A methyltransferase targeting assay reveals silencer-telomere interactions in budding yeast.
We have designed a modified version of the Dam identification technique and used it to probe higher-order chromatin structure in Saccharomyces cerevisiae. We fused the bacterial DNA methyltransferase Dam to the DNA-binding domain of TetR and targeted the resulting chimera to Tet operators inserted in the yeast genome at the repressed locus HML. We then monitored the methylation status of HML and other sequences by a quantitative technique combining methylation-sensitive restriction and real-time PCR. As expected, we found that TetR-Dam efficiently methylated HML in cis. More strikingly, when TetR-Dam was present at HML, we observed increased methylation in the III-L subtelomeric region but not in intervening sequences. This effect was lost when the HML silencers were inactivated by mutations. When the HM silencers and the Tet operators were transferred to a plasmid, strong methylation was clearly observed not only in the III-L subtelomeric region but also at other telomeres. These data indicate that HM silencers can specifically associate with telomeres, even those located on different chromosomes.
Lebrun
Eleonore
E
Laboratoire de Biologie Moléculaire de la Cellule, UMR5665, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France.
Fourel
Geneviève
G
Defossez
Pierre-Antoine
PA
Gilson
Eric
E
D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors; D008780:Methyltransferases / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D016615:Telomere / Q000378:metabolism*
D009963:Orotic Acid; C001242:5-fluoroorotic acid; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)