The PHA-4/FoxA forkhead transcription factor in nematodes plays an integral role in all aspects of pharyngeal development, from the earliest specification of pharyngeal precursors to the determination of definitive cell types (1-3). Previous studies in the Mango lab have suggested that PHA-4 activates the expression of many pharyngeal genes directly (4). Our previous studies revealed that binding site affinity for PHA-4 contributes to target diversity in vivo. In fact, the affinity between PHA-4 and its target promoters contributes to the timing of gene expression (4). Mutation of a high affinity PHA-4 binding site to a lower affinity site results in a later onset of expression; conversely, switching a low affinity site to a higher affinity advances gene activation (4). However, the exact mechanism by which PHA-4 temporally regulates all of its targets through binding site affinity remains unclear. To address this question, we employed the NSA (Nuclear Spot Assay) which uses LacO/LacI::CFP to mark pseudochromosome arrays and PHA-4::YFP to track
pha-4 (5-7). By tracking both YFP and CFP signals and their distributed domains in the nucleus, we can follow patterns of association between PHA-4 and its target promoters in living embryos with precise spatial and temporal resolution. Our data indicate that PHA-4::YFP binds to target promoters hours before the transcription firing and that the level of PHA-4::YFP association is significantly different between high versus low affinity sites at early embryonic stages. In addition, manipulation of PHA-4 levels at early embryonic stages (2E to 8E) by heatshock (over expression) or RNAi (reduced expression) can alter the onset of pharyngeal expression. These data suggest that PHA-4 binds to pharyngeal target promoters through different affinity sites at early embryonic stages and this binding may prime the timing of gene expression at later stages. We will further investigate the PHA-4 occupancy at endogenous loci during development by chromatin immunoprecipitation. Together, this study will help clarify how binding site affinity influences temporal regulation of transcription. Reference 1.Mango S.E., Lambie E.J., and Kimble J. Development 120 : 3019. (1994) 2.Horner M., Quintin S., Domeier M.E., Kimble J., Labouesse M. and Mango S.E. Gene & Development 12:1947. (1998). 3.Gaudet J., Muttumu S., Horner M., and Mango S.E. PLoS Biol 2 :
e352. (2004) 4.Gaudet J., and Mango S.E. Science 295 : 821. (2002) 5.Carmi I., JKopczynski J.B. and Meyer B.J. Nature 396:168. (1998) 6.Belmont A.S., Straight A.F. Trends Cell Biol 8:121. (1998) 7.Fakhouri T.H., Stevenson J., Chisholm A.D., Mango S.E. PLos Genet 6:
e1001060. (2010).