RESUMO
Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.
Assuntos
Desenvolvimento Embrionário/genética , Proteínas de Ligação a RNA/genética , Animais , Diferenciação Celular/genética , Autorrenovação Celular/genética , Regulação da Expressão Gênica , Mamíferos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
A central enigma in epigenetics is how epigenetic factors are guided to specific genomic sites for their function. Previously, we reported that a Piwi-piRNA complex associates with the piRNA-complementary site in the Drosophila genome and regulates its epigenetic state. Here, we report that Piwi-piRNA complexes bind to numerous piRNA-complementary sequences throughout the genome, implicating piRNAs as a major mechanism that guides Piwi and Piwi-associated epigenetic factors to program the genome. To test this hypothesis, we demonstrate that inserting piRNA-complementary sequences to an ectopic site leads to Piwi, HP1a, and Su(var)3-9 recruitment to the site as well as H3K9me2/3 enrichment and reduced RNA polymerase II association, indicating that piRNA is both necessary and sufficient to recruit Piwi and epigenetic factors to specific genomic sites. Piwi deficiency drastically changed the epigenetic landscape and polymerase II profile throughout the genome, revealing the Piwi-piRNA mechanism as a major epigenetic programming mechanism in Drosophila.
Assuntos
Proteínas Argonautas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila/genética , Epigenômica , Genoma , Metiltransferases/genética , RNA Interferente Pequeno/genética , Animais , Animais Geneticamente Modificados , Cromatina/genética , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Elementos de DNA Transponíveis/genética , Genômica , Mutação/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Canalization, also known as developmental robustness, describes an organism's ability to produce the same phenotype despite genotypic variations and environmental influences. In Drosophila, Hsp90, the trithorax-group proteins and transposon silencing have been previously implicated in canalization. Despite this, the molecular mechanism underlying canalization remains elusive. Here using a Drosophila eye-outgrowth assay sensitized by the dominant Kr(irregular facets-1)(Kr(If-1)) allele, we show that the Piwi-interacting RNA (piRNA) pathway, but not the short interfering RNA or micro RNA pathway, is involved in canalization. Furthermore, we isolated a protein complex composed of Hsp90, Piwi and Hop, the Hsp70/Hsp90 organizing protein homolog, and we demonstrated the function of this complex in canalization. Our data indicate that Hsp90 and Hop regulate the piRNA pathway through Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization.