DUX4 negatively regulates the activity of the progesterone receptor while gonadal steroids protects cells from DUX4-mediated toxicity

GATICA LAURA VIRGINIA, PROIETTI CECILIA , ELIZALDE PATRICIA, CORONA EDGARDO DANIEL, JACQUELIN DANIELA, PELLIZAS CLAUDIA, ROSA ALBERTO LUIS

Workshop; FSH Society FSHD International Research Consortium Workshop - Boston; 2013

  FSHD seemed to run a milder clinical course in females than males. Females, however, appear to be more affected following menopause. No explanation is available on this gender difference in severity and progression of FSHD. In this work we present preliminary evidence suggesting a possible connection between this clinical observation and a potential physiological role we propose here for DUX4, the main candidate pathogenic protein in FSHD (1,2). In previous studies on molecular determinants for nuclear import and pathogenicity of DUX4 (3), we performed a detailed in silico analyses of the C-terminal domain of DUX4 and DUX4c proteins. We found that DUX4, but no DUX4c, contains a functional motif (LXXLL or NR box) previously recognized in co-regulators of nuclear receptors (4). This domain is highly conserved among DUX4-homologous proteins during evolution. We hypothesized and propose here that DUX4 is a potential hormone receptor co-regulator and its cell toxicity may be connected with its potential role at the endocrine pathway. Interestingly, DUX4 is highly expressed in gonadal tissues (4). In our first set of experiments we used a gene reporter strategy to study if DUX4 regulates the activity of the progesterone receptor (PR). In these studies, T47D cells (breast cancer) were co-transfected, in culture media supplemented with physiological doses of progesterone, with a MMTV-luciferase reporter gene, pRL-CMV Renilla (internal control), and different amounts of a vector expressing DUX4. In these studies we observed that ligand activated- PR transcriptional activation was inhibited in a DUX4-dose dependent manner. These preliminary results highlight a potential novel function for DUX4 as a negative co-regulator of the progesterone receptor. Additional studies concerning DUX4 as a potential regulator of the androgen receptor (AR) are currently being performed. In our second set of experiments we studied if treatment of cultured cells with different doses of sex steroids (i.e. progesterone, estrogen and testosterone) modifies DUX4-mediated cytotoxicity. In these studies, DUX4 toxicity was measured using a DUX4/GFP co-transfection assay developed in our laboratory (1,3). Cells were pre-treated with various amounts of the analyzed hormones (or vehicle) and then co-transfected with plasmid vectors expressing GFP and DUX4 (or empty vector). A 2-fold increase in GFP-positive cells (i.e. diminished toxicity of DUX4) was observed when cells were pre-treated with physiologically doses of progesterone or supra-physiological doses of estrogen or testosterone. These results indicate that sex hormones protect cells from DUX4 toxicity. Our studies show for the first time that DUX4 modulates the activity of a nuclear hormone receptor and that gonadal steroids modulate DUX4-mediated cytotoxicity. These findings may have physiological relevance concerning the normal/pathological role of DUX4 contributing to explain the gender difference observed in FSHD clinical manifestations.     (1) Kowaljow,V., Marcowytz, A., Ansseau, E., Conde, C., Sauvage, S., Mattéotti, C., Arias, C., Corona, E.D., Nuñez, N.G., Leo, O. et al. (2007) The DUX4 gene at FSHD1A locus encodes a pro-apoptotic protein. Neuromuscul. Disord., 17, 611-623. (2) Lemmers, R.J., van der Vliet, P.J., Klooster, R., Sacconi, S., Camaño, P., Dauwerse, J.G., Snider, L., Straasheijm, K.R., van Ommen, G.J., Padberg, G.W. et al (2010) A unifying genetic model for facioscapulohumeral muscular dystrophy. Science, 329, 1650-1653. (3) Corona, ED, Jacquelin, D, Gatica, L. and Rosa, A.L. (2013) Multiple protein domains contribute to nuclear import and cell toxicity of DUX4, a candidate pathogenic protein for facioscapulohumeral muscular dystrophy. PLoS ONE (in press) (4) Leo, C. and Chen, J.D. (2000) The SRC family of nuclear receptor coactivators. Gene., 245, 1-11. (5) Snider, L., Geng, L.N., Lemmers, R.J., Kyba, M., Ware, C.B., Nelson, A.M., Tawil, R., Filippova, G.N., van der Maarel, S.M., Tapscott, S.J. et al. (2010) Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. PLoS. Genet., 6, e1001181.