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 G(2), M, and G(1) 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.