Abstract
Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGFβ signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGFβ pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGFβ pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.
Highlights
Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified
Expression Pattern of repressor element 1 (RE1)-silencing transcription factor (REST)—We first investigated the expression of REST in porcine oocytes, nuclear transfer (NT), and parthenogenetic activation (PA) embryos by real-time PCR and Western blotting analysis
REST was dispersed in the MII oocyte cytoplasm (Fig. 2, A and AЈ, n ϭ 17), and was incorporated into transferred donor nuclei in NT embryos when the nuclei were condensed at 2 h post-NT (Fig. 2, B and BЈ, n ϭ 15) and decondensed at 6 h post-NT (Fig. 2, C and CЈ, locked nucleic acid; Q-PCR, quantitative PCR; iPS, induced pluripotent stem; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane1,3-diol
Summary
Expression Pattern of REST—We first investigated the expression of REST in porcine oocytes, nuclear transfer (NT), and parthenogenetic activation (PA) embryos by real-time PCR and Western blotting analysis. By RT-PCR analysis, the expressions of TGFR1, ACVR2A, ACVR2B, Smad, and Smad were not detected in oocytes and were high in donor cell PFFs. In onecell NT embryos, the expressions of these genes were at relatively low levels but dramatically up-regulated after REST deficiency (Fig. 4B). Smad3-p in normal and REST-deficient NT embryos detected by Western blotting and immunofluorescence analysis (Fig. 4, D and E), indicating the TGF pathway was efficiently inhibited by SB431542. The development of NT embryos was significantly enhanced by SB431542 treatment (Con ϩ DMSO-NT versus Con ϩ SB-NT, 18.14 versus 28.93%, respectively; p Ͻ 0.05; Table 1) These results indicate that the failure of NT-mediated reprogramming in REST-deficient NT embryos can, at least to some extent, be attributed to TGF pathway up-regulation and TGF pathway may block nuclear reprogramming. Our results indicate the REST repressing TGF pathway regulates NT-mediated reprogramming (Fig. 5C)
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