RESUMO
Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mecanotransdução Celular , Camundongos , Camundongos Transgênicos , Fosfoproteínas/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.
Assuntos
Ciclina E/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Antimetabólitos/farmacologia , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina E/deficiência , Ciclina E/genética , Fluoruracila/farmacologia , Genótipo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Transplante HomólogoRESUMO
Transposons present an acute challenge to the germline, and mechanisms that repress their activity are essential for transgenerational genomic integrity. LINE1 (L1) is the most successful retrotransposon and is epigenetically repressed by CpG DNA methylation. Here, we identify two additional important mechanisms by which L1 is repressed during spermatogenesis. We demonstrate that the Piwi protein Mili and the piRNA pathway are required to posttranscriptionally silence L1 in meiotic pachytene cells even in the presence of normal L1 DNA methylation. Strikingly, in the absence of both a functional piRNA pathway and DNA methylation, L1 elements are normally repressed in mitotic stages of spermatogenesis. Accordingly, we find that the euchromatic repressive histone H3 dimethylated lysine 9 modification cosuppresses L1 expression therein. We demonstrate the existence of multiple epigenetic mechanisms that in conjunction with the piRNA pathway sequentially enforce L1 silencing and genomic stability during mitotic and meiotic stages of adult spermatogenesis.
Assuntos
Epigênese Genética , Inativação Gênica , Elementos Nucleotídeos Longos e Dispersos/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Espermatogênese/genética , Fatores Etários , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Western Blotting , Metilação de DNA , Expressão Gênica , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Mitose/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatócitos/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.