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Update by Jocelyn O Eidahl PhD.
Aim 1: To define a subset of DUX4 post-translational modifications (PTMs) from affected models.
We have finalized our list of DUX4 PTMs found on the DUX4 protein when we overexpress our CMV.DUX4.V5 construct in the HEK293 cell line. We are now beginning to perform the same experiments in human myoblasts and possibly a DUX4-inducible myoblast cell line, where we can examine DUX4 modification in differentiated myotubes. Additionally, I plan to test optimized conditions for immunoprecipitating endogenous DUX4 from monkey testes.
Aim 2: To examine the role of post-translational modifications on DUX4 function.
Our submitted manuscript examined 34 DUX4 modification mutant constructs in HEK293 cells. We used caspase-3/7 activation and expression of a DUX4-specific transactivation reporter to monitor toxicity and transcription factor function of each. In addition, we monitored steady-state protein production, nuclear localization and biomarker expression levels of all mutants displaying a differential phenotype.
Based on the location and biochemical properties of some of our DUX4 modification mutants, we hypothesize they have a reduced DNA binding affinity which could be altering their toxicity/function. In the past 6 months, I have optimized conditions to monitor the chromatin binding affinity of wildtype DUX4 in HEK293 cells. I will next begin to monitor the DUX4 mutants that display a differential phenotype using this method.
Aim 3: To identify modifying enzymes that regulate DUX4 function.
Our manuscript summarizes results of the two methods used to identify modifying enzymes of DUX4; (1) radioactive kinase screen and (2) RIME. Scott has spent a significant amount of time finalizing a list of compounds that are commercially available that target some of the identified modifying enzymes, specifically the kinase class of enzymes. As I described in my proposal, our plan is to test the ability of these compounds to reduce toxicity in DUX4 expressing cells. We are also designing experiments to overexpress some of these modifying enzymes and monitor DUX4 toxicity/function.
We identified a modifying enzyme that associates with DUX4 using the RIME methodology. In the past 6 months, we have shown two inhibitors of this enzyme can misregulate DUX4 toxicity and function. We will continue to narrow down the IC50 of these two compounds.