Nuclear location of DUX4 is required for its corepressor activity on the progesterone nuclear receptor

GATICA LV; ROSA AL

Boston, MA

Congreso; FSH Society Facioscapulohumeral Muscular Dystrophy [FSHD] 2015 International Research Consortium & Research Planning Meetings; 2015

Our laboratory first presented evidence indicating that DUX4 is a negative co-regulator of the human progesterone receptor (PR). We also showed that progesterone protects cultured cells from DUX4-mediated cytotoxicity. DUX4 is normally expressed in gonadal tissues of healthy individuals, coincidental with a potential role for DUX4 in the endocrine pathway. Because DUX4 is a toxic protein, we hypothesize that cells from germinal tissues have specific regulatory and protective mechanism allowing normal expression of the DUX4 gene and bypassing the toxic effect of DUX4, respectively. The hypothetical protective mechanism(s) would not be present in tissues where ectopically/improperly expressed DUX4 leads to cell death. We have previously shown that DUX4-mediated toxicity is dependent on the subcellular location of DUX4. In this work we explored if alternative subcellular locations of DUX4 regulates/modifies its coregulatory activity on the PR. Mutations at the more relevant NLSs from DUX4 (i.e. NLS1 and NLS2) partially delocalized DUX4 from the cell nucleus and were analyzed in these studies. It was observed that DUX4 with altered transit to the nuclei lose its corepressor activity on the PR. Thus, re-located DUX4 (i.e. mostly cytoplasmic) does not have any effect on the activity of the PR. These results indicate that the corepressor effect of DUX4 on the activity of the PR is exerted at the cell nucleus. DUX4-NLS mutants carrying the NLS from the virus SV40 would allow to re-direct DUX4 to the nuclei to analyze if the DUX4 NLS sequences per se participate in the DUX4 regulatory activity on the PR. To explore if alternative macromolecular structures of DUX4 disturb its activity on the PR, DUX4 fusions to GFP were analyzed. All the studies were performed on a reconstructed PR gene reporter system, using HepG2 cells, as well as on breast cancer cells endogenously expressing the PR. In this work we also analyzed the protective effect of progesterone on the toxicity of DUX4 NLS mutants. The toxic effect of DUX4 mutants was analyzed using a modified assay described in our laboratory and quantified using FACS. Results from these experiments indicate that progesterone synergize the low-toxicity of DUX4 NLS mutants. Taken together these studies strongly support our previous contention about the negative co-regulatory activity of DUX4 on the PR as well as the protective effect of progesterone on DUX4 toxicity. These results are relevant to both the normal and pathological function of DUX4 as well as the future rational therapies in FSHD.