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Molecular and Cellular Biology, March 2003, p. 1498-1508, Vol. 23, No. 5
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.5.1498-1508.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Methyltransferase Targeting Assay Reveals Silencer-Telomere Interactions in Budding Yeast

Eleonore Lebrun, Geneviève Fourel, Pierre-Antoine Defossez, and Eric Gilson*

Laboratoire de Biologie Moléculaire de la Cellule, UMR5665, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France,

Received 10 June 2002/ Returned for modification 29 July 2002/ Accepted 10 December 2002

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.

* Corresponding author. Mailing address: Laboratoire de Biologie Moléculaire de la Cellule, UMR5665, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. Phone: 33 472728453. Fax: 33 472728080. E-mail: Eric.Gilson{at}ens-lyon.fr.

Molecular and Cellular Biology, March 2003, p. 1498-1508, Vol. 23, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.5.1498-1508.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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