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Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

ABSTRACT: Cis-regulatory elements are critical for the precise spatiotemporal regulation of genes during development. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Here, we identified putative regulatory elements during sex determination by performing ATAC-seq and ChIP-seq for H3K27ac in purified XX and XY gonadal supporting cells before and after sex determination in mice. We show that XX and XY supporting cells initiate sex determination with similar chromatin landscapes and acquire sex-specific regulatory elements as they commit to the male or female fate. To validate our approach, we identified a functional gonad-specific enhancer downstream of Bmp2, an ovary-promoting gene. This work increases our understanding of the complex regulatory network underlying mammalian sex determination and provides a powerful resource for identifying non-coding regulatory elements that could harbor mutations that lead to Disorders of Sexual Development.

SUBMITTER: Garcia-Moreno SA

PROVIDER: S-EPMC6368449 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

Garcia-Moreno S Alexandra SA Futtner Christopher R CR Salamone Isabella M IM Gonen Nitzan N Lovell-Badge Robin R Maatouk Danielle M DM

Developmental biology 20181227 2

Cis-regulatory elements are critical for the precise spatiotemporal regulation of genes during development. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Here, we identified putative regulatory elements during sex determination by performing ATAC-seq and ChIP-seq for H3K27ac in purified XX and XY gonadal supporting cells before and after sex determination in m ...[more]

PMID: 30594505

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Project description:BackgroundThe American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD.ResultsOur characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted.ConclusionsGonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

Project description:A critical transcription factor required for mammalian male sex determination is SRY (sex determining region on the Y chromosome). The expression of SRY in precursor Sertoli cells is one of the initial events in testis development. The current study was designed to determine the impact of environmentally induced epigenetic transgenerational inheritance on SRY during gonadal sex determination in the male. The agricultural fungicide vinclozolin and vehicle control (DMSO) exposed gestating females (F0 generation) during gonadal sex determination promoted the transgenerational inheritance of differential DNA methylation in sperm of the F3 generation (great grand-offspring). The fetal gonads in F3 generation males were used to identify potential alterations in SRY binding sites in the developing Sertoli cells. Chromatin immunoprecipitation with an SRY antibody followed by genome-wide promoter tiling array (ChIP-Chip) was used to identify alterations in SRY binding. A total of 81 adjacent oligonucleotide sites and 173 single oligo SRY binding sites were identified to be altered transgenerationally in the Sertoli cell vinclozolin lineage F3 generation males. Observations demonstrate the majority of the previously identified normal SRY binding sites were not altered and the altered SRY binding sites were novel and new additional sites. The chromosomal locations, gene associations and potentially modified cellular pathways were investigated. In summary, environmentally induces epigenetic transgenerational inheritance of germline epimutations appears to alter the cellular differentiation and development of the precursor Sertoli cell SRY binding during gonadal sex determination that influence the developmental origins of adult onset testis disease. The experimental design involved the intraperitoneal exposure of gestating female rats to vinclozolin or a vehicle control (dimethyl sulfoxide, DMSO) transiently from embryonic days E8-E14. Sister littermates were divided into control and vinclozolin treatment groups and mated to similar males to minimize the genetic variation between the control and vinclozolin lineages. Sufficient females were used so no inbreeding (sibling or cousin) occurred in any generation. The F1 generation was bred within the lineage to generate the F2 generation and these F2 generation bred to generate the F3 generation. The only exposure was the F0 generation female. The F3 generation control and vinclozolin lineage embryonic day 13 (E13) embryos were collected and the gonads micro-dissected and then sexed with an SRY PCR protocol. The male gonads were pooled from a minimum of three different litters and the pools used to collect DNA. Three different experiments were performed to collect 3 control and vinclozolin E13 F3 generation testis pools, each with different animals (n=25 gonads/pool). The chromatin DNA (not denatured) from each pool was fragmented and used in an SRY chromatin immunoprecipitation (ChIP) procedure for each pool separately. The control and vinclozolin SRY ChIP DNA were paired for a competitive hybridization on a genome-wide promoter tiling array (ChIP-Chip) assay. The hybridization data obtained was used to identify the SRY binding sites that were different in the F3 generation vinclozolin versus control lineage in E13 testis.

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Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

Publications

Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

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