81 Oral - Gene Regulation
Friday April 08, 10:45 AM - 11:00 AM

Authors:
Samuel Manning ^1,2; Benjamin Kroeger ^1,2; Elizabeth Hinde ³; Kieran Harvey ^1,2

Affiliations:
1) Monash University, Melbourne, Australia; 2) Peter MacCallum Cancer Centre, Melbourne, Australia; 3) The University of Melbourne, Australia

Keywords:
c. activators/coactivators; d. histone variants and modifications

The Hippo signalling pathway is a highly conserved regulator of cell fate and organ size, which regulates the nuclear-cytoplasmic shuttling of the transcriptional co-activator Yorkie (Yki), and thereby regulates gene expression. We currently have very little understanding of how the Hippo pathway modifies the nuclear biophysical behaviour and genomic interactions of Yki or its DNA binding partner Scalloped (Sd) to regulate transcription.
To address this, we used cutting-edge ex vivo fluorescence microscopy approaches including Raster Image Correlation Spectroscopy (RICS), Fluorescence Recovery After Photobleaching (FRAP) and Single Molecule Tracking (SMT) to investigate the biophysical behaviour of Sd and Yki in the nuclei of living Drosophila tissues. We found that Yki moves more rapidly through the nuclear environment than Sd, and that Yki’s movement is highly sensitive to Sd abundance, demonstrating the importance of Sd for Yki’s navigation of the genome. Sd and Yki were highly enriched at regions of active chromatin, consistent with their role as a heterodimeric activator of transcription. Further, enrichment of Sd at these loci is driven in part by increased chromatin residence times. Yki interacts more transiently with chromatin than Sd at these sites, and the duration of this interaction is dependent on Sd levels, further highlighting the importance of Sd levels for recruitment of Yki. FRAP and SMT demonstrate that Yki and the transcriptional repressor Nerfin-1 alter Sd chromatin residence times, providing mechanistic insight in to how these proteins alter Hippo pathway output.
Taken together we show that a transcriptional co-activator and repressor are able to modify the binding duration of a transcription factor, with subsequent impacts on transcription. These results also indicate that altering binding duration is one mechanism by which Hippo pathway transcription factors can be enriched at a specific locus to regulate gene expression. These results have implications for understanding how the Hippo pathway regulates normal development and disease, and also provide mechanistic frameworks for understanding how transcription may be regulated by other signalling pathways.