PI: Joel Chamberlain PhD
The milestones for year one were to perform an initial screen for Platform 1 and prepare and validate Platform 2.
Platform 1: Non-specific readout for DUX4 activity.
Expressing DUX4 in muscle cells results in cell death. Therefore agents that prevent cell death have therapeutic potential. Screening for agents that facilitate survival of DUX4 expressing cells might yield relatively non-specific agents, but is an efficient way to quickly prioritize agents for more specific screens. We evaluated three cell types for DUX4 induced toxicity. We determined a time course of death and percentage of death following DUX4 induction and identified a cell line that had a highly reproducible profile suitable for high throughput screening. The format used and the cell death assay met the criteria for the Genomics Shared Resource Screening Facility to initiate the screen; however, the method of DUX4 induction did not. Therefore, we sought to develop an inducible construct that would satisfy the requirement of the screening facility. Although we thought our new strategy to produce the inducible construct was rather straightforward, it proved much more difficult than we had anticipated. However, at this point we do not anticipate any further problems with preparing the cells for the Genomics facility.
Platform 2: Specific readout for DUX4 activity.
We currently believe that any intervention that blocks the activity of the DUX4 transcription factor will be a candidate therapeutic for FSHD. Our recent work has identified genes regulated by DUX4 and the DUX4 binding regions that transcriptionally activate these genes. We have made a construct that uses the regulatory elements from one of the DUX4 regulated genes to drive expression of the luciferase reporter gene and demonstrated that this construct shows robust activation by DUX4. The next step is to introduce this construct into FSHD cells. We have made substantial progress on cloning and validating additional DUX4 responsive promoters and now have several candidate second or third reporter genes that can be co-introduced into cells.
See also 2011 year 1 grant for Modulators of Epigenetic Disease.