Figure 6. Reporter activities near Met4-binding sites are enhanced over a ~40-kb range.

(A) Schematics of measuring the co-localization of the GFP reporter with Met4 puncta. S.kud MET3pr-fsGFP (frameshifted GFP) reporter gene and a tetO array (196x) are inserted side by side into the genome, in this case near the MET6 gene. (B) Averaged Max intensity projections (MIPs) of mCherry and GFP intensities near MET6 (Met4 target) and PUT1/ATG36 (not Met4 targets) in the presence of nearby reporter. These images are generated using the same method as in Figure 3F. Scale bars represent 1 µm. (C) The GFP intensity profile across the dot center in panel B. For MET6 and PUT1, the same type of data without the GFP reporter (−rep) are also included. Number of cells analyzed (for panels B and C): PUT1 +/−rep (318/524), ATG36 +rep (386), and MET6 +/−rep (330/381). (D) Schematic of GFP reporter insertion near three additional Met4-bound loci, MET17, RMD6, and MET6. The distance and orientation of the insertion are labeled in the diagram. (E) Mean GFP fluorescent intensities when S.kud MET3pr-GFP are inserted near indicated genes. The genes labeled in red have adjacent Met4-bound sites, while the ones labeled in gray do not. Error bars represent standard error among cells (collected in three independent experiments), and the asterisks *** represents P<0.001 (same for F, H). (F) Same as in panel E except with MET17pr-GFP and GAL1Spr-GFP reporter. Strains with MET17pr-GFP were grown in −met, and the ones with GAL1Spr-GFP were pre-grown in raffinose and induced by galactose for 6 hr. (G) Schematic of the MET3pr-GFP reporter inserted at various distances from the Met4-binding site near the MET6 gene. The same orientation was used for all the loci as indicated. (H) Mean GFP fluorescent intensities with MET3pr-GFP and GAL1Spr-GFP reporters inserted into locations indicated in panel G, in comparison to the same reporter inserted into two control loci far from Met4-binding sites.