The ETS Factors Pnt and Yan Regulate Distinct Cell Fates in <i>Drosophila</i>

Development of properly patterned tissues requires cells to transit from a multipotent state to diverse differentiated states in a precise spatiotemporal manner. These transitions are directed by a limited number of signaling pathways via their transcriptional effectors that regulate the necessary changes in gene expression. How repeated use of the same signaling machinery confers specificity to the spectrum of fate transitions it controls is still poorly understood. I focused on this broad question from the viewpoint of Receptor Tyrosine Kinase/Mitogen Activated Protein Kinase (RTK/MAPK) signaling pathway and its downstream transcriptional effectors, the repressor Yan and the activator Pointed (Pnt). My research centered on two scales of this process: primarily how MAPK signaling regulate the activator Pnt for diverse cell fate transitions; secondly how Yan and Pnt control target gene expression. I first studied the MAPK/Pnt response in the Drosophila eye, which consists of a highly ordered array of ommatidia. Each ommatidium contains eight distinct photoreceptors, R1-R8, specified sequentially. Recruitment of R1-R7 fates all require reiterative RTK/MAPK signaling-mediated activation of the transcriptional effector Pnt. However, R1-R7 can be divided into two rounds of specification defined by different MAPK activation levels, raising the question of how the Pnt response is tailored to the distinctive MAPK signaling contexts. I discovered a previously unstudied Pnt isoform, PntP3, and showed that its respective inclusion or exclusion in the RTK transcriptional effector circuitry defines two distinct Pnt regulatory networks for the two rounds of specification. I also mapped out the cross- and auto-regulatory interactions between the three Pnt isoforms that define the two networks. Considering the conservation of the RTK/MAPK/Pnt pathway, the interconnected transcriptional effector network that my work uncovered is likely to be exploited in diverse developmental scenarios. As the recruitment of cell fates is driven directly by the activation of gene expression, I then studied Pnt/Yan regulation on gene transcription at the molecular level. Precise regulation of gene expression from transcription factors is organized by the DNA regulatory elements, which contain clusters of transcription factor binding sites. The number, affinity and organization of the binding sites, termed the cis-regulatory syntax, coordinate transcription factors binding at the regulatory element and determine the transcription output. I used the Muscle Heart Enhancer (MHE), a well-defined enhancer that activates even-skipped (eve) expression in the Drosophila embryo, as an example to study the cis-regulatory syntax organizing Yan and Pnt. I developed a thermodynamic model to study how this cis-regulatory syntax organizes the exact Yan and Pnt binding at each site in order to generate hypothesis for future experimental investigations. Using this model I demonstrated how three-dimensional long-range interactions among the three strong-affinity sites could facilitate Yan occupancy and transcription repression, and showed that such regulation would be dependent on the self-association ability of Yan. This work sets a theoretical basis for future experimental studies on a general principle for Yan and Pnt regulation on different enhancers