DNA methylation at promoter CpG islands (CGI) is an epigenetic modification 
associated with inappropriate gene silencing in multiple tumor types. In the 
absence of a human pituitary tumor cell line, small interfering RNA-mediated 
knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 
5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. 
Sustained knockdown induced reexpression of the fully methylated and normally 
imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite 
restriction analysis (COBRA) revealed that reexpression of Nnat was associated 
with partial CGI demethylation, which was also observed at the H19 
differentially methylated region. Subsequent genome-wide microarray analysis 
identified 91 genes that were significantly differentially expressed in Dnmt1 
knockdown cells (10% false discovery rate). The analysis showed that genes 
associated with the induction of apoptosis, signal transduction, and 
developmental processes were significantly overrepresented in this list (P < 
0.05). Following validation by reverse transcription-PCR and detection of 
inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and 
S100A10) were analyzed in primary human pituitary tumors, each displaying 
significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For 
two of these genes, NNAT and S100A10, decreased expression was associated with 
increased promoter CGI methylation. Induced expression of Nnat in stable 
transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is 
the first report of array-based "epigenetic unmasking" in combination with Dnmt1 
knockdown and reveals the potential of this strategy toward identifying genes 
silenced by epigenetic mechanisms across species boundaries.