Fig. 2
From: Identification of protein lysine methylation readers with a yeast three-hybrid approach
Principle of Y3H approach for detection of methylation readers. a Bait, GAL4-BD fused to H3 short polypeptide (H3N) recognizes and binds the promoter region of the reporter genes. The bait is methylated by the additionally expressed catalytic domain of G9a methyltransferase (G9a-SET). Interaction between methylated bait (H3K9me3) and prey recruits the GAL4-AD to the promoter upstream of the reporter genes, thereby reconstituting an active TF and as a result activating transcription of the HIS3-auxotrophic selection marker (which allows growth on media lacking histidine) and the MEL1-color selection marker (encodes α-galactosidase which in the presence of the chromagenic substrate X-α-Gal cause cells turn into blue). b In negative control settings, G9a-SET is not expressed, hence the bait is not modified and not recognized by the prey. As a result, GAL4 is not reconstituted, and the selection markers are not expressed