Figure 1: Strategy to observe PHA-4 associated with target promoters in vivo. Transgenic arrays are generated, with multiple copies of the candidate target promoter and the Lac operator in worms that also express PHA-4::YFP and lacI::CFP. LacI::CFP bound to the Lac operator reveals the position of the extrachromosomal array within the nucleus. YFP fluorescence is observed throughout the nucleus because there are PHA-4 target genes throughout the genome, but is enriched on the transgenic array if PHA-4::YFP binds the candidate target promoter.
THE TRANSCRIPTIONAL LOGIC OF PHARYNX ORGANOGENESIS
pha-4 specifies organ identity in the foregut. Our analysis of the organ identity gene pha-4 revealed how a single transcription factor could coordinate gene expression throughout an organ and at different developmental stages. Classical studies in developmental biology had discovered genes that establish cell type (e.g. muscle) or positional (e.g. anterior) fate. Our genetic studies identified the pha-4 locus and suggested PHA-4 was essential to specify "organ identity" for cells within the pharynx. PHA-4 encodes a FoxA transcription factor, indicating that it functions by regulating gene expression. Using a microarray-based approach, we identified direct PHA-4 target genes to distinguish between two possible mechanisms of PHA-4 action: PHA-4 could activate a few genes at the top of a hierarchy or PHA-4 could activate many genes at multiple stages throughout development. Our analysis demonstrated that PHA-4 functions globally to activate both early and late-expressed pharyngeal genes. Moreover, the affinity of PHA-4 for its DNA binding site contributes to the timing of expression onset of these targets. High affinity DNA binding sites promote expression during early organogenesis whereas low affinity sites are typically restricted to later development. An intriguing possibility is that direct regulation of an entire gene network may be a hallmark of many developmental transcription factors (e.g. vertebrate FoxA factors, myoD).
The regulatory network controlling pharyngeal development: We expanded the pharynx network to discover cis-regulatory elements that function in combination with PHA-4 sites for pharyngeal gene expression. In collaboration with Jim Kent (UC Santa Cruz), we created an algorithm to identify new cis-regulatory sites within pharyngeal genes and tested those sites to discover their activity in vivo. These elements, in combination with PHA-4, establish a code that can account for the onset of expression of approximately half the pharyngeal genes and that can be used in genome-wide searches to discover new early or late-expressed pharyngeal genes. We identified candidate transcription factors that bind these cis-regulatory elements, in an on-going collaboration with Marian Walhout's lab (U Mass Med, Worcester). One of these factors is the nuclear hormone receptor DAF-12.
We are complementing the cis-regulatory studies with functional analyses of the candidate pharyngeal genes identified by gene profiling. We have inactivated 376 pharyngeal genes by RNAi using a sensitized screen, and discovered 88 genes with phenotypes that reflect cell fate specification, morphogenesis or function within the pharynx. The RNAi studies were undertaken in collaboration with Marc Vidal's lab (Dana Farber), which performed a high-throughput yeast two-hybrid screen of the pharyngeal genes to construct a pharynx interactomeG. The combined analyses of expression and function of the pharyngeal genes established a network for pharynx organogenesis that relies heavily on transcription factors (25 phenotypes) and predicted plasma membrane-associated proteins (18 phenotypes). Intriguingly, the pharynx network depends preferentially on genes conserved among multicellular organisms or worms, at the expense of ancient genes that have orthologs in S. cerevisiae. This result suggests that evolution of a metazoan structure, an organ, depends on inventing genes that are metazoan specific.
Future Directions: An ongoing goal of the lab is to understand the mechanisms of temporal control during organ formation. During organogenesis, pluripotent precursor cells acquire a defined identity such as visceral muscle or nerve. The transition from 'naïve' precursor towards the differentiated state is characterized by sequential waves of gene expression that are determined by a host of transcription factors. A key question is how transcriptional circuitry dictates the succession of events that accompanies developmental competence, cell fate specification and ultimately differentiation. The de-differentiation of cells during somatic cell transfer, regeneration and possibly cancer illustrates that the progression of developmental states is not inexorable, but can also run backwards. What mechanisms ensure that, under normal conditions, developmental time runs forwards? Our studies indicate that PHA-4 contributes to temporal regulation according to the affinity model described above. Genome-wide analysis of pharyngeal genes suggests additional mechanisms. We are in the process of investigating developmental progression during organogenesis and the contribution of PHA-4 to those events.
Figure 2: Example of PHA-4::YFP binding its target myo-2. myo-2 carries a high affinity PHA-4 site and is expressed in the pharynx, but not the midgut. Accordingly, we observe PHA-4 bound to the myo-2 promoter in early and late pharyngeal cells (yellow arrows) but not midgut cells (white arrow). This result reveals PHA-4::YFP association tracks with affinity and not gene expression, since myo-2 is expressed late in pharyngeal development but carries a high affinity PHA-4 site. Association of PHA-4::YFP with a transgenic array is lost if the PHA-4 binding site is mutated (not shown).
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