The ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome is a severe developmental disorder affecting males caused by mutations in the chromatin remodelling gene ATRX. 80% of patients display genital abnormalities which include hypospadias and ambiguous genitalia. These patients have small poorly formed testes containing few seminiferous tubules. Previously, we generated a mouse model for these testicular defects with Atrx specifically inactivated in the Sertoli cells (ScAtrxKO). ScAtrxKO mice develop small testes with discontinuous tubules due to a prolonged G2/M phase and apoptosis of Sertoli cells during fetal life.1
Here, we investigate further the cellular and molecular mechanisms underlying the defects in ATRX-deficient Sertoli cells. In wildtype Sertoli cells, GATA4 is present at PML nuclear bodies (PML-NBs). Intriguingly, in ScAtrxKO mice, the size of a single GATA4-PML-NBs within each Sertoli cell nucleus was increased about 3-fold. Giant PML-NB have been observed in ICF syndrome, in which the giant PML contains hypomethylated DNA and undergoes specific chromosome 1, 9, and 16 breakage.2
In the wildtype Sertoli cells, HP1, DAXX, and FOG2 markers co-localized with GATA4 at a single PML nuclear body. In contrast, in scAtrxKO, DAXX and HP1 were no longer present. Normally ATRX, DAXX and HP1 may facilitate reestablishment of condensed heterochromatin within PML-NBs. Furthermore, the presence of HP1 indicated that this PML-NB is associated with chromatin. At these giant PML NBs PH3, a marker of late G2 and mitosis, was absent suggesting that chromosome condensation was affected at G2/M phase. The giant PML-NBs also co-localised with γ-H2AX, a marker of DNA double strand breaks. We propose that ATRX loss at a single PML-NB in Sertoli cells leads to a failure in chromosome condensation with subsequent DNA damage and apoptosis. Future studies are directed at defining the precise protein and DNA components of these giant PML NBs.