Divergent gene pairs (DGPs) are abundant in eukaryotic genomes. Since two genes in a DGP potentially share the same regulatory sequence, one might expect that they should be co-regulated. However, an inspection of yeast DGPs containing cell-cycle or stress response genes revealed that most DGPs are differentially-regulated. The mechanism underlying DGP differential regulation is not understood. Here, we showed that co-versus differential regulation cannot be explained by genetic features including promoter length, binding site orientation, TATA elements, nucleosome distribution, or presence of non-coding RNAs. Using time-lapse fluorescence microscopy, we carried out an in-depth study of a differentially regulated DGP, PFK26-MOB1. We found that their differential regulation is mainly achieved through two DNA-binding factors, Tbf1 and Mcm1. Similar to 'enhancer-blocking insulators' in higher eukaryotes, these factors shield the proximal promoter from the action of more distant transcription regulators. We confirmed the blockage function of Tbf1 using synthetic promoters. We further presented evidence that the blockage mechanism is widely used among genome-wide DGPs. Besides elucidating the DGP regulatory mechanism, our work revealed a novel class of insulators in yeast.
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