Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.
Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.
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DOI：
10.1093/nar/gkx601
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发表时间：
2017-09-19
影响因子：
14.9
通讯作者：
Wirth D
中科院分区：
文献类型：
--
作者：
Gödecke N;Zha L;Spencer S;Behme S;Riemer P;Rehli M;Hauser H;Wirth D
转基因在细胞培养物和小鼠中的忠实表达经常受到基因座依赖性表观遗传沉默的挑战。我们研究了小鼠ROSA 26基因座内Tet-controlled表达盒的沉默。我们观察到明显的DNA甲基化的泰特启动子伴随着mES细胞以及分化的细胞和转基因动物中的表达丧失。引人注目的是，ROSA 26启动子保持活性且无甲基化，这表明这种沉默机制专门影响转基因，但不会扩散到宿主的染色体邻近区域。为了重新激活Tet盒，构建合成融合蛋白并在沉默细胞中表达。该蛋白质包括10 - 11易位甲基胞嘧啶双加氧酶1（泰特-1）的酶结构域以及泰特阻遏物DNA结合结构域。合成融合蛋白的表达和多西环素处理允许ROSA 26基因座和另一个基因组位点中的泰特启动子的靶向去甲基化，从而挽救细胞和转基因小鼠中的转基因表达。因此，诱导的，可逆的和位点特异性的表观遗传调节是一个有前途的策略沉默的转基因表达的重新激活，独立的整合位点。
Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
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在单细胞水平上表观遗传调节的动力学。
DOI：
10.1126/science.aab2956
发表时间：
2016-02-12
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bintu L;Yong J;Antebi YE;McCue K;Kazuki Y;Uno N;Oshimura M;Elowitz MB
通讯作者：
Elowitz MB
影响因子：
12.3
作者：
Klug M;Heinz S;Gebhard C;Schwarzfischer L;Krause SW;Andreesen R;Rehli M
通讯作者：
Rehli M
影响因子：
62.1
作者：
Lu X;Zhao BS;He C
通讯作者：
He C
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
3
作者：
Backman, Cristina M.;Zhang, YaJun;Tomac, Andreas C.
通讯作者：
Tomac, Andreas C.