RESUMO
TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.
Assuntos
Embrião não Mamífero/citologia , Células-Tronco Embrionárias/citologia , Proteína Smad7/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/metabolismo , Gastrulação/genética , Humanos , Células Jurkat , Camundongos , Proteólise , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12â»/â» knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.