Project : Deciphering the role of the heterochromatin mark H3K9me3 in mammalian testicular determination

In mammals, the embryonic bipotential gonad can develop either as a testis or an ovary in XY and XX embryos respectively and depends on antagonistic pro-testis and pro-ovarian pathways. Testis development is initiated by the expression of the SRY gene carried by the Y chromosome, that in turn induces the expression of the master transcription factor SOX9 leading to the differentiation of Sertoli cells. These supporting cells produce crucial factors that further orchestrate testis morphogenesis and differentiation. If the "pro-testicular" effects of SRY and SOX9 transcription factors are well documented, little is known about how the ovarian program is repressed during testis development. This is an important issue as the abnormal activation of the female program in XY individuals can lead to sex reversal, one type of disorders of sexual development (DSD). A genetic diagnosis is achieved in only 40% of human DSD patients analyzed by exome sequencing, indicating that additional levels of regulation control gonadal development.

We have recently discovered that sex-determination induces a strong sexual dimorphism for the tri-methylation on the lysine 9 of the histone H3, a marker of heterochromatin regions in mice. While being maintained in testis supporting cells, the repressive mark H3K9me3 is strongly reduced in their female counterpart, the granulosa cells. We hypothesize that an unexpected level of genomic regulation involving heterochromatin and H3K9me3 deposition contributes to sex-determination through the lock-down of the female genes in the testis. Moreover, as H3K9me3 is an important component of DNA repeats, a sexual dimorphism might exist at the level of the repetitive fraction of the genome composing crucial chromosomal structures such as the telomeric and pericentromeric regions. 

For the present project we have defined several objectives that propose to investigate:

- The role of heterochromatin on sex determination through the study of multiple sex-reversed mutants and the role of the histone methylase SETDB1 as a central actor of male-specific heterochromatin. 

- Using genomic profiling by the CUT&RUN technique on purified Sertoli or granulosa cells, we will test a model where SOX9 would induce the redeployment of SETDB1 and thus of H3K9me3 on chromatin via their common partner TRIM28. In addition, we will analyse the histone marks associated with transcriptional activation or repression.

This project will unravel a new level of regulation of gonadal sex-determination. It will determine whether male-specific heterochromatin formation is a cause or a consequence of sex determination. Moreover, it will provide new perspectives for the analysis of unresolved DSD patients in human. Insights we gain from this project might also help explaining the differences of some disease incidences between man and woman.