Trapped in action: direct visualization of DNA methyltransferase activity in living cells.
DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.
Schermelleh
Lothar
L
Ludwig Maximilians University Munich, Department of Biology II, Planegg-Martinsried, Germany.
Spada
Fabio
F
Easwaran
Hariharan P
HP
Zolghadr
Kourosh
K
Margot
Jean B
JB
Cardoso
M Cristina
MC
Leonhardt
Heinrich
H
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D000077209:Decitabine; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D051379:Mice; D010777:Photochemistry / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; D001374:Azacitidine