A single high-zinc activation enhancer can control two genes orientated head-to-head in .

Enhancers play critical roles in gene expression, but a full understanding of their complex functions has yet to be defined. The cellular response to excess zinc levels in requires the HIZR-1 transcription factor, which binds the high-zinc activation (HZA) enhancer in the promoters of multiple target genes. Cadmium hijacks the excess zinc response by binding and activating HIZR-1. By analyzing the genome-wide transcriptional response to excess zinc and cadmium, we identified two positions in the genome where head-to-head oriented genes are both induced by metals. In both examples, a single predicted HZA enhancer is positioned between the two translational start sites. We hypothesized that a single enhancer can control both head-to-head genes, an arrangement that has not been extensively characterized. To test this hypothesis, we used CRISPR genome editing to precisely delete the HZA enhancer positioned between and ; in this mutant, both head-to-head genes display severely reduced zinc-activated transcription, whereas zinc-activated transcription of more distant genes was not strongly affected. Deleting the HZA enhancer positioned between and caused both head-to-head genes to display reduced cadmium-activated transcription, whereas cadmium-activated transcription of more distant genes was not strongly affected. These studies rigorously document that a single HZA enhancer can control two head-to-head genes, advancing our understanding of the diverse functions of enhancers.