RESUMO
Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/ß-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/ß-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote ß-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and ß-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.
Assuntos
Epigênese Genética/fisiologia , Gônadas/embriologia , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Ovário/embriologia , Ovário/metabolismo , Caracteres Sexuais , Diferenciação Sexual/genética , Testículo/embriologia , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
XX and XY fetal gonads are initially bipotential, poised between the ovary and testis fate. Multiple lines of evidence suggest that commitment to testis fate requires the repression of genes associated with ovary fate. It was previously shown that loss of CBX2, the subunit of the Polycomb Repressive Complex 1 (PRC1) that binds H3K27me3 and mediates silencing, leads to ovary development in XY mice and humans. While it had been proposed that CBX2 is an activator of the testis-determining gene Sry, we investigated the alternative possibility that CBX2 has a direct role as a repressor of the antagonistic ovary-promoting pathway. To investigate this possibility, we developed a quantitative genome-wide profile of the repressive histone mark H3K27me3 and its active counterpart H3K4me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that testis and ovary sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue testis development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, an ovary-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development.
Assuntos
Epigênese Genética , Ovário/metabolismo , Complexo Repressor Polycomb 1/genética , Processos de Determinação Sexual , Testículo/metabolismo , Via de Sinalização Wnt/genética , Animais , Embrião de Mamíferos , Feminino , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Ovário/citologia , Ovário/crescimento & desenvolvimento , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Complexo Repressor Polycomb 1/deficiência , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Sexual , Testículo/citologia , Testículo/crescimento & desenvolvimento , Proteína Wnt4/genética , Proteína Wnt4/metabolismoRESUMO
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.
Assuntos
Cromatina/genética , Gônadas/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Processos de Determinação Sexual/genética , Acetilação , Animais , Cromatina/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos TransgênicosRESUMO
Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9 Although others are redundant, enhancer 13 (Enh13), a 557-base pair element located 565 kilobases 5' from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with Sox9 transcript levels equivalent to those in XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans.
Assuntos
Elementos Facilitadores Genéticos/genética , Disgenesia Gonadal 46 XY/genética , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Testículo/embriologia , Animais , Sequência Conservada , Feminino , Humanos , Masculino , Camundongos , Deleção de Sequência , Proteína da Região Y Determinante do Sexo/genética , Sítio de Iniciação de TranscriçãoRESUMO
A fundamental goal in biology is to understand how distinct cell types containing the same genetic information arise from a single stem cell throughout development. Sex determination is a key developmental process that requires a unidirectional commitment of an initially bipotential gonad towards either the male or female fate. This makes sex determination a unique model to study cell fate commitment and differentiation in vivo. We have focused this review on the accumulating evidence that epigenetic mechanisms contribute to the bipotential state of the fetal gonad and to the regulation of chromatin accessibility during and immediately downstream of the primary sex-determining switch that establishes the male fate.
