Gödecke Natascha, Zha Lisha, Spencer Shawal, Behme Sara, Riemer Pamela, Rehli Michael, Hauser Hansjörg, Wirth Dagmar
Helmholtz Centre for Infection Research, RG Model Systems for Infection and Immunity (MSYS), Braunschweig, Germany.
University Hospital, Dept. Internal Medicine III, Regensburg, Germany.
Nucleic Acids Res. 2017 Sep 19;45(16):e147. doi: 10.1093/nar/gkx601.
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.
转基因在细胞培养物和小鼠中的忠实表达常常受到位点依赖性表观遗传沉默的挑战。我们研究了小鼠ROSA26位点内Tet控制的表达盒的沉默情况。我们观察到Tet启动子出现明显的DNA甲基化，同时在小鼠胚胎干细胞、分化细胞和转基因动物中表达丧失。引人注目的是，ROSA26启动子保持活性且无甲基化，这表明这种沉默机制特异性地影响转基因，但不会扩散到宿主的染色体邻域。为了重新激活Tet表达盒，构建了一种合成融合蛋白并在沉默细胞中表达。该蛋白包含十一易位甲基胞嘧啶双加氧酶1（TET-1）的酶结构域以及Tet阻遏物DNA结合结构域。合成融合蛋白的表达和强力霉素处理使得ROSA26位点以及另一个基因组位点的Tet启动子发生靶向去甲基化，挽救了细胞和转基因小鼠中的转基因表达。因此，可诱导、可逆且位点特异性的表观遗传调控是重新激活沉默转基因表达的一种有前景的策略，且与整合位点无关。
