Hippo pathway target genes and transcription factors in organ growth control

© 2020 Katrina MitchellThe Hippo pathway, first discovered in the fruit fly Drosophila melanogaster, is an evolutionarily conserved regulator of organ growth. The Hippo pathway controls organ growth by regulating the nuclear activity of the transcriptional coactivator Yorkie (Yki). Yki binds to sequence-specific transcription factors to regulate target gene expression, including the TEAD/TEF family transcription factor Scalloped (Sd). It was recently discovered that the Hippo pathway controls the growth of organs in Drosophila by balancing transcriptional activation mediated by Yki and Sd, and transcriptional repression mediated by Tondu-domain- containing Growth Inhibitor (Tgi) and Sd. If this fine balance is disrupted and Yki is hyperactivated, organs can grow beyond their normal size. Importantly, deregulation of Hippo signalling drives tumorigenesis in a variety of human cancers. The high degree of conservation of the Hippo pathway throughout the animal kingdom makes the fruit fly a powerful model organism to understand how this pathway regulates tissue growth during development and cancer. The Hippo pathway regulates the expression of genes involved in cell proliferation, survival, stemness, differentiation, and migration. However, we lack a clear understanding of the full transcriptional program that the Hippo pathway regulates in growing tissues beyond a small number of well-studied target genes. In this thesis, I aimed to deepen our understanding of the transcriptional program that the Hippo pathway controls in the growing Drosophila eye. To do this, I addressed three key questions: 1) What are the target genes of Yki, Sd and Tgi? 2) What gene expression changes occur as a result of hyperactive Yki? 3) What additional transcriptional regulators control Hippo pathway target gene expression and eye growth? 1) Using targeted DamID-seq, I identified target genes of Yki, Sd and Tgi in growing Drosophila eye discs. I found a very high degree of overlapping target genes between Yki, Sd, and Tgi, indicating that Sd is the key transcription factor that mediates binding of both Yki and Tgi to their target gene loci. Additionally, I found a strong enrichment of the AP-1 binding motif in Yki, Sd, and Tgi target genes, suggesting that these transcription factors co-regulate many target genes. 2) I performed comprehensive analyses of Yki, Sd, and Tgi target genes, and the changes in gene expression and chromatin accessibility caused by Yki hyperactivation. This highlighted key biological processes and signalling pathways that the Hippo pathway could regulate during eye growth, including: MAPK pathway, glutathione S-transferases (GSTs), cuticle development, extracellular matrix (ECM), and retinal determination and differentiation. 3) Using targeted DamID-seq, I found that the Drosophila AP-1 transcription factor Jra/Jun shares 71% of its target genes with Yki and/or Sd. However, using genetic studies, I showed that AP-1 transcription factors do not regulate physiological Drosophila eye growth, and are not essential regulators of Hippo pathway target gene expression. The results presented in this thesis provide new insights into the target genes and transcriptional program that is regulated by the Hippo pathway during Drosophila eye growth. They have identified previously unexplored biological processes that the Hippo pathway might regulate in the eye (e.g. cuticle development), and reinforced known roles for the Hippo pathway (e.g. cell fate specification). My findings also extend our understanding of the functional links between AP-1 and the Hippo pathway in growing organs. Importantly, the data generated in this thesis will provide a rich resource for researchers interested in studying the direct target genes of both Hippo signalling and AP-1 transcription factors