The preservation of germ cell sexual identity is essential for reproduction. Defects in sex-specific programs leads to infertility and germ cell tumors. Despite its importance to human health, how germ cell sexual identity is maintained is unknown. The Drosophila female germline is an ideal model for studying how this important cell fate decision is maintained. The connection between failures to maintain sexual identity, aberrant expression of spermatogenesis genes and germ cell tumor development enabled the assignment of genes to a sex fate maintenance pathway. These new studies demonstrate that an epigenetic regulatory pathway in which SXL is the upstream female-specific regulator, SETDB1 is the required chromatin writer and phf7 is one of the key downstream SETDB1 target genes, maintains sexual identity. SETDB1 trimethylates H3K9 (H3K9me3), an histone modification associated with gene silencing. The evidence suggests a hypothesis in which Sxl dependent transcription factors direct SETDB1 to its spermatogenesis target genes, where it deposits a local H3K9me3 mark, leading to HP1a-dependent silencing. Disruption of this pathway leads to ectopic PHF7 expression. This testis-specific protein is sufficient to reprogram transcription of its target genes (including itself), leading to spermatogenesis gene activation and a germ cell tumor. The studies in this proposal will test this model and advance our understanding of this newly identified pathway, in two Aims.
In Aim 1, the focus is on SETDB1. The hypothesis that SETDB1 silences spermatogenesis genes by installing gene specific silencing chromatin on its target genes will be tested at a global level by comparing the distribution of H3K9me3 and its ligand HP1a in wild-type and SETDB1 depleted germ cells. A key question raised by this hypothesis is how SETDB1 is recruited to its spermatogenesis target genes. Genetic studies have identified two Sxl dependent transcription factors that are likely to be the critical targeting factors. Combined genetic, cytological and genomic approaches will be used to determine how these newly identified pathway members contribute to female fate maintenance.
In Aim 2, the focus is on PHF7. PHF7 is a chromatin reader that binds to H3K4me2, a mark associated with active or poised genes. Thus ectopic PHF7 may reprogram sexual fate by activating silent, but poised, spermatogenesis genes, and/or by repressing active genes required for spermatogenesis gene silencing. Testing this model requires identification of PHF7 target genes. We will first build on evidence that PHF7 reprograms its own transcription, and then take a more global approach. These studies will lead to an understanding of how forced expression is able to disrupt female fate, and will serve as a discovery tool for assigning new genes to this novel pathway. The striking similarities in germ cell biology between humans and flies suggest that the knowledge gained from the studies in this proposal will provide insight into how human germ cells maintain their sexual identity, and how errors in this process interferes with reproduction.

Public Health Relevance

Whether germ cells succeed in making eggs or sperm depends on a number of factors, including preservation of sexual identity. Evidence suggests that the mechanism uncovered in Drosophila ovarian germ cells is used by other organisms, including humans. Thus the knowledge gained from the studies in this proposal will impact our understanding of how human germ cells maintain their sexual identity, and why errors in this process leads to infertility and germ cell tumors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM129478-03
Application #
9986796
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Salazar, Desiree Lynn
Project Start
2018-09-01
Project End
2022-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Genetics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Smolko, Anne E; Shapiro-Kulnane, Laura; Salz, Helen K (2018) The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells. Nat Commun 9:4155