Genome Biology Laboratory • Kohara Group

A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21, and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification.

Expression analysis of gcy-8 and gcy-18 promoter GFP constructs with CEH-14 and/or TTX-1 binding site mutations.
GFP reporter expression of gcy-8 (A-D) and gcy-18 (E-H) promoter sequences with CEH-14 and/or TTX-1 binding site mutations; (wild-type: A, E) wild-type 300-bp promoter, (ΔCEH: B, F) CEH-14 binding site mutation, (ΔTTX: C, G) TTX-1 binding site mutation, and (ΔCEHΔTTX: D, H) CEH-14 and TTX-1 binding site mutation. Images were acquired using an exposure time of 0.2 s (A, E) and 1 s (B-D, F-H). The inset shows digitally enhanced images of the AFD cell body (shown by the dotted line).

CEH-14 and TTX-1 binding sites are essential for gcy-8 and gcy-18 expression in AFD
CEH-14 and TTX-1 directly bind to promoters of the AFD genes (CEH-14: CTAAT, TTX-1: TAA(T/G)CTT) and co-regulate downstream genes. The expression was reduced in GFP reporters carrying either of CEH-14 or TTX-1 binding site mutation, whereas reporter carrying double mutation (ΔCEHΔTTX) completely lost GFP expression.