Temporal control of organogenesis by PHA-4/FOXA

FoxA forkhead are conserved transcription factors that function in regulating foregut development of animals. FoxA factors regulate organogenesis from early cell fate specification to cell differentiation and functional morphogenesis by orchestrating timely transcriptional programs that drive these events at each developmental stage. Using the PHA-4/FoxA-dependent pharynx development in C. elegans as a model, we revealed that PHA-4 modulates temporal pharyngeal gene expression partly through DNA binding affinity. Pharyngeal genes with high affinity PHA-4 binding sites are competent to activate earlier when compared to genes with low affinity sites. We further demonstrated that affinity affects the level of PHA-4 occupancy at pharyngeal promoters and the decompaction of chromatin induced after PHA-4 binding. We tested the effect of affinity in response to changes in PHA-4 level and showed that temporal pharyngeal gene expression was sensitive to PHA-4 levels but was maintained properly within a range of PHA-4 level fluctuation. The dynamic level of PHA-4 during embryogenesis coordinates with the different binding affinity between PHA-4 and its targets to mediate the proper temporal order of pharyngeal gene expression. Sequence-specific transcription factors are involved in regulating gene expression by recruiting RNA Pol II and/or mediating the release of paused RNA iv Pol II at target genes. Poised Pol II at developmental regulated genes is shown to play an important role in controlling gene expression during embryogenesis in different organisms. However, poised Pol II is relatively rare in C. elegans. By examining the dynamic genome-wide Pol II occupancy at different embryonic stages, we discovered that poised Pol II was temporally regulated at different genes. Among poised pharyngeal genes, Pol II is found at genes that are either activated at later stages or highly expressed at earlier stages with a reduction in expression later. This result suggests that poised Pol II serves as a preparation for future gene expression or as a memory of past gene expression. Moreover, we showed that PHA-4 activity was required to load Pol II at poised pharyngeal genes. We propose that PHA-4/FoxA functions as a pioneer factor that primes gene activation by regulating Pol II. In summary, PHA-4 utilizes DNA binding affinity by differentially binding its targets to control the timing of activation. The binding of PHA-4 also regulates Pol II occupancy at pharyngeal promoters. Our studies delineate the role of PHA- 4 in regulating temporal gene expression during pharynx development and suggest that FoxA factors might function similarly in other systems.