain

    Amparo Cano

  9. Department of Genetics, School of Science, University of Navarra, Pamplona, Spain

    Maria J Calasanz

  10. Hematopathology Unit, Hospital Clinic, University of Barcelona, Catalonia, Spain

    Dolors Colomer

  11. Department of Chemistry and Biochemistry, The Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, USA

    Natalie Ahn

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  1. Mario F Fraga
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Correspondence to Manel Esteller.

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Supplementary information

Supplementary Fig. 1

Relative levels of monoacetylated and trimethylated histone H4 in human colon primary tumors and corresponding normal tissue from the same patient were quantified by HPCE. (PDF 60 kb)

Supplementary Fig. 2

Modification patterns of histone H4 according to doubling time and cell cycle. (PDF 25 kb)

Supplementary Fig. 3

HPCE quantification of histone H4 monoacetylation in human normal colon, wild type HCT116 colon cancer cells and HCT116 cells deficient in p300. (PDF 11 kb)

Supplementary Fig. 4

Quantitative RT-PCR measurement of the RNA levels of the histone methyltransferase Suv4-20h2 after knock-down with RNAi specific for Suv4-20h2. (PDF 11 kb)

Supplementary Fig. 5

Relative abundance of non- and monoacetylated forms in their different methylated status (non-, mono-, di- and trimethylated) of histone H4 in normal lymphocytes and the human cancer cell line HL60. (PDF 17 kb)

Supplementary Fig. 6

Representative HPCE electropherograms and western blots showing the acetylation status of histone H4 in HL60 before and after treatment with 10 mM Sodium Butyrate for 6h. (PDF 29 kb)

Supplementary Fig. 7

Acetylation patterns of histone H4 according to chromosomal translocations in leukemia. (PDF 17 kb)

Supplementary Table 1

Primer sequences and annealing temperatures for bisulfite sequencing, methylation-specific PCR, chromatin immunoprecipitation and RT-PCR reactions. (PDF 18 kb)

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Fraga, M., Ballestar, E., Villar-Garea, A. et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37, 391–400 (2005). https://doi.org/10.1038/ng1531

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