The Retinoblastoma Protein Regulates Pericentric Heterochromatin

Abstract

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G₁-to-S-phase transition.

We thank numerous investigators for reagents and advice during the course of this study: A. McClatchey, E. Li, S. Mittnacht, M. Classen, B. Kaelin, E. Hernando, C. Cordon-Cardo, T. Jenuwein, R. Bremner, and G. DiMattia. Many thanks to M. Keeney and W. Brown for expert assistance in flow cytometry and to J. Seabrook for help with statistical analysis. We gratefully acknowledge the advice of many colleagues at the MGH Cancer Centre, Dana-Farber, and the London Regional Cancer Program that helped to make this research possible.

C.E.I., S.M.F., L.A.S., and L.M.J. have all received support from the Strategic Training Program in Cancer Research. L.A.S. and S.M.F. also acknowledge funding from OGSST. N.G.B. is a recipient of a CIHR New Investigator Award. F.A.D. is a Research Scientist of the Canadian Cancer Society through an award from the National Cancer Institute of Canada. This work was supported by grants from the National Institutes of Health to N.J.D. (CA64402) and to L.H. (AG024398) and from the Canadian Institutes of Health Research to F.A.D. (MOP64253).