Methylation of CpG sites in the control regions of tumor suppressor genes may be 
an important mechanism for their heritable, yet reversible, transcriptional 
inactivation. These changes in methylation may impair the proper expression 
and/or function of cell cycle regulatory genes and confer a selective growth 
advantage to affected cells. Detailed methylation analysis using genomic 
bisulfite sequencing was performed on a series of subclones of a bladder cancer 
cell line in which a hypermethylated p16 gene had been reactivated by transient 
treatment with 5-aza-2'-deoxycytidine. Methylation of the CpG island in the 
promoter of the p16 gene in human bladder cancer cells did not stop the 
formation of a transcript initiated 20 kb upstream by the p19 promoter but did 
prevent the expression of a p16 transcript. Furthermore, we show that reactivant 
clones that expressed p16 at varying levels contained heterogeneous methylation 
patterns, suggesting that p16 expression can occur even in the presence of a 
relatively heavily methylated coding region. We also present the first 
functional evidence that methylation of only a small number of CpG sites can 
significantly down-regulate p16 promoter activity, thus providing support for 
the model of progressive inactivation of this tumor suppressor gene by DNA 
methylation.