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David Zarkower - One of the best experts on this subject based on the ideXlab platform.
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DMRT1 an ancient sexual regulator required for human gonadogenesis
Sexual Development, 2021Co-Authors: David Zarkower, Mark W MurphyAbstract:Transcriptional regulators related to the invertebrate sexual regulators doublesex and mab-3 occur throughout metazoans and control sex in most animal groups. Seven of these DMRT genes are found in mammals, and mouse genetics has shown that one, DMRT1, plays a crucial role in testis differentiation, both in germ cells and somatic cells. Deletions and, more recently, point mutations affecting human DMRT1 have demonstrated that its heterozygosity is associated with 46,XY complete gonadal dysgenesis. Most of our detailed knowledge of DMRT1 function in the testis, the focus of this review, derives from mouse studies, which have revealed that DMRT1 is essential for male somatic and germ cell differentiation and maintenance of male somatic cell fate after differentiation. Moreover, ectopic DMRT1 can reprogram differentiated female granulosa cells into male Sertoli-like cells. The ability of DMRT1 to control sexual cell fate likely derives from at least 3 properties. First, DMRT1 functionally collaborates with another key male sex regulator, SOX9, and possibly other proteins to maintain and reprogram sexual cell fate. Second, and related, DMRT1 appears to function as a pioneer transcription factor, binding "closed" inaccessible chromatin and promoting its opening to allow binding by other regulators including SOX9. Third, DMRT1 binds DNA by a highly unusual form of interaction and can bind with different stoichiometries.
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the conserved sex regulator DMRT1 recruits sox9 in sexual cell fate reprogramming
Nucleic Acids Research, 2021Co-Authors: Robin E Lindeman, Mark W Murphy, Vivian J. Bardwell, Micah D Gearhart, Kellie S Agrimson, Rachel L Gewiss, David ZarkowerAbstract:Mammalian sexual development commences when fetal bipotential progenitor cells adopt male Sertoli (in XY) or female granulosa (in XX) gonadal cell fates. Differentiation of these cells involves extensive divergence in chromatin state and gene expression, reflecting distinct roles in sexual differentiation and gametogenesis. Surprisingly, differentiated gonadal cell fates require active maintenance through postnatal life to prevent sexual transdifferentiation and female cell fate can be reprogrammed by ectopic expression of the sex regulator DMRT1. Here we examine how DMRT1 reprograms granulosa cells to Sertoli-like cells in vivo and in culture. We define postnatal sex-biased gene expression programs and identify three-dimensional chromatin contacts and differentially accessible chromatin regions (DARs) associated with differentially expressed genes. Using a conditional transgene we find DMRT1 only partially reprograms the ovarian transcriptome in the absence of SOX9 and its paralog SOX8, indicating that these factors functionally cooperate with DMRT1. ATAC-seq and ChIP-seq show that DMRT1 induces formation of many DARs that it binds with SOX9, and DMRT1 is required for binding of SOX9 at most of these. We suggest that DMRT1 can act as a pioneer factor to open chromatin and allow binding of SOX9, which then cooperates with DMRT1 to reprogram sexual cell fate.
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an ancient protein dna interaction underlying metazoan sex determination
Nature Structural & Molecular Biology, 2015Co-Authors: Mark W Murphy, Ken Mcelreavey, Micah D Gearhart, John K Lee, Sandra Rojo, Kayo Kurahashi, Surajit Banerjee, Guy Andre Loeuille, Anu Bashamboo, David ZarkowerAbstract:DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.
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sexual cell fate reprogramming in the ovary by DMRT1
Current Biology, 2015Co-Authors: Robin E Lindeman, Vivian J. Bardwell, Anthony D Krentz, Micah D Gearhart, Anna Minkina, David ZarkowerAbstract:Summary Transcription factors related to the insect sex-determination gene doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans and are implicated in transitions between sex-determining mechanisms during vertebrate evolution [1]. In mice, DMRT1 is required for male gonadal differentiation in somatic cells and germ cells [2–4]. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual cell-fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents—granulosa cells—and testicular tissue reorganizes to a more ovarian morphology [5]. Here we use a conditional DMRT1 transgene to show that DMRT1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single-cell RNA sequencing in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single-gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sex development (DSDs) and empirically support evolutionary models in which loss or gain of DMRT1 function promotes establishment of new vertebrate sex-determination systems.
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the mammalian doublesex homolog dmrt6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis
Development, 2014Co-Authors: Teng Zhang, Vivian J. Bardwell, Mark W Murphy, Micah D Gearhart, David ZarkowerAbstract:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here, we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) in mice and find that Dmrt6 plays a crucial role in directing germ cells through the mitotic-to-meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain, a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing inappropriate expression of spermatogonial differentiation factors, including SOHLH1, SOHLH2 and DMRT1 as well as the meiotic initiation factor STRA8, and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant germ cells can complete spermatogonial differentiation and enter meiosis, but they show defects in meiotic chromosome pairing, establishment of the XY body and processing of recombination foci, and they mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 chromatin immunoprecipitation sequencing suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis.
Vivian J. Bardwell - One of the best experts on this subject based on the ideXlab platform.
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the conserved sex regulator DMRT1 recruits sox9 in sexual cell fate reprogramming
Nucleic Acids Research, 2021Co-Authors: Robin E Lindeman, Mark W Murphy, Vivian J. Bardwell, Micah D Gearhart, Kellie S Agrimson, Rachel L Gewiss, David ZarkowerAbstract:Mammalian sexual development commences when fetal bipotential progenitor cells adopt male Sertoli (in XY) or female granulosa (in XX) gonadal cell fates. Differentiation of these cells involves extensive divergence in chromatin state and gene expression, reflecting distinct roles in sexual differentiation and gametogenesis. Surprisingly, differentiated gonadal cell fates require active maintenance through postnatal life to prevent sexual transdifferentiation and female cell fate can be reprogrammed by ectopic expression of the sex regulator DMRT1. Here we examine how DMRT1 reprograms granulosa cells to Sertoli-like cells in vivo and in culture. We define postnatal sex-biased gene expression programs and identify three-dimensional chromatin contacts and differentially accessible chromatin regions (DARs) associated with differentially expressed genes. Using a conditional transgene we find DMRT1 only partially reprograms the ovarian transcriptome in the absence of SOX9 and its paralog SOX8, indicating that these factors functionally cooperate with DMRT1. ATAC-seq and ChIP-seq show that DMRT1 induces formation of many DARs that it binds with SOX9, and DMRT1 is required for binding of SOX9 at most of these. We suggest that DMRT1 can act as a pioneer factor to open chromatin and allow binding of SOX9, which then cooperates with DMRT1 to reprogram sexual cell fate.
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sexual cell fate reprogramming in the ovary by DMRT1
Current Biology, 2015Co-Authors: Robin E Lindeman, Vivian J. Bardwell, Anthony D Krentz, Micah D Gearhart, Anna Minkina, David ZarkowerAbstract:Summary Transcription factors related to the insect sex-determination gene doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans and are implicated in transitions between sex-determining mechanisms during vertebrate evolution [1]. In mice, DMRT1 is required for male gonadal differentiation in somatic cells and germ cells [2–4]. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual cell-fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents—granulosa cells—and testicular tissue reorganizes to a more ovarian morphology [5]. Here we use a conditional DMRT1 transgene to show that DMRT1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single-cell RNA sequencing in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single-gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sex development (DSDs) and empirically support evolutionary models in which loss or gain of DMRT1 function promotes establishment of new vertebrate sex-determination systems.
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the mammalian doublesex homolog dmrt6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis
Development, 2014Co-Authors: Teng Zhang, Vivian J. Bardwell, Mark W Murphy, Micah D Gearhart, David ZarkowerAbstract:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here, we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) in mice and find that Dmrt6 plays a crucial role in directing germ cells through the mitotic-to-meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain, a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing inappropriate expression of spermatogonial differentiation factors, including SOHLH1, SOHLH2 and DMRT1 as well as the meiotic initiation factor STRA8, and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant germ cells can complete spermatogonial differentiation and enter meiosis, but they show defects in meiotic chromosome pairing, establishment of the XY body and processing of recombination foci, and they mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 chromatin immunoprecipitation sequencing suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis.
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interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line
Developmental Biology, 2013Co-Authors: Anthony D Krentz, Mark W Murphy, Michael D Griswold, Vivian J. Bardwell, Aaron L Sarver, Teng Zhang, S Jain, David ZarkowerAbstract:DMRT1 (doublesex and mab-3 related transcription factor (1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis DMRT1 regulates germ cell pluripotency in a strain-dependent manner. Loss of DMRT1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in DMRT1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between DMRT1 and genetic background we compared mRNA expression in wild type and DMRT1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of DMRT1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of DMRT1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although DMRT1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in DMRT1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between DMRT1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease.
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genome wide analysis of dna binding and transcriptional regulation by the mammalian doublesex homolog DMRT1 in the juvenile testis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Mark W Murphy, David Zarkower, Aaron L Sarver, Daren A Rice, Katerina Hatzi, Ari Melnick, Leslie L Heckert, Vivian J. BardwellAbstract:The DM domain proteins Doublesex- and MAB-3–related transcription factors (DMRTs) are widely conserved in metazoan sex determination and sexual differentiation. One of these proteins, DMRT1, plays diverse and essential roles in development of the vertebrate testis. In mammals DMRT1 is expressed and required in both germ cells and their supporting Sertoli cells. Despite its critical role in testicular development, little is known about how DMRT1 functions as a transcription factor or what genes it binds and regulates. We combined ChIP methods with conditional gene targeting and mRNA expression analysis and identified almost 1,400 promoter-proximal regions bound by DMRT1 in the juvenile mouse testis and determined how expression of the associated mRNAs is affected when DMRT1 is selectively mutated in germ cells or Sertoli cells. These analyses revealed that DMRT1 is a bifunctional transcriptional regulator, activating some genes and repressing others. ChIP analysis using conditional mutant testes showed that DNA binding and transcriptional regulation of individual target genes can differ between germ cells and Sertoli cells. Genes bound by DMRT1 in vivo were enriched for a motif closely resembling the sequence DMRT1 prefers in vitro. Differential response of genes to loss of DMRT1 corresponded to differences in the enriched motif, suggesting that other transacting factors may modulate DMRT1 activity. DMRT1 bound its own promoter and those of six other Dmrt genes, indicating auto- and cross-regulation of these genes. Many of the DMRT1 target genes identified here are known to be important for a variety of functions in testicular development; the others are candidates for further investigation.
Mark W Murphy - One of the best experts on this subject based on the ideXlab platform.
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DMRT1 an ancient sexual regulator required for human gonadogenesis
Sexual Development, 2021Co-Authors: David Zarkower, Mark W MurphyAbstract:Transcriptional regulators related to the invertebrate sexual regulators doublesex and mab-3 occur throughout metazoans and control sex in most animal groups. Seven of these DMRT genes are found in mammals, and mouse genetics has shown that one, DMRT1, plays a crucial role in testis differentiation, both in germ cells and somatic cells. Deletions and, more recently, point mutations affecting human DMRT1 have demonstrated that its heterozygosity is associated with 46,XY complete gonadal dysgenesis. Most of our detailed knowledge of DMRT1 function in the testis, the focus of this review, derives from mouse studies, which have revealed that DMRT1 is essential for male somatic and germ cell differentiation and maintenance of male somatic cell fate after differentiation. Moreover, ectopic DMRT1 can reprogram differentiated female granulosa cells into male Sertoli-like cells. The ability of DMRT1 to control sexual cell fate likely derives from at least 3 properties. First, DMRT1 functionally collaborates with another key male sex regulator, SOX9, and possibly other proteins to maintain and reprogram sexual cell fate. Second, and related, DMRT1 appears to function as a pioneer transcription factor, binding "closed" inaccessible chromatin and promoting its opening to allow binding by other regulators including SOX9. Third, DMRT1 binds DNA by a highly unusual form of interaction and can bind with different stoichiometries.
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the conserved sex regulator DMRT1 recruits sox9 in sexual cell fate reprogramming
Nucleic Acids Research, 2021Co-Authors: Robin E Lindeman, Mark W Murphy, Vivian J. Bardwell, Micah D Gearhart, Kellie S Agrimson, Rachel L Gewiss, David ZarkowerAbstract:Mammalian sexual development commences when fetal bipotential progenitor cells adopt male Sertoli (in XY) or female granulosa (in XX) gonadal cell fates. Differentiation of these cells involves extensive divergence in chromatin state and gene expression, reflecting distinct roles in sexual differentiation and gametogenesis. Surprisingly, differentiated gonadal cell fates require active maintenance through postnatal life to prevent sexual transdifferentiation and female cell fate can be reprogrammed by ectopic expression of the sex regulator DMRT1. Here we examine how DMRT1 reprograms granulosa cells to Sertoli-like cells in vivo and in culture. We define postnatal sex-biased gene expression programs and identify three-dimensional chromatin contacts and differentially accessible chromatin regions (DARs) associated with differentially expressed genes. Using a conditional transgene we find DMRT1 only partially reprograms the ovarian transcriptome in the absence of SOX9 and its paralog SOX8, indicating that these factors functionally cooperate with DMRT1. ATAC-seq and ChIP-seq show that DMRT1 induces formation of many DARs that it binds with SOX9, and DMRT1 is required for binding of SOX9 at most of these. We suggest that DMRT1 can act as a pioneer factor to open chromatin and allow binding of SOX9, which then cooperates with DMRT1 to reprogram sexual cell fate.
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an ancient protein dna interaction underlying metazoan sex determination
Nature Structural & Molecular Biology, 2015Co-Authors: Mark W Murphy, Ken Mcelreavey, Micah D Gearhart, John K Lee, Sandra Rojo, Kayo Kurahashi, Surajit Banerjee, Guy Andre Loeuille, Anu Bashamboo, David ZarkowerAbstract:DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.
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the mammalian doublesex homolog dmrt6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis
Development, 2014Co-Authors: Teng Zhang, Vivian J. Bardwell, Mark W Murphy, Micah D Gearhart, David ZarkowerAbstract:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here, we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) in mice and find that Dmrt6 plays a crucial role in directing germ cells through the mitotic-to-meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain, a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing inappropriate expression of spermatogonial differentiation factors, including SOHLH1, SOHLH2 and DMRT1 as well as the meiotic initiation factor STRA8, and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant germ cells can complete spermatogonial differentiation and enter meiosis, but they show defects in meiotic chromosome pairing, establishment of the XY body and processing of recombination foci, and they mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 chromatin immunoprecipitation sequencing suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis.
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interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line
Developmental Biology, 2013Co-Authors: Anthony D Krentz, Mark W Murphy, Michael D Griswold, Vivian J. Bardwell, Aaron L Sarver, Teng Zhang, S Jain, David ZarkowerAbstract:DMRT1 (doublesex and mab-3 related transcription factor (1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis DMRT1 regulates germ cell pluripotency in a strain-dependent manner. Loss of DMRT1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in DMRT1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between DMRT1 and genetic background we compared mRNA expression in wild type and DMRT1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of DMRT1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of DMRT1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although DMRT1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in DMRT1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between DMRT1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease.
Leslie L Heckert - One of the best experts on this subject based on the ideXlab platform.
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a wt1 DMRT1 transgene restores DMRT1 to sertoli cells of DMRT1 testes a novel model of DMRT1 deficient germ cells
Biology of Reproduction, 2013Co-Authors: Valentine A. Agbor, Shixin Tao, Ning Lei, Leslie L HeckertAbstract:DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of DMRT1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding DMRT1-null (DMRT1(-/-)) mice with Wt1-DMRT1 transgenic (DMRT1(+/-;tg)) mice, which express a rat DMRT1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like DMRT1(-/-) mice, male DMRT1(-/-) transgenic mice (DMRT1(-/-;tg)) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supporting cells of the newborn gonads of both sex and in Sertoli cells of the testis afterbirth. Sertoli cells were evaluated by electron microscopy, revealing that maturation of DMRT1(-/-;tg) Sertoli cells was incomplete. Morphological analysis of testes from 42-day-old mice showed that, compared to DMRT1(-/-) mice, DMRT1(-/-;tg) mice have improved seminiferous tubule structure, with lumens present in many. Immunohistochemistry of the polarity markers ESPIN and NECTIN-2 showed that DMRT1 in Sertoli cells is required for NECTIN-2 expression and influences organization of ectoplasmic specializations. Further functional analyses of the transgene on a DMRT1(-/-) background showed that it did not rescue the decrease in DMRT1(-/-) testis size, but when expressed on a wild-type background, exogenous DMRT1 prevented the normal age-related decline in testis size and enhanced sperm progressive motility. The studies suggest that DMRT1 in Sertoli cells regulates tubule morphology, spermatogenesis, and sperm function via its effects on Sertoli cell maturation and polarity. Furthermore, expression and function of transgenic DMRT1 in Sertoli cells establishes a novel mouse model of DMRT1-deficient germ cells generated by breeding DMRT1-null mice with Wt1-DMRT1 transgenic mice (rescue; DMRT1(-/-;tg)).
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A Wt1-DMRT1 transgene restores DMRT1 to Sertoli cells of DMRT1−/− testes: a novel model of DMRT1deficient germ cells
2013Co-Authors: Valentine A. Agbor, Shixin Tao, Ning Lei, Leslie L HeckertAbstract:DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of DMRT1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding DMRT1-null (DMRT1/) mice with Wt1-DMRT1 transgenic (DMRT1+/;tg) mice, which express a rat DMRT1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like DMRT1/ mice, male DMRT1/ transgenic mice (DMRT1/;tg) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supportin
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genome wide analysis of dna binding and transcriptional regulation by the mammalian doublesex homolog DMRT1 in the juvenile testis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Mark W Murphy, David Zarkower, Aaron L Sarver, Daren A Rice, Katerina Hatzi, Ari Melnick, Leslie L Heckert, Vivian J. BardwellAbstract:The DM domain proteins Doublesex- and MAB-3–related transcription factors (DMRTs) are widely conserved in metazoan sex determination and sexual differentiation. One of these proteins, DMRT1, plays diverse and essential roles in development of the vertebrate testis. In mammals DMRT1 is expressed and required in both germ cells and their supporting Sertoli cells. Despite its critical role in testicular development, little is known about how DMRT1 functions as a transcription factor or what genes it binds and regulates. We combined ChIP methods with conditional gene targeting and mRNA expression analysis and identified almost 1,400 promoter-proximal regions bound by DMRT1 in the juvenile mouse testis and determined how expression of the associated mRNAs is affected when DMRT1 is selectively mutated in germ cells or Sertoli cells. These analyses revealed that DMRT1 is a bifunctional transcriptional regulator, activating some genes and repressing others. ChIP analysis using conditional mutant testes showed that DNA binding and transcriptional regulation of individual target genes can differ between germ cells and Sertoli cells. Genes bound by DMRT1 in vivo were enriched for a motif closely resembling the sequence DMRT1 prefers in vitro. Differential response of genes to loss of DMRT1 corresponded to differences in the enriched motif, suggesting that other transacting factors may modulate DMRT1 activity. DMRT1 bound its own promoter and those of six other Dmrt genes, indicating auto- and cross-regulation of these genes. Many of the DMRT1 target genes identified here are known to be important for a variety of functions in testicular development; the others are candidates for further investigation.
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sex specific differences in mouse DMRT1 expression are both cell type and stage dependent during gonad development
Biology of Reproduction, 2007Co-Authors: Ning Lei, Daren A Rice, Valentine A. Agbor, Kaori I Hornbaker, Tatiana S Karpova, Leslie L HeckertAbstract:Immunohistochemistry was used to examine GCNA1, a germ cell-specific protein, together with DMRT1 (Doublesex and Mab-3-related transcription factor-1), a transcription factor implicated in Sertoli cell and germ cell function, in order to resolve DMRT1's cellular profile during pre- and postnatal gonad development in the mouse. In the indifferent gonad (10.5-11.5 days postcoitus [dpc]), DMRT1 localized to somatic cells and GCNA1(+) germ cells and was indistinguishable in males and females. By 12.5 dpc, a clear sexual preference for DMRT1 in male somatic cells was observed, with male DMRT1 localized to testicular cords and more abundant in Sertoli cells than in germ cells and female DMRT1 diffusely labeled and markedly lower in somatic cells than in germ cells. A male somatic preference continued throughout development, with DMRT1 evident in Sertoli cells at all ages examined and absent in ovarian somatic cells from 13.5 dpc onward. In contrast, expression in primordial germ cells was not sexually distinct, and both sexes showed DMRT1 increasing through 13.5 dpc and absent by 15.5 dpc. Notably, sexual differences in germ cell DMRT1 were detected after birth, when it was detected only in spermatogonia of the testis. Colocalization of DMRT1 with proliferation markers KI67 and proliferating cell nuclear antigen (PCNA) and stem cell markers OCT4 (also known as POU5F1) and NGN3 indicated that, in postnatal testes, DMRT1 was present in both stem and proliferating spermatogonia. Together, the findings implicate opposite functions for DMRT1 in somatic and germ cells of the testis. In Sertoli cells, DMRT1 expression correlated with differentiation, whereas in germ cells, it suggested a role in expansion and maintenance of undifferentiated spermatogonia.
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sp1 and egr1 regulate transcription of the DMRT1 gene in sertoli cells
Biology of Reproduction, 2002Co-Authors: Ning Lei, Leslie L HeckertAbstract:DMRT1 is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating the DMRT1 proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the rat DMRT1 gene and more than 9 kilobases of 5' flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. The DMRT1 5' flanking region from -5000 to +74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for full DMRT1 promoter function. These regions were located between -3200 and -2000 base pairs (bp) and downstream of -150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of -150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate the DMRT1 promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role in DMRT1 regulation in the testis.
Manfred Schartl - One of the best experts on this subject based on the ideXlab platform.
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DMRT1 genes at the crossroads: a widespread and central class of sexual development factors in fish
FEBS Journal, 2011Co-Authors: Amaury Herpin, Manfred SchartlAbstract:A plethora of corroborative genetic studies led to the view that, across the animal kingdom, the gene-regulatory cascades triggering sexual development bear little resemblance to each other. As a result, the common emerging picture is that the genes at the top of the cascade are not conserved, whereas the downstream genes have homologues in a much broader spectrum of species. Among these downstream effectors, a gene family involved in sex differentiation in organisms as phylogenetically divergent as corals, Caenorhabditis elegans, Drosophila, frogs, fish, birds and mammals is the dmrt gene family. Despite the attention that DMRT1 factors have received, to date it has not been elucidated how DMRT1s mediate their activities and putative downstream targets have yet to be characterized. However, a remarkable amount of descriptive expression data has been gathered in a large variety of fish, particularly with respect to early gonadal differentiation and sex change. This minireview aims at distilling the current knowledge of fish DMRT1s, in terms of expression and regulation. It is shown how gonadal identities correlate with dimorphic DMRT1 expression in gonochoristic and hermaphroditic fish species. It is also described how sex steroid hormones affect gonadal identity and DMRT1 expression. Emphasis is also given to recent findings dealing with transcriptional, post-transcriptional, post-translational and functional regulations of the DMRT1a/DMRT1bY gene pair in medaka.
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Transcriptional rewiring of the sex determining DMRT1 gene duplicate by transposable elements
PLoS Genetics, 2010Co-Authors: Amaury Herpin, Cornelia Schmidt, Ingo Braasch, Michael Kraeussling, Eva C. Thoma, Shuhei Nakamura, Minoru Tanaka, Manfred SchartlAbstract:Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE) might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish (Oryzias latipes) a duplicated copy of DMRT1, designated DMRT1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the DMRT1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal DMRT1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target DMRT1 site nested within the DMRT1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of DMRT1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of DMRT1bY, this contributed to create new hierarchies of sex-determining genes.
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developmentally regulated and non sex specific expression of autosomal dmrt genes in embryos of the medaka fish oryzias latipes
Mechanisms of Development, 2004Co-Authors: Christoph Winkler, Ute Hornung, Mariko Kondo, Akihiro Shima, Cordula Neuner, Jutta Duschl, Manfred SchartlAbstract:The dmrtgene family of vertebrates comprises several transcription factors that share a highly conserved DNA-binding domain, the DM domain. Like some of their invertebrate counterparts, e.g. Drosophila doublesex (dsx) and the Caenorhabditis elegans Mab3, several are implicated in sex determination and differentiation. Thus far, dmrt genes represent the only factors involved in sexual development that are conserved across phyla. In the teleost Medaka (Oryzias latipes), a duplicated copy of DMRT1, designated DMRT1bY or dmy, has recently been postulated to be the master regulator of male development in this species. Here, we have analyzed the expression of four additional Medaka dmrt genes during embryonic and larval development. In contrast to other vertebrates, the autosomally located DMRT1a gene of Medaka is not expressed at detectable levels during embryogenesis. On the other hand, dmrt2, dmrt3 and dmrt4 show highly restricted and non-overlapping expression patterns during embryogenesis. While dmrt2 is expressed in early somites, dmrt3 transcripts are found in dorsal interneurons and dmrt4 is expressed in the developing olfactory system. Other than in mouse, they do not show any sex specific expression and no transcription could be detected in the early developing gonads. However, all four analyzed dmrt genes share expression in the differentiating gonad of larvae and in adult testis.
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cloning of the DMRT1 gene of xiphophorus maculatus dmy DMRT1y is not the master sex determining gene in the platyfish
Gene, 2003Co-Authors: Annemarie Veith, Manfred Schartl, Indrajit Nanda, Michael Schmid, Alexander Froschauer, Cornelia Korting, Reinhold Hanel, Jean Nicolas VolffAbstract:In contrast to the situation observed in mammals and birds, a switching between different sex determination systems frequently occurred during the evolution of the teleost fish lineage. This might be due to a frequent turnover of sex-determining signals at the top of the sex determination cascade (master sex-determining genes in the case of genetic sex determination). Alternatively, different variations of a same master gene might decide the sex of individuals in different sex determination systems. In the medaka Oryzias latipes, a Y-specific copy of the putative transcription factor gene DMRT1 very likely corresponds to the master sex-determining gene inducing male formation [Nature 417 (2002) 559; Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 11778]. This gene, alternately called dmY and DMRT1Y, has been formed by duplication of the autosomal DMRT1. In order to determine if an orthologue of DMRT1Y was also located in the sex determination region of the related platyfish Xiphophorus maculatus, Southern blot analysis was performed on genomic DNA from XY and YY males and WY and XX females using both medaka and human DMRT1 cDNA as probes. Using different restriction enzymes, no evidence for sex-specific dmrt-containing genomic fragments could be found in the platyfish. No dmrt gene could be detected by low-stringency Southern blot analysis of genomic inserts from 60 bacterial artificial chromosome (BAC) clones linked to the sex-determining locus on the X and Y chromosomes. Six different groups of BAC clones containing dmrt genes were isolated from a platyfish genomic library. X. maculatus DMRT1 gene and cDNA were characterised. In adult, expression of DMRT1 was detected only in testis. Phylogenetic analysis indicated that the duplication of DMRT1 that led to the formation of dmY/DMRT1Y within the medaka lineage clearly occurred after its divergence from the platyfish lineage. Hence, a dmY/DMRT1Y orthologue generated by this particular event of duplication does not correspond to the master sex-determining gene in the platyfish.
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absence of the candidate male sex determining gene DMRT1b y of medaka from other fish species
Current Biology, 2003Co-Authors: Mariko Kondo, Ute Hornung, Indrajit Nanda, Akihiro Shima, Michael Schmid, Shuichi Asakawa, Nobuyoshi Shimizu, Hiroshi Mitani, Manfred SchartlAbstract:Abstract Although the sex-determining genes are known in mammals, Drosophila , and C. elegans , little is known in other animals. Fishes are an attractive group of organisms for studying the evolution of sex determination because they show an amazing variety of mechanisms, ranging from environmental sex determination and different forms of hermaphroditism to classical sex chromosomal XX/XY or WZ/ZZ systems and modifications thereof. In the fish medaka, DMRT1b(Y) has recently been found [1, 2] to be the candidate male sex-determining gene. It is a duplicate of the autosomal DMRT1a gene, a gene acting in the sex determination/differentiation cascade of flies, worms, and mammals. Because in birds DMRT1 is located on the Z-chromosome [3], both findings led to the suggestion that DMRT1b(Y) is a "non-mammalian Sry " with an even more widespread distribution. However, although Sry was found to be the male sex-determining gene in the mouse and some other mammalian species, in some it is absent [4] and has obviously been replaced by other genes that now fulfil the same function. We have asked if the same might be true of the DMRT1b(Y) gene. We find that the gene duplication generating DMRT1b(Y) occurred recently during the evolution of the genus Oryzias . The gene is absent from all other fish species studied. Therefore, it may not be the male-sex determining gene in all fishes.