The role of chromatin modifications in the efficiency of reprogramming

涉及将分化的哺乳动物细胞重编程为胚胎样多能细胞的分子机制尚不清楚。在第一个资助期，我们建立了一个基于转座子的诱导重编程试验，使用神经干细胞（NS），并分析了组蛋白甲基转移酶和组蛋白脱甲基酶的作用。NS细胞中甲基转移酶Mll 1的条件诱变导致诱导多能干细胞（iPS）集落的8倍增加。相反，在M112的条件失活后观察到iPS集落数量的减少。我们还获得了更多的iPS集落后，四环素调节组蛋白去甲基化酶Jmjd 1a（H3 K9 me 2）和Utx（H3 K27 me 3）的过表达。在第二个资助期，我们的目标是确定NS细胞中上述组蛋白甲基转移酶和脱甲基酶的靶点，作为解开重编程分子机制的第一步。此外，我们将继续努力为其他现有的脱甲基酶产生条件诱变和诱导表达/RNAi系统，并扩展分析，除了诱导重编程测定，ES细胞融合和核转移测定。

The molecular mechanisms involved in reprogramming of a differentiated mammalian cell to an embryonic like pluripotent cell are poorly defined. In the first funding period we established a transposon based induced reprogramming assay using Neural Stem (NS) cells and analysed the role of histone methyltransferases and histone demethylases. Conditional mutagenesis of the methyltransferase Mll1 in NS cells resulted in a 8-fold increase of induced pluripotent stem cell (iPS) colonies. In contrast a reduction in the number of iPS colonies was observed after conditional inactivation of Mll2. We also obtained more iPS colonies after tetracycline-regulated overexpression of the histone demethylases Jmjd1a (H3K9me2) and Utx (H3K27me3). In the second funding period we aim at identifying the targets of the above-mentioned histone methyltransferases and demethylases in NS cells as a first step for unravelling the molecular mechanisms of reprogramming. Furthermore we will continue our efforts for generating conditional mutagenesis and inducible expression/RNAi systems for the other existing demethylases and extend the analysis using in addition to induced reprogramming assays, ES-cell fusion and nuclear transfer assays.