BioloGical BloG

Dam methylation

2007. 3. 30. 12:31

Protein-DNA interactions are the essence of gene regulation, but many of them are transient and extremely difficult to study within living cells. Moreover, the techniques used to catch proteins in the act—such as chemical cross-linking and in situ hybridizaton with fluorescent probes—can create artefactual results. But there's another option: adenine methylation, not normally found in eukaryotes, can be used to "mark" DNA, and the methyl group, unlike most proteins, will remain attached no matter how harshly any particular procedure treats the DNA. Adenine methylation has previously been used to mark accessible, or comparatively 'open', regions of DNA throughout the genomes of yeast and Drosophila. In this month's issue of Nature Biotechnology, Bas van Steensel and Steven Henikoff report the development of a new technique, which they call DamID (for Dam identification), to identify the target sites of chromatin proteins in living cells. They tethered Escherichia coli Dam methylase to different chromatin proteins, and then detected the molecular 'fingerprints'—methylated adenines within the GATC recognition site—left behind by the enzyme. They were able to reconstruct the chromatin proteins' interactions with specific DNA sequences. Providing proof-of-principle, van Steensel and Henikoff showed that Dam methylase tethered to GAL4 resulted in adenine methylation—detected by methylation-specific restriction enzymes or antibodies—exclusively in the vicinity of a GAL4 binding site in fruit flies. When the methylase was tethered to endogenous Drosophila HP1 (heterochromatin protein 1), the interaction revealed a number of expected as well as unexpected target loci. These results suggest that DamID will be a useful tool in reconstructing protein-DNA interactions in monitoring access of proteins to highly condensed sequences.