In mammals, DNA methylation is an important epigenetic mark that is associated 
with gene silencing, particularly in constitutive heterochromatin. However, the 
effect of DNA methylation on other epigenetic properties of chromatin is 
controversial. In this study, we show that inhibition of DNA methylation in 
mouse fibroblast cells affects histone modification and the subnuclear 
localization of histone H3.3 in a cell cycle-dependent manner. Using a DNA 
methyltransferase (Dnmt) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we found 
that reduced levels of DNA methylation were associated with the activation of 
transcription from centromeric and pericentromeric satellite repeats. The 
de-repressed pericentromeric chromatin was enriched in euchromatic histone 
modifications such as acetylation of histone H4, and di- and tri-methylation of 
lysine 4 on histone H3. Spatio-temporal analysis showed that the accumulation of 
these euchromatic histone modifications occurred during the second S phase 
following 5-aza-dC treatment, corresponding precisely with a shift in 
replication timing of the pericentromeric satellite repeats from middle/late S 
phase to early S phase. Moreover, we found that histone H3.3 was deposited on 
the pericentromeric heterochromatin prior to the accumulation of the euchromatic 
histone modifications. These results suggest that DNA CpG methylation is 
essential for the proper organization of pericentromeric heterochromatin in 
differentiated mouse cells.