Interaction between PKA signalling and TRIM28 in ovarian maintenance and pathology – OvaryProtect

Gonadal sex fate in mammals is determined during embryonic development and must be actively maintained through adulthood. Therefore, gonadal sex-specific transcription factors are required to prevent transdifferentiation of adult gonadal somatic cells into the alternate sex fate. In mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying the protective effect of these transcription factors is unknown.
We have shown that in the female mouse that the nuclear scaffold protein TRIM28 is a cofactor of FOXL2 in the maintenance of ovarian cell fate through its E3-SUMO ligase activity. In absence of TRIM28, granulosa cells are reprogrammed in Sertoli cells, leading to a post-natal sex-reversal. Remarkably, we also found that a mouse model of the human CARNEY complex syndrome recapitulates the ovarian deletion of Trim28 by inducing a female-to-male sex-reversal. In these mice, the deletion in granulosa cells of the Prkar1a gene, encoding the regulatory subunit of the protein kinase A (PKA), leads to the constitutive activation of the PKA signalling pathway, driving to their transdifferentiation into Sertoli cells

In light of the unprecedented phenotypic convergence of these two models developed by our consortium, we hypothesize that PKA signalling might converge on TRIM28/FOXL2 complexes to inactivate their repressing effect on the male genetic pathway and induce trans differentiation of granulosa into Sertoli cells. Indeed, our preliminary in vitro and in vivo results shows that PKA signalling induces phosphorylation of TRIM28 and repress its transcriptional activity.

The main objective of the present project is to explore the link between PKA pathway and TRIM28 in the ovarian physiology as well as in mutated background and pathological situations. Using in vitro approaches and an important toolkit of mouse genetics, we will decipher at the cellular, genomic and transcriptional levels the interconnection between the PKA pathway and the TRIM28-dependent SUMOylation. This will allow us to describe the molecular transition occurring between physiological and mutant SUMOylomes. We will determine how gonadotropic hormones/PKA-TRIM28 axis influence SUMOylation and the landscape of sex-maintaining genes to prevent trans differentiation of ovarian somatic cells by using single-cell and bulk RNA sequencing, chromatin remodelling, epigenomic analyses in combination with genetic models of post-natal sex reversal.

Our consortium puts together the skills of three groups specialized in reproduction and endocrine signalling. With elaborate genetic models and state-of-the-art chromatin, transcriptomic and proteomic analyses, OvaryProtect will decipher the functional interactions between PKA signalling and the nuclear effector Trim28, the SUMO-E3 ligase activity of which is crucial for ovarian identity. The data generated by this project should lead to revisit our vision of genetic diseases of PKA such as Carney complex by considering epigenetic and transdifferentiating actions as an integral part of the pathogenicity of PKA.

At a broader level, by analysing the deregulation of ovarian identity maintenance, in the CNC syndrome, this project will extend the knowledge on frequent ovarian pathologies as Polycystic ovary syndrome (affecting 6 to 8% of women worldwide) where hyperactive PKA signalling has been observed in Granulosa cells of PCOS patients.