Using ROADMAP Data to Identify Enhancers Associated with Disorders of Sex Development

Ohnesorg, Thomas; Croft, Brittany; Tan, Jacqueline; Sinclair, Andrew H.

doi:10.1159/000445398

Cited by 12 publications

(10 citation statements)

References 23 publications

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“…All luciferase assays were performed in COS7. Briefly, cells were seeded at 80–90% confluency in 96 well plate for 2–4 h. Each well was transfected with a combination of enhancer reporter plasmid pgl4-beta-globin (75 ng) and transcription factors in pcDNA3.1 (100 ng) with 15 ng of Renilla luciferase pLR-TK (Promega) as a control using lipofecamine 2000 for 24 h. The cells were lysed using 1.25x passive lysis buffer (Promega) and luciferase was determined using Dual-Luciferase® Reporter Assay System (Promega) as previously described 40 . All enhancers were further tested in HEK293T, TM3 and TM4 using the same method as COS7 (Supplementary Figure 2).…”

Section: Methodsmentioning

confidence: 99%

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

et al. 2018

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Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

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Section: Methodsmentioning

confidence: 99%

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

et al. 2018

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“…While no significant gonadal expression was detected for these genes at d34-44 in controls, either could be expressed earlier, perhaps before RSPO1 expression normally begins in ovaries. Functionality of potential transcription factor binding sites introduced by the G+ insertion near these genes could be tested by in vitro luciferase assays [ 50 ]. Briefly, luciferase reporter constructs containing a potential site and two controls, one containing the native canine sequence, the other being an empty plasmid, could be prepared such that each site is located at an appropriate distance from a minimal promoter.…”

Section: Discussionmentioning

confidence: 99%

XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris)

et al. 2017

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Remarkable progress has been achieved in understanding the mechanisms controlling sex determination, yet the cause for many Disorders of Sex Development (DSD) remains unknown. Of particular interest is a rare XX DSD subtype in which individuals are negative for SRY, the testis determining factor on the Y chromosome, yet develop testes or ovotestes, and both of these phenotypes occur in the same family. This is a naturally occurring disorder in humans (Homo sapiens) and dogs (C. familiaris). Phenotypes in the canine XX DSD model are strikingly similar to those of the human XX DSD subtype. The purposes of this study were to identify 1) a variant associated with XX DSD in the canine model and 2) gene expression alterations in canine embryonic gonads that could be informative to causation. Using a genome wide association study (GWAS) and whole genome sequencing (WGS), we identified a variant on C. familiaris autosome 9 (CFA9) that is associated with XX DSD in the canine model and in affected purebred dogs. This is the first marker identified for inherited canine XX DSD. It lies upstream of SOX9 within the canine ortholog for the human disorder, which resides on 17q24. Inheritance of this variant indicates that XX DSD is a complex trait in which breed genetic background affects penetrance. Furthermore, the homozygous variant genotype is associated with embryonic lethality in at least one breed. Our analysis of gene expression studies (RNA-seq and PRO-seq) in embryonic gonads at risk of XX DSD from the canine model identified significant RSPO1 downregulation in comparison to XX controls, without significant upregulation of SOX9 or other known testis pathway genes. Based on these data, a novel mechanism is proposed in which molecular lesions acting upstream of RSPO1 induce epigenomic gonadal mosaicism.

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“…Recent studies have attempted to identify human enhancers for this purpose, using publicly available data from the NIH Roadmap Epigenomics Program (Roadmap Epigenomics Consortium et al, 2015). DNaseIseq datasets for human fetal ovary and testis tissue have been used to identify putative enhancers (Ohnesorg et al, 2016). These tissues were presumably collected well after 4-6 weeks of gestation, the stage at which sex determination occurs in humans, and it is currently unclear whether enhancers that operate at the sexdetermining stage are maintained in the late fetal stages.…”

Section: Regulatory Sites May Harbor Non-coding Mutations That Cause mentioning

confidence: 99%

Genome-wide identification of regulatory elements in Sertoli cells

et al. 2017

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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.

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Using ROADMAP Data to Identify Enhancers Associated with Disorders of Sex Development

Cited by 12 publications

References 23 publications

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

XX Disorder of Sex Development is associated with an insertion on chromosome 9 and downregulation of RSPO1 in dogs (Canis lupus familiaris)

Genome-wide identification of regulatory elements in Sertoli cells

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