A current goal of molecular biology is to identify transcriptional networks that regulate cell differentiation. However, identifying functional gene regulatory elements has been challenging in the context of developing tissues where material is limited and cell types are mixed. To identify regulatory sites during sex determination, we subjected Sertoli cells from mouse fetal testes to DNaseI-seq and ChIP-seq for H3K27ac. DNaseI-seq identified putative regulatory sites around genes enriched in Sertoli and pregranulosa cells; however, active enhancers marked by H3K27ac were enriched proximal to only Sertoli-enriched genes. Sequence analysis identified putative binding sites of known and novel transcription factors likely controlling Sertoli cell differentiation. As a validation of this approach, we identified a novel Sertoli cell enhancer upstream of Wt1, and used it to drive expression of a transgenic reporter in Sertoli cells. This work furthers our understanding of the complex genetic network that underlies sex determination and identifies regions that potentially harbor non-coding mutations underlying disorders of sexual development.
The authors declare no competing or financial interests.
D.M.M., A.N., G.E.C., U.O. and B.C. designed the experiments. D.M.M., A.N., L.S. and Y.S. carried out the experiments. D.M.M. and A.N. prepared the manuscript with input from all authors.
Funding was provided by the Duke University School of Medicine for use of the sequencing facility and the National Institutes of Health (5R01-HD039963-13 to B.C.). Deposited in PMC for release after 12 months.
Datasets (including DNaseI-seq, ChIP-seq and RNA-seq) are publicly available in GEO (Accession Number GSE53076).
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.142554.supplemental
- Received August 1, 2016.
- Accepted December 30, 2016.