Certain and Progressive Methylation of Histone H4 at Lysine 20 during the Cell Cycle

Abstract

Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G₂, M, and G₁ phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.

ACKNOWLEDGMENTS

Funding from the Packard Foundation and the Sloan Foundation, a Cottrell Scholar Award, and an NIH grant (GM 067193) to N.L.K. are gratefully acknowledged. C.A.M. thanks the Roy J. Carver Charitable Trust (grant 04-76) and the March of Dimes (Basil O'Connor Scholar Award FY05-1232) for funding. J.J.P. was a recipient of an NIH Institutional NRSA in Molecular Biophysics (5T32 GM 08276).