Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Clin Invest ; 131(22)2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34779407

RESUMO

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

2.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
3.
Nature ; 593(7860): 602-606, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33953397

RESUMO

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer1,2. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important1,3,4, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis5-7. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES) cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

4.
Sci Transl Med ; 12(566)2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087503

RESUMO

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.


Assuntos
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/tratamento farmacológico , Animais , Apoptose , Calmodulina , Eritropoese , Humanos , Proteína Supressora de Tumor p53 , Peixe-Zebra
5.
Nat Commun ; 11(1): 2619, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457326

RESUMO

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Exorribonucleases/metabolismo , Macrossomia Fetal/microbiologia , RNA Mensageiro/metabolismo , Tumor de Wilms/microbiologia , Animais , Sinalização do Cálcio/genética , Diferenciação Celular , Células-Tronco Embrionárias , Exorribonucleases/deficiência , Exorribonucleases/genética , Macrossomia Fetal/enzimologia , Macrossomia Fetal/genética , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Camundongos , Biossíntese de Proteínas , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Uridina Monofosfato/metabolismo , Tumor de Wilms/enzimologia , Tumor de Wilms/genética
6.
Nat Struct Mol Biol ; 26(6): 490-500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160785

RESUMO

Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name '7SB' rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.


Assuntos
Exorribonucleases/metabolismo , RNA Ribossômico 5,8S/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Macrossomia Fetal/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Transporte de RNA , Ribossomos/metabolismo , Uridina/metabolismo , Tumor de Wilms/metabolismo
7.
Methods ; 155: 10-19, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30395968

RESUMO

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.


Assuntos
Biologia Computacional/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Uridina/metabolismo , Região 3'-Flanqueadora , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/deficiência , Exorribonucleases/genética , Edição de Genes/métodos , Camundongos , Células-Tronco Embrionárias Murinas , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Guia/genética , RNA Guia/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo
8.
Stem Cell Reports ; 10(6): 1734-1750, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29779894

RESUMO

During early cortical development, neural stem cells (NSCs) divide symmetrically to expand the progenitor pool, whereas, in later stages, NSCs divide asymmetrically to self-renew and produce other cell types. The timely switch from such proliferative to differentiative division critically determines progenitor and neuron numbers. However, the mechanisms that limit proliferative division in late cortical development are not fully understood. Here, we show that the BAF (mSWI/SNF) complexes restrict proliferative competence and promote neuronal differentiation in late corticogenesis. Inactivation of BAF complexes leads to H3K27me3-linked silencing of neuronal differentiation-related genes, with concurrent H3K4me2-mediated activation of proliferation-associated genes via de-repression of Wnt signaling. Notably, the deletion of BAF complexes increased proliferation of neuroepithelial cell-like NSCs, impaired neuronal differentiation, and exerted a Wnt-dependent effect on neocortical and hippocampal development. Thus, these results demonstrate that BAF complexes act as both activators and repressors to control global epigenetic and gene expression programs in late corticogenesis.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Ribonucleoproteínas/metabolismo , Via de Sinalização Wnt , Animais , Diferenciação Celular , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Hipocampo/embriologia , Hipocampo/metabolismo , Camundongos , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Ribonucleoproteínas/genética
9.
Cell Stem Cell ; 22(6): 851-864.e5, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29804889

RESUMO

The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras. Loss- and gain-of-function experiments reveal that ISY1 promotes exit from the naive state and is necessary and sufficient to induce and maintain poised pluripotency, and that persistent ISY1 overexpression inhibits the transition from the naive to the primed state. We identify a large subset of ISY1-dependent miRNAs that can rescue the inability of miRNA-deficient ESCs to establish the poised state and transition to the primed state. Thus, dynamic ISY1 regulates poised pluripotency through miRNAs to control ESC fate.


Assuntos
Células-Tronco Embrionárias/metabolismo , MicroRNAs/metabolismo , Células A549 , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Masculino , Camundongos , Proteínas de Ligação a RNA/metabolismo , Transfecção
10.
Stem Cell Reports ; 8(4): 813-821, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28330620

RESUMO

The chromatin of naive embryonic stem cells (ESCs) has a largely open configuration, as evident by the lack of condensed heterochromatin and the hypomethylation of DNA. Several molecular mechanisms promoting this constellation were previously identified. Here we present evidence for an important epigenetic function of MAD2L2, a protein originally known for its role in DNA damage repair, and for its requirement in germ cell development. We demonstrate using super-resolution microscopy that numerous MAD2L2 microfoci are exclusively associated with euchromatin, similar to other factors of the DNA damage response. In the absence of MAD2L2 the amount of heterochromatin demarcated by H3K9me2 was significantly increased. Among the most strongly suppressed genes was Dppa3, an ESC- and germ-cell-specific gene regulating DNA methylation. In Mad2l2-deficient ESCs 5-methylcytosine levels were globally increased, while several imprinted genes became hypomethylated and transcriptionally activated. Our results emphasize the important function of MAD2L2 for the open chromatin configuration of ESCs.


Assuntos
Epigênese Genética , Eucromatina/metabolismo , Proteínas Mad2/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteínas Repressoras/genética , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona , Dano ao DNA , Metilação de DNA , Regulação para Baixo , Eucromatina/genética , Deleção de Genes , Loci Gênicos , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas Mad2/análise , Proteínas Mad2/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ativação Transcricional
11.
Cell Rep ; 16(7): 1861-73, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27498873

RESUMO

Mutations in the 3'-5' exonuclease DIS3L2 are associated with Perlman syndrome and hypersusceptibility to Wilms tumorigenesis. Previously, we found that Dis3l2 specifically recognizes and degrades uridylated pre-let-7 microRNA. However, the widespread relevance of Dis3l2-mediated decay of uridylated substrates remains unknown. Here, we applied an unbiased RNA immunoprecipitation strategy to identify Dis3l2 targets in mouse embryonic stem cells. The disease-associated long noncoding RNA (lncRNA) Rmrp, 7SL, as well as several other Pol III-transcribed noncoding RNAs (ncRNAs) were among the most highly enriched Dis3l2-bound RNAs. 3'-Uridylated Rmrp, 7SL, and small nuclear RNA (snRNA) species were highly stabilized in the cytoplasm of Dis3l2-depleted cells. Deep sequencing analysis of Rmrp 3' ends revealed extensive oligouridylation mainly on transcripts with imprecise ends. We implicate the terminal uridylyl transferases (TUTases) Zcchc6/11 in the uridylation of these ncRNAs, and biochemical reconstitution assays demonstrate the sufficiency of TUTase-Dis3l2 for Rmrp decay. This establishes Dis3l2-mediated decay (DMD) as a quality-control pathway that eliminates aberrant ncRNAs.


Assuntos
Exorribonucleases/genética , MicroRNAs/genética , Clivagem do RNA , RNA Citoplasmático Pequeno/genética , RNA não Traduzido/genética , Partícula de Reconhecimento de Sinal/genética , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Exorribonucleases/deficiência , Edição de Genes , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Camundongos , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Citoplasmático Pequeno/metabolismo , RNA não Traduzido/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Transdução de Sinais , Uridina/metabolismo
12.
Cell Rep ; 13(9): 1842-54, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26655900

RESUMO

BAF (Brg/Brm-associated factors) complexes play important roles in development and are linked to chromatin plasticity at selected genomic loci. Nevertheless, a full understanding of their role in development and chromatin remodeling has been hindered by the absence of mutants completely lacking BAF complexes. Here, we report that the loss of BAF155/BAF170 in double-conditional knockout (dcKO) mice eliminates all known BAF subunits, resulting in an overall reduction in active chromatin marks (H3K9Ac), a global increase in repressive marks (H3K27me2/3), and downregulation of gene expression. We demonstrate that BAF complexes interact with H3K27 demethylases (JMJD3 and UTX) and potentiate their activity. Importantly, BAF complexes are indispensable for forebrain development, including proliferation, differentiation, and cell survival of neural progenitor cells. Our findings reveal a molecular mechanism mediated by BAF complexes that controls the global transcriptional program and chromatin state in development.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Córtex Cerebelar/metabolismo , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Regulação para Baixo , Embrião de Mamíferos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo
13.
Cell Rep ; 13(2): 260-6, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26440890

RESUMO

Let-7 microRNAs (miRNAs) are critical regulators of animal development, stem cell differentiation, glucose metabolism, and tumorigenesis. Mammalian genomes contain 12 let-7 isoforms that suppress expression of a common set of target mRNAs. LIN28 proteins selectively block let-7 biogenesis in undifferentiated cells and in cancer. The current model for coordinate let-7 repression involves the LIN28 cold-shock domain (CSD) binding the terminal loop and the two CCHC-type zinc fingers recognizing a GGAG sequence motif in precursor let-7 (pre-let-7) RNAs. Here, we perform a systematic analysis of all let-7 miRNAs and find that a single let-7 family member, human let-7a-3 (and its murine ortholog let-7c-2), escapes LIN28-mediated regulation. Mechanistically, we find that the pre-let-7c-2 loop precludes LIN28A binding and regulation. These findings refine the current model of let-7 regulation by LIN28 proteins and have important implications for understanding the LIN28/let-7 axis in development and disease.


Assuntos
MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química
14.
Cell Cycle ; 14(10): 1596-610, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25928475

RESUMO

The induction and maintenance of pluripotency requires the expression of several core factors at appropriate levels (Oct4, Sox2, Klf4, Prdm14). A subset of these proteins (Oct4, Sox2, Prdm14) also plays crucial roles for the establishment of primordial germ cells (PGCs). Here we demonstrate that the Mad2l2 (MAD2B, Rev7) gene product is not only required by PGCs, but also by pluripotent embryonic stem cells (ESCs), depending on the growth conditions. Mad2l2(-/-) ESCs were unstable in LIF/serum medium, and differentiated into primitive endoderm. However, they could be stably propagated using small molecule inhibitors of MAPK signaling. Several components of the MAPK cascade were up- or downregulated even in undifferentiated Mad2l2(-/-) ESCs. Global levels of repressive histone H3 variants were increased in mutant ESCs, and the epigenetic signatures on pluripotency-, primitive endoderm-, and MAPK-related loci differed. Thus, H3K9me2 repressed the Nanog promoter, while the promoter of Gata4 lost H3K27me3 and became de-repressed in LIF/serum condition. Promoters associated with genes involved in MAPK signaling also showed misregulation of these histone marks. Such epigenetic modifications could be indirect consequences of mutating Mad2l2. However, our previous observations suggested the histone methyltransferases as direct (G9a) or indirect (Ezh2) targets of Mad2l2. In effect, the intricate balance necessary for pluripotency becomes perturbed in the absence of Mad2l2.


Assuntos
Proteínas Mad2/metabolismo , Animais , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator Inibidor de Leucemia/farmacologia , Proteínas Mad2/deficiência , Proteínas Mad2/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog , Regiões Promotoras Genéticas , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
PLoS Genet ; 9(8): e1003712, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24009519

RESUMO

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2(-/-) embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.


Assuntos
Diferenciação Celular/genética , Epigênese Genética , Células Germinativas/crescimento & desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Proteínas Mad2/genética , Animais , Proteína Quinase CDC2/genética , Feminino , Fertilidade/genética , Histona Metiltransferases , Histonas/metabolismo , Humanos , Proteínas Mad2/metabolismo , Masculino , Camundongos , Ativação Transcricional/genética
16.
J Assist Reprod Genet ; 30(3): 325-32, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23274510

RESUMO

PURPOSE: Spermatogonial stem cells are affected by the interactions of extrinsic signals produced by components of the microenvironment niche, in addition to the chemical and physical properties of the extracellular matrix. Therefore, this study was initiated to assess the interaction of these cells on a synthetic nanofibrillar extracellular matrix that mimicked the geometry and nanotopography of the basement membrane for cellular growth. METHODS: This study has used a variety of experimental approaches to investigate the interaction of mouse neonatal-derived spermatogonial stem-like cells on a synthetic random oriented three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web™). RESULTS: Spermatogonial stem-like cell colonies were characterized by their ability to express α6-integrin, Thy-1, PLZF, and ß1-integrin. After culture of cells on the nanofibrillar surfaces for 7 days, the number of colonies, the number of cells in each colony, and the average area of colonies were increased (P < 0.05). However, the expression difference of related markers in both groups was not significant. A significantly higher proliferation and survival was observed in the nanofibrillar group (P < 0.05). After transplantation into the testes of busulfan-treated adult mice, spermatogonial stem-like cell colonies that were cultured on the nanofibrillar surface demonstrated functionality, as verified by their ability to migrate to the seminiferous basal membrane, where they produced additional colonies. CONCLUSIONS: These results have suggested that electrospun nanofibrillar surfaces could provide a more favorable microenvironment for in vitro short term culture of spermatogonial stem-like cell colonies.


Assuntos
Técnicas de Cultura de Células/métodos , Nanofibras/química , Espermatogônias/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Proliferação de Células , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Masculino , Camundongos , Gravidez , Células-Tronco/metabolismo , Propriedades de Superfície
17.
J Mol Med (Berl) ; 90(7): 753-61, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22584374

RESUMO

Primordial germ cells (PGCs) are induced in the epiblast early in mammalian development. They develop their specific fate separate from somatic cells by the generation of a unique transcriptional profile and by epigenetic modifications of histones and DNA. PGCs are related to pluripotent cells in many respects, both on a molecular and a cell biological level. Mimicking their in vivo development, PGCs can be derived in culture from pluripotent cells. Vice versa, PGCs can be converted in vitro into pluripotent embryonic germ cells. Recent evidence indicates that the derivation of pluripotent embryonic stem cells from explanted inner cell mass cells may pass through a germ cell-like state, but that this intermediate is not obligatory. In this review, we discuss PGC development and its relevance to pluripotency in mammalian embryos. We outline possibilities and problems connected to the application of in vitro-derived germ cells in reproductive medicine.


Assuntos
Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Epigênese Genética , Células Germinativas/citologia , Humanos , Células-Tronco Pluripotentes/citologia , Medicina Reprodutiva
18.
Int J Fertil Steril ; 5(4): 217-24, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25210606

RESUMO

BACKGROUND: This study compared neonatal and adult mice-derived Sertoli cells (NSCs and ASCs) to examine the influence of feeder cells derived from donors of different ages on the maintenance of mouse spermatogonial stem cells (SSCs) in vitro. MATERIALS AND METHODS: SSCs were derived from the testes of six-day-old mice. They were subsequently transferred to Sertoli cells which were isolated by datura stramonium agglutinin (DSA) lectin from neonatal and adult mice for five days. RESULTS: The numbers of spermatogonial colonies, the numbers of cells per colony, and cloning efficiency were assessed in presence of NSCs and ASCs. The expression of α6- and ß1-integrin- positive cells was evaluated. Moreover, the functionality of the cells was assessed by their transplantation into the testes of busulfan-induced infertile mice. Colony efficiency assay showed that the number of colonies derived from single spermatogonial cells were significantly higher on NSCs. Additionally, the transplantation of dissociated colonies into the testes of busulfan-induced infertile mice showed their migration to the seminiferous basal membrane. CONCLUSION: These results show that NSCs may provide a more favorable microenvironment in comparison with ASCs for in vitro culture of spermatogonial colonies.

19.
Cell Biol Int ; 32(2): 278-86, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18023369

RESUMO

Parallel to the importance of the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function like primary islets. To increase the efficiency of endocrine pancreatic-like cell differentiation from mouse embryonic stem cells (ESCs), we applied activin-B to nestin-positive selection (protocol 1) and spontaneous differentiation (protocol 2) in different groups including: [A] activin-B, or [B] basic fibroblast growth factor (bFGF), and/or [C] activin-B+bFGF. The differentiated cells expressed most pancreatic-related genes. The number of insulin- and C peptide-positive cells, as well as dithizone-positive clusters in group A of protocol 1 was higher than in the other groups. Significant insulin concentrations in protocol 1 were produced when glucose was added to the medium, in comparison with protocol 2. Moreover, insulin release was increased significantly in group A of protocol 1 even with lower glucose. In conclusion, Addition of activin-B in a nestin-positive selection protocol increased the insulin-secreting cells in comparison with the same protocol with bFGF and/or spontaneous differentiation in presence of bFGF and/or activin-B alone. However, improvements of the current method are required to generate a sufficient source of true beta-cells for the treatment of diabetes mellitus.


Assuntos
Ativinas/farmacologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Insulina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nestina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...