We have previously demonstrated proteasomal degradation of DNMT1 in mammalian 
cells following treatment with several DNA hypomethylating agents. Here, we 
demonstrate dose-dependent degradation of Dnmt1 in mouse embryonic stem (ES) 
cells expressing catalytic site mutant (cys-ser), confirming that the covalent 
bond formation between Dnmt1 and decitabine-incorporated DNA is not essential 
for this process. DNMT1o, the oocyte-specific isoform that lacks the N-terminal 
118-amino acid domain, did not undergo decitabine-mediated degradation, which 
further proves the requirement of multiple domains including nuclear 
localization signal, KEN box, and BAH domains for this process. Analysis of 
glycerol density gradient fractions of micrococcal nuclease-digested nuclei 
showed that both nucleosomal and nucleoplasmic DNMT1 are degraded upon 
decitabine treatment. Among different inhibitors tested, the inhibitors of the 
proteasomal pathway and several protein kinases impeded decitabine-induced DNMT1 
degradation. The maximal effect caused by inhibiting protein kinase C (PKC) 
persuaded us to investigate further its role in decitabine-mediated DNMT1 
degradation. Blockage of the degradation process after treatment with rottlerin, 
an inhibitor of PKCδ, or after siRNA-mediated depletion of PKCδ, indicated that 
this protein kinase is involved in decitabine-mediated depletion of DNMT1. PKCδ 
interacted with and phosphorylated DNMT1 in vitro. Moreover, rottlerin inhibited 
both basal and decitabine-induced phosphorylation of DNMT1. These studies 
provide substantial evidence that decitabine-induced degradation of the 
maintenance methyltransferase DNMT1 does not require covalent bond formation 
with the substrate and also elucidate its underlying molecular mechanism.