During development of the C. elegans foregut, pluripotent precursors acquire a defined identity. This process depends on cascades of gene expression, which are orchestrated by combinations of transcription factors, including
pha-4/Foxa [1]. Our previous in vitro studies with
pha-4 revealed that binding site affinity contributes to target diversity within the foregut. Genes with high affinity PHA-4 binding sites are competent to fire earlier than genes with low affinity sites. A simple model is that binding site affinity dictates when in development PHA-4 associates with a target promoter. We hypothesize that PHA-4 binding does not trigger transcription by itself. Rather, PHA-4 may render a target promoter competent to associate with additional transcription factors, which then allows transcription initiation. To test this model, we used the Nuclear Spot Assay [NSA; 2, 3] to examine PHA-4 association with pharyngeal genes. For this approach, pseudochromosomes (transgene arrays) were constructed that carried multiple copies of PHA-4 target promoters and the Lac operator. The pseudochromosomes also expressed PHA-4::YFP and LacI::CFP. LacI::CFP bound to the Lac operator revealed the position of the pseudochromosome. YFP fluorescence was observed throughout the nucleus, but was enriched on pseudochromosomes because of the high density of target promoters there. To examine PHA-4 - promoter association, we examined PHA-4::YFP with the
myo-2 promoter, which carries high affinity PHA-4 binding sites but is transcribed late in embryogenesis [1]. As predicted by the Affinity Model, PHA-4::YFP associated with the
myo-2 promoter early, hours before
myo-2 transcription. Binding was specific, since inactivation of the PHA-4 binding sites within the
myo-2 promoter abrogated association. Pseudochromosomes underwent dramatic morphological changes during development, and these changes were dependent on PHA-4. Fluorescence In Situ Hybridization revealed that endogenous pharyngeal genes underwent similar morphological behaviors as genes in pseudochromosomes. These and other data indicate that PHA-4::YFP behaved as predicted by the Affinity Model. We suggest that chromatin decompaction by PHA-4 renders genes susceptible to binding by additional transcription factors, and thereby primes genes for transcription initiation. If we extrapolate from one PHA-4 target to hundreds of targets, then we envision that PHA-4 poises a cell to acquire pharynx fate by priming hundreds of pharyngeal genes for activation. [1]Mango, S.E.(2007) WormBook [2]Fukushige et al.(1999) PNAS [3]Carmi I et al.(1998) Nature.