An early pharyngeal muscle enhancer from the Caenorhabditis elegans ceh-22 gene is targeted by the Forkhead factor PHA-4

AbstractCaenorhabditis elegans pharyngeal muscle development involves ceh-22, an NK-2 family homeobox gene related to genes controlling heart development in other species. ceh-22 is the earliest known gene expressed in the pharyngeal muscles and is likely regulated directly by factors specifying pharyngeal muscle fate. We have previously implicated the ceh-22 distal enhancer in initiating ceh-22 expression. Here we analyze the distal enhancer using functional and comparative assays. The distal enhancer contains three subelements contributing additively to its activity, and functionally important regulatory sequences are highly conserved in Caenorhabditis briggsae. One subelement, termed DE3, is strongly active in the pharyngeal muscles, and we identified two short oligonucleotides (de199 and de209) contributing to DE3 activity. Multimerized de209 enhances transcription similarly to DE3 specifically in the pharyngeal muscles, suggesting it may be an essential site regulating ceh-22. de209 binds the pan-pharyngeal Forkhead factor PHA-4 in vitro and responds to ectopic pha-4 expression in vivo, suggesting that PHA-4 directly initiates ceh-22 expression through de209. Because de209 enhancer activity is primarily limited to the pharyngeal muscles, we hypothesize that de209 also binds factors functioning with PHA-4 to specifically activate ceh-22 expression in pharyngeal muscle