RESUMO
Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitina/metabolismo , Dano ao DNA , Ligação Proteica , Proteases Específicas de UbiquitinaRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) facilitates DNA damage response (DDR). While the Ewing's sarcoma breakpoint region 1 (EWS) protein fused to FLI1 triggers sarcoma formation, the physiological function of EWS is largely unknown. Here, we investigate the physiological role of EWS in regulating PARP1. We show that EWS is required for PARP1 dissociation from damaged DNA. Abnormal PARP1 accumulation caused by EWS inactivation leads to excessive Poly(ADP-Ribosy)lation (PARylation) and triggers cell death in both in vitro and in vivo models. Consistent with previous work, the arginine-glycine-glycine (RGG) domain of EWS is essential for PAR chain interaction and PARP1 dissociation from damaged DNA. Ews and Parp1 double mutant mice do not show improved survival, but supplementation with nicotinamide mononucleotides extends Ews-mutant pups' survival, which might be due to compensatory activation of other PARP proteins. Consistently, PARP1 accumulates on chromatin in Ewing's sarcoma cells expressing an EWS fusion protein that cannot interact with PARP1, and tissues derived from Ewing's sarcoma patients show increased PARylation. Taken together, our data reveal that EWS is important for removing PARP1 from damaged chromatin.
Assuntos
Sarcoma de Ewing , Animais , Cromatina/genética , Dano ao DNA , Transtornos Dissociativos , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1 , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/genéticaRESUMO
Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.
Assuntos
Elementos Facilitadores Genéticos/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Camadas Germinativas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição/genética , Ativinas/farmacologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA , Epigênese Genética , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica , Proteínas de Ligação a RNA , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation.
Assuntos
Blastocisto/enzimologia , Metilação de DNA , Instabilidade Genômica , Óvulo/enzimologia , Proteínas Metiltransferases/fisiologia , Espermatozoides/enzimologia , Animais , Apoptose , Blastocisto/citologia , Células Cultivadas , Dano ao DNA , Elementos de DNA Transponíveis , Desenvolvimento Embrionário , Células-Tronco Embrionárias/enzimologia , Feminino , Histonas/metabolismo , Masculino , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-MetiltransferasesRESUMO
Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a RNA , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Células-Tronco Fetais/citologia , Células-Tronco Fetais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Dosagem de Genes , Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologiaRESUMO
Germ-line hypomorphism of the pleiotropic transcription factor Myc in mice, either through Myc gene haploinsufficiency or deletion of Myc enhancers, delays onset of various cancers while mice remain viable and exhibit only relatively mild pathologies. Using a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will, we asked whether this resistance to tumour progression is also emplaced when Myc hypomorphism is acutely imposed in adult mice. Indeed, adult Myc hypomorphism profoundly blocked KRasG12D-driven lung and pancreatic cancers, arresting their evolution at the early transition from indolent pre-tumour to invasive cancer. We show that such arrest is due to the incapacity of hypomorphic levels of Myc to drive release of signals that instruct the microenvironmental remodelling necessary to support invasive cancer. The cancer protection afforded by long-term adult imposition of Myc hypomorphism is accompanied by only mild collateral side effects, principally in haematopoiesis, but even these are circumvented if Myc hypomorphism is imposed metronomically whereas potent cancer protection is retained.
Assuntos
Genes ras , Neoplasias Pancreáticas , Camundongos , Animais , Fatores de Transcrição/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular TumoralRESUMO
Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation.
Assuntos
Proteínas de Ligação a DNA , Proteínas Nucleares , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNARESUMO
Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1beta accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose/fisiologia , Cromossomos Sexuais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , Homologia de Sequência , Células de Sertoli/fisiologia , Fatores de Transcrição/genéticaRESUMO
Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteínas Nucleares/metabolismo , Animais , Linhagem Celular , Centríolos/metabolismo , Segregação de Cromossomos , Humanos , Camundongos , Ligação Proteica , Proteína de Replicação C/metabolismo , Fase SRESUMO
There are hundreds of copies of rDNA repeats in mammalian chromosomes and the ratio of active, poised, or inactive rDNA is regulated in epigenetic manners. Recent studies demonstrated that a post-DNA replication repair enzyme, SHPRH affects rRNA transcription by recognizing epigenetic markers on rDNA promoters and unveiled potential links between DNA repair and ribosome biogenesis. This study suggests that SHPRH could be a link between mTOR-mediated epigenetic regulations and rRNA transcription, while concomitantly affecting genomic integrity.
Assuntos
DNA Helicases/metabolismo , DNA Ribossômico/genética , Epigênese Genética , RNA Ribossômico/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA Helicases/química , Humanos , Regiões Promotoras Genéticas , Domínios Proteicos , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/químicaRESUMO
Naive hypomethylated embryonic pluripotent stem cells (ESCs) are developmentally closest to the preimplantation epiblast of blastocysts, with the potential to contribute to all embryonic tissues and the germline, excepting the extra-embryonic tissues in chimeric embryos. By contrast, epiblast stem cells (EpiSCs) resembling postimplantation epiblast are relatively more methylated and show a limited potential for chimerism. Here, for the first time, we reveal advanced pluripotent stem cells (ASCs), which are developmentally beyond the pluripotent cells in the inner cell mass but with higher potency than EpiSCs. Accordingly, a single ASC contributes very efficiently to the fetus, germline, yolk sac and the placental labyrinth in chimeras. Since they are developmentally more advanced, ASCs do not contribute to the trophoblast. ASCs were derived from blastocysts in two steps in a chemically defined medium supplemented with Activin A and basic fibroblast growth factor, followed by culturing in ABCL medium containing ActA, BMP4, CHIR99021 and leukemia inhibitory factor. Notably, ASCs exhibit a distinct transcriptome with the expression of both naive pluripotency genes, as well as mesodermal somatic genes; Eomes, Eras, Tdgf1, Evx1, hand1, Wnt5a and distinct repetitive elements. Conversion of established ESCs to ASCs is also achievable. Importantly, ASCs exhibit a stable hypermethylated epigenome and mostly intact imprints as compared to the hypomethylated inner cell mass of blastocysts and naive ESCs. Properties of ASCs suggest that they represent cells at an intermediate cellular state between the naive and primed states of pluripotency.
Assuntos
Células-Tronco Embrionárias Murinas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Quimera , Metilação de DNA , Fator de Crescimento Epidérmico/genética , Camadas Germinativas/citologia , Proteínas de Homeodomínio/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Neoplasias/genética , Proteína Oncogênica p21(ras)/genética , Análise de Sequência de RNA , Proteínas com Domínio T/genética , Proteína Wnt-5a/genéticaRESUMO
The only molecular similarity shown so far for sexual regulatory genes among different phyla involves doublesex (dsx) of Drosophila, mab-3 and mab-23 of Caenorhabditis elegans, and Dmrt1 of vertebrates. These genes encode DM domain transcription factors (DM = dsx and mab-3) and are required for sexual differentiation. In the case of dsx and mab-3, the two genes control analogous aspects of sexual development, bind similar DNA sequences, and are capable of functional substitution in vivo. All three phyla have multiple DM domain genes, but it is unknown how many of these are involved in sexual development. Mammals, for example, have at least seven DM domain genes, but embryonic expression has only been examined in detail for Dmrt1(dsx- and mab-3 related transcription factor 1). We have identified additional murine DM domain genes and have examined their expression in the mouse embryo, with emphasis on the developing gonad. At least three murine DM domain genes in addition to Dmrt1 are expressed in the embryonic gonad: Dmrt4 is expressed at similar levels in gonads of both sexes; Dmrt3 is more highly expressed in males; and Dmrt7 is more highly expressed in females. Expression of three other genes is low or absent in the embryonic gonad. Two of these, Dmrt5 and Dmrt6, are expressed primarily in the brain, and the third, Dmrt2, is expressed in presomitic mesoderm and developing somites. Our data suggest that multiple DM domain genes may be involved in mammalian sexual development, and that they may function in both testis and ovary development.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Perfilação da Expressão Gênica , Ovário/embriologia , Testículo/embriologia , Animais , Proteínas de Caenorhabditis elegans/genética , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Feminino , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ovário/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.
Assuntos
Células Germinativas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Reprogramação Celular , Implantação do Embrião , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Germinativas/citologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fatores de Transcrição/deficiênciaRESUMO
Genes containing the DM domain, a conserved DNA binding motif first found in Doublesex of Drosophila and mab-3 of Caenorhabditis elegans, regulate sexual differentiation in multiple phyla. The DM domain gene Dmrt1 is essential for testicular differentiation in vertebrates. In the mouse, Dmrt1 is expressed in pre-meiotic germ cells and in Sertoli cells, which provide essential support for spermatogenesis. Dmrt1 null mutant mice have severely dysgenic testes in which Sertoli cells and germ cells both fail to differentiate properly after birth. Here we use conditional gene targeting to identify the functions of Dmrt1 in each cell type. We find that Dmrt1 is required in Sertoli cells for their postnatal differentiation, and for germ line maintenance and for meiotic progression. Dmrt1 is required in germ cells for their radial migration to the periphery of the seminiferous tubule where the spermatogenic niche will form, for mitotic reactivation and for survival beyond the first postnatal week. Thus Dmrt1 activity is required autonomously in the Sertoli and germ cell lineages, and Dmrt1 activity in Sertoli cells is also required non-autonomously to maintain the germ line. These results demonstrate that Dmrt1 plays multiple roles in controlling the remodeling and differentiation of the juvenile testis.
Assuntos
Testículo/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Diferenciação Celular , Primers do DNA/genética , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Receptores Androgênicos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Espermatogênese , Testículo/citologia , Testículo/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Sex determination in C. elegans is controlled by the TRA-1 zinc finger protein, a Ci/GLI homolog that promotes female cell fates throughout the body. The regulatory hierarchy that controls TRA-1 is well established, but the downstream effectors that establish sexual dimorphism during larval development remain largely unknown. Here, we describe the use of cDNA microarrays to identify sex-enriched transcripts expressed during three stages of C. elegans larval development. By excluding previously identified germline-enriched transcripts, we focused on somatic sexual development. This approach identified a large number of sex-enriched transcripts that are good candidates to encode regulators of somatic sexual development. We found little overlap between genes with sex-enriched expression in early versus late larval development, indicating that distinct sexual regulatory programs operate at these times. Genes with sex-enriched expression are found throughout the genome, with no strong bias between autosomes and X chromosomes. Reporter gene analysis revealed that these genes are expressed in highly specific patterns in a variety of sexually dimorphic cells. We searched for TRA-1 consensus DNA binding sites near genes with sex-enriched expression, and found that most strongly sex-enriched mRNAs are likely to be regulated indirectly by TRA-1. These results suggest that TRA-1 controls sexual dimorphism through a small number of intermediary regulators rather than by acting directly on the full constellation of genes involved in sex-specific differentiation.