ATRX mutations cause X-linked α-thalassemia/mental retardation syndrome (ATR-X) characterized by intellectual disability, developmental delay, facial dysmorphism, seizures, and genital abnormalities. To study genital abnormalities, we deleted Atrx from mouse testicular Sertoli cells; knockout mice develop small testes with discontinuous unbranched tubules and apoptosis of Sertoli cells during fetal life.
Aims: To investigate the mechanism underlying the ATR-X syndrome gonadal phenotype.
Methods: Mouse embryonic gonads from wildtype and Atrx knockout E16.5 embryos were processed sectioned and Immunofluorescence (IF) or Immuno-FISH performed followed by quantification using ImageJ.
Results: PML nuclear bodies (PML-NBs) are spherical structures in the nucleus with 1 micron in diameter; they vary in protein composition and have been implicated in gene expression, chromatin assembly, telomere lengthening and DNA repair. Wildtype Sertoli cell nuclei showed unexpectedly two types of PML-NBs that contain ATRX, its binding partner DAXX and heterochromatin protein HP1a. Firstly, classic PML-NBs and secondly a single novel “GATA4 PML-NB” that strongly expressed the somatic cell marker GATA4. Immuno-FISH showed that the GATA4 PML-NBs localize on the short arm of the Y chromosome. In knockout mice, ATRX-deficient PML-NBs showed loss of DAXX and HP1a, suggesting a failure in heterochromatin formation. In addition, ~30% of ATRX-deficient Sertoli cells showed GATA4 PML-NBs that were 2-3 times larger, and 84% of them (vs 4.1% in control) showed DNA damage (gH2Ax staining) either within the GATA4 PML-NBs or widespread throughout the nucleus of apoptotic cells.
Conclusion: A novel PML-NB, bearing GATA4 and the short arm of the Y chromosome, was identified in Sertoli cells. Within these PML-NBs, ATRX mediates chromatin condensation of the Y chromosome, which is required for Sertoli cell survival, and testis development.