RNF12 Controls Embryonic Stem Cell Fate and Morphogenesis in Zebrafish Embryos by Targeting Smad7 for Degradation

Abstract

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β 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-β signaling. Depletion of RNF12 dramatically reduced TGF-β/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-β family signaling.

Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling

“…Immunoprecipitation and immunoblot was performed as described previously 9,31,42 . Cells were lysed with 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 25 mM NaF, 1% Triton X-100) plus protease inhibitors (Sigma) for 10 min at 4°C.…”

“…ChIP assay was performed as described previously 9,43 . Cells grown to 100% confluence were crosslinked with 1% formaldehyde for 15 min at 37°C; crosslinking reactions were quenched with 0.125 M glycine for 5 min at room temperature.…”

“…SMAD7 functions as an inhibitory SMAD by recruiting the E3 ligase SMURF1/2 to the type I receptor and subsequently degrading TbRI and mitigating TGF-b signalling 6,7 . ARKADIA and RNF12 potentiate TGF-b signalling by targeting SMAD7 for polyubiquitination and degradation 8,9 .…”

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

“…Smad7 itself is subjected to proteolysis-based regulation by the ubiquitin ligase Rnf12, which acts as an indirect activator of BMP and TGF-β signaling (13,26). Given the known function of Rnf12 as a regulator of XIST expression during embryonic development (14, 27), we assessed whether Rnf12 might also be involved in maintenance of the Xi in adult cells by regulating BMP/TGF-β signaling.…”

“…We found that the BMP signaling pathway is required for maintenance of XIST expression. Furthermore, we show that Rnf12, a known regulator of BMP signaling (13), modulates XIST expression in adult cells, suggesting that the role of Rnf12 as a regulator of XIST during initiation of X-chromosome inactivation in ES cells (14,15) is retained in differentiated cells through control of BMP signaling.…”