Mark W. Murphy

DMRT1 prevents female reprogramming in the postnatal mammalian testis

Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/β-catenin signalling. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates and Dmrt1-related sexual regulators are conserved throughout metazoans. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation and testicular cancer.Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/β-catenin signalling. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates and Dmrt1-related sexual regulators are conserved throughout metazoans. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation and testicular cancer.

The Mammalian Doublesex Homolog DMRT1 Is a Transcriptional Gatekeeper that Controls the Mitosis versus Meiosis Decision in Male Germ Cells

The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers, including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related transcription factor DMRT1 in adult spermatogenesis using conditional gene targeting in the mouse. Loss of Dmrt1 causes spermatogonia to precociously exit the spermatogonial program and enter meiosis. Therefore, DMRT1 determines whether male germ cells undergo mitosis and spermatogonial differentiation or meiosis. Loss of Dmrt1 in spermatogonia also disrupts cyclical gene expression in Sertoli cells. DMRT1 acts in spermatogonia to restrict RA responsiveness, directly repress Stra8 transcription, and activate transcription of the spermatogonial differentiation factor Sohlh1, thereby preventing meiosis and promoting spermatogonial development. By coordinating spermatogonial development and mitotic amplification with meiosis, DMRT1 allows abundant, continuous production of sperm.

The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage.

DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary.

Dmrt1 belongs to the DM domain gene family of conserved sexual regulators. In the mouse Dmrt1 is expressed in the genital ridge (the gonadal primordium) in both sexes and then becomes testis-specific shortly after sex determination. The essential role of DMRT1 in testicular differentiation is well established, and includes transcriptional repression of the meiotic inducer Stra8. However Dmrt1 mutant females are fertile and the role of Dmrt1 in the ovary has not been studied. Here we show in the mouse that most Dmrt1 mutant germ cells in the fetal ovary have greatly reduced expression of STRA8, and fail to properly localize SYCP3 and γH2AX during meiotic prophase. Lack of DMRT1 in the fetal ovary results in the formation of many fewer primordial follicles in the juvenile ovary, although these are sufficient for fertility. Genome-wide chromatin immunoprecipitiation (ChIP-chip) and quantitative ChIP (qChIP) combined with mRNA expression profiling suggests that transcriptional activation of Stra8 in fetal germ cells is the main function of DMRT1 in females, and that this regulation likely is direct. Thus DMRT1 controls Stra8 sex-specifically, activating it in the fetal ovary and repressing it in the adult testis.

Genome-wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis

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.

Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA.

Background:The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences.Results:We used a random oligonucleotide in vitro selection method to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence we determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. We also find that some DMRT proteins can bind DNA as heterodimers.Conclusion:Our results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers.

mRNA Expression Analysis and the Molecular Basis of Neonatal Testis Defects in Dmrt1 Mutant Mice

Transcriptional regulators containing the DM domain DNA binding motif have been found to control sexual differentiation in a diverse group of metazoan animals including vertebrates, insects, and nematodes, suggesting that these proteins may comprise a very ancient group of sexual regulators. Dmrt1, 1 of 7 mammalian DM domain genes, is essential for several aspects of testicular differentiation in mice. The Dmrt1 mutant phenotype becomes apparent shortly after birth, and culminates in severe testicular dysgenesis. To better understand the roles of Dmrt1 in testicular development we have performed a more detailed analysis of its mutant phenotypes, and we have used mRNA expression profiling to identify genes misregulated in the neonatal Dmrt1 mutant testis. We find that Dmrt1 mutant germ cells fail to undergo several of the normal postnatal events of germ cell development, including radial movement, mitotic proliferation, differentiation into spermatogonia, and initiation of meiosis, and they die by P14. During this period Dmrt1 mutant Sertoli cells fail to polarize and form tight junctions, and fail to cease proliferation, eventually filling the seminiferous tubules. Expression profiling at P1 and P2 in Dmrt1 mutant testes indicates defects in several important testicular signaling pathways (Gdnf, retinoic acid, TGFβ, FSH), and detects elevated expression of the pluripotency marker Stella/Dppa3/Pgc7, providing insight into the molecular basis of Dmrt1 testis defects. This work also identifies a number of new candidate testicular regulators for further investigation.

An ancient protein-DNA interaction underlying metazoan sex determination

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.

Interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line

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.

The mammalian Doublesex homolog DMRT6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis.

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.