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Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin

Nature Cell Biology volume 7, pages 420–428 (2005)Cite this article

Abstract

Here, we show a role for the RB1 family proteins in directing full heterochromatin formation. Mouse embryonic fibroblasts that are triply deficient for RB1 (retinoblastoma 1), RBL1 (retinoblastoma-like 1) and RBL2 (retinoblastoma-like 2) — known as TKO cells — show a marked genomic instability, which is coincidental with decreased DNA methylation, increased acetylation of histone H3 and decreased tri-methylation of histone H4 at lysine 20 (H4K20). Chromatin immunoprecipitation showed that H4K20 tri-methylation was specifically decreased at pericentric and telomeric chromatin. These defects are independent of E2F family function. Indeed, we show a direct interaction between the RB1 proteins and the H4K20 tri-methylating enzymes Suv4-20h1 and Suv4-20h2, indicating that the RB1 family has a role in controlling H4K20 tri-methylation by these histone methyltransferases. These observations indicate that the RB1 family is involved in maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin, linking tumour suppression and the epigenetic definition of chromatin.

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Figure 1: Genomic instability in mouse embryonic fibloblasts (MEFs) deficient for different retinoblastoma 1 (RB1) family members.
Figure 2: Centromeric defects in mouse embryonic fibloblasts (MEFs) deficient for retinoblastoma 1 (RB1) family members.
Figure 3: Global chromatin changes in cells lacking the retinoblastoma 1 (RB1) family.
Figure 4: Role of the retinoblastoma 1 (RB1) family in heterochromatin assembly.
Figure 5: Retinoblastoma 1 (RB1) family members interact with Suv4-20h1 and Suv4-20h2 HMTases and stabilize histone methylation at constitutive heterochromatin.

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Acknowledgements

We thank A. Kohlmaier for help with the RT-PCR assays. We are very grateful to J. Sage and T. Jacks for the donation of numerous vials of DKO and TKO MEFs, as well as for their helpful advice. We are indebted to D. Peeper and W. Krek for providing the pBabePuro–E2F1-DB construct. We thank K. Wikenheiser, S. Wong and M. Serrano for advice and helpful comments. S.G. was supported by the National Institutes of Health and by the National Science Foundation; M.G.-C. by the Spanish Ministry of Science and Technology (MCYT); and R.E. by the Regional Government of Madrid (CAM). Research in the laboratory of T.J. is supported by the Institute of Molecular Pathology through Boehringer Ingelheim and by grants from the Vienna Economy Promotion Fund, the European Union and the GEN-AU initiative, which is financed by the Austrian Ministry of Education, Science and Culture. Research in M.A.B.'s laboratory is funded by the MCYT (SAF2001-1869, GEN2001-4856-C13-08), by the Regional Government of Madrid, CAM (08.1/0054/01), by the European Union (TELOSENS FIGH-CT.2002-00217, INTACT LSHC-CT-2003-506803, ZINCAGE FOOD-CT-2003-506850, RISC-RAD F16R-CT-2003-508842) and by the Josef Steiner Cancer Award 2003.

Author information

Author notes

  1. Douglas C. Dean

    Present address: Cancer Center University of Louisville School of Medicine, Louisville, KY 40202, USA

  2. Susana Gonzalo and Marta García-Cao: These two authors contributed equally to this work.

Authors and Affiliations

  1. Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, E-28029, Spain

    Susana Gonzalo, Marta García-Cao, Raúl Eguía & María A. Blasco

  2. Epigenetics Group, Molecular Pathology Program, Spanish National Cancer Centre (CNIO), Madrid, E-28029, Spain

    Mario F. Fraga & Manel Esteller

  3. Research Institute of Molecular Pathology (IMP), Vienna Biocenter, Dr. Bohrgasse 7, Vienna, A-1030, Austria

    Gunnar Schotta, Antoine H.F.M. Peters & Thomas Jenuwein

  4. Division of Molecular Oncology, Washington University School of Medicine, St. Louis, MO 63110, USA

    Shane E. Cotter & Douglas C. Dean

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Correspondence to María A. Blasco.

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Gonzalo, S., García-Cao, M., Fraga, M. et al. Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin. Nat Cell Biol 7, 420–428 (2005). https://doi.org/10.1038/ncb1235

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