PHA-4 is a FoxA/forkhead transcription factor that is essential for pharyngeal organogenesis in C. elegans. PHA-4 is expressed in all five classes of pharyngeal cells as well as in the rectum, intestine and somatic gonad. The
pha-4 gene produces three PHA-4 protein isoforms called PHA-4A, B and C that differ only at their N-termini. To identify potential PHA-4 target genes expressed both inside and outside of the pharynx, the preferred PHA-4 binding sites of all three isoforms have been determined. Using baculovirus-produced PHA-4A, B or C, we have completed six rounds of DNA site selection from a pool of random oligonucleotides 20 bp long. PHA-4C selects the sequence TRTTKRY (where R=A/G, K=T/G and Y=T/C); this sequence matches the known TGTTTG PHA-4 binding site in the promoter of
myo-2, pharyngeal myosin (1), and the high and low PHA-4 affinity sites in the promoters of most pharyngeal expressed genes identified by Gaudet and Mango (2). The most common selected sequence of PHA-4C is TGTTTAC, matching a high affinity binding site. Surprisingly, PHA-4B selects TGTGGY, a novel sequence that has never been previously associated with PHA-4/FoxA activity. Our hypothesis is that PHA-4C regulates gene expression in the pharynx through the TRTTKRY site whereas PHA-4B regulates gene expression somewhere outside of the pharynx (in the rectum, intestine or somatic gonad) through this newly identified binding site. We wish to determine if this PHA-4B selected site functions in vivo. Transgenic worms were created carrying the PHA-4B selected sequence (multimerized for greater sensitivity) regulating a GFP reporter gene to determine if reporter gene expression could be detected in the same cells or in a subset of the cells in which
pha-4 is expressed. No reporter gene expression is detected in these animals. This result could be due to a lack of sensitivity; thus, we have begun to test the activity of the PHA-4B selected sequence in a yeast one-hybrid assay. We have also started other experiments to determine the role of each PHA-4 protein isoform in pharyngeal development by using RNA interference. We are injecting transcript-specific-double-stranded RNA into worms in order to knock out specific PHA-4 isoforms and assaying for pharyngeal development. (1) Kalb et al., Development 125, 2171-2180 (1998) (2) Gaudet and Mango, Science 295, 821-825 (2002)