Mechanisms controlling cell type-specific transcription factor activity in the development of serially homologous structures in Drosophila

Project summary/abstract To ensure proper morphogenesis and cell fate specification, animals must generate highly stereotyped spatial and temporal patterns of gene expression. To this end, transcription factors (TFs) bind DNA regulatory elements such as enhancers to activate or repress transcription in particular cell types at particular developmental stages. Many TFs are expressed in several cell types at multiple stages throughout animal development. These TFs modulate different gene regulatory networks (GRNs) in different cell types, allowing a single TF to specify multiple cell fates. Despite decades of research, it remains unclear how individual TFs are able to perform distinct functions in different cell types. One example of such a TF is the Drosophila Hox TF Ultrabithorax (Ubx). Ubx specifies third thoracic (T3) segmental identity by binding thousands of enhancers to regulate hundreds of genes, modifying the ground-state second thoracic segment (T2) GRN. T3 is highly modified at all positions along the proximal-distal (PD) axis relative to the serially homologous T2, including morphological changes to the body wall, hinge, and appendage proper. All of these changes must ultimately be due to Ubx activity. A primary mechanism by which TFs such as Ubx enact changes in GRNs is through modification of chromatin structure. This is largely mediated by TF interaction with chromatin remodeling enzymes, leading to histone post-translational modifications and changes in chromatin accessibility at targeted genomic loci. My proposal outlines a series of experiments to identify cell type-specific molecular interactions that underlie cell type-specific Ubx binding, chromatin-modifying activities, and transcriptional regulatory activities. Recent evidence from our lab suggests that Ubx chromatin remodeling activity is spatially segregated along the PD axis in T3. In Aim 1 of my Research Strategy, I will test the hypothesis that Ubx functions predominantly to either promote a more open chromatin state in intermediate positions along the PD axis or a more closed chromatin state in proximal and distal positions along the PD axis. I will use a novel technique developed in our lab called SpyChIP to assay Ubx binding in these distinct populations of cells along the PD axis in T3. I will also perform ATAC-seq in these populations to identify changes in chromatin accessibility downstream of Ubx binding. These experiments will provide information as to how TF binding leads to differential chromatin landscapes in different populations of cells. In Aim 2, I will test the hypothesis that the zinc finger TF Teashirt (Teashirt) mediates Ubx repressive activity in the proximal domain of T3. In this Aim, I describe both gain- and loss-of-function experiments to characterize the role of Tsh in regulating Ubx repressive activity. I will also perform protein-protein interaction experiments to determine if Ubx interacts with Tsh and other coregulators in a cell type-specific manner. Overall, these experiments will elucidate the mechanisms by which a single TF modulates different GRNs in different populations of cells throughout animal development to give rise to multiple cell types.