Role of Sox2 in the direct lineage reprogramming of astroglia into neurons

我们在上一个资助期的研究表明，未成熟的星形胶质细胞可以完全和稳定的谱系重编程为功能性神经元的一个单一的神经源性转录因子（TF）。相反，在更成熟的星形胶质细胞中，单一神经源性TF不能诱导重编程。然而，前神经基因诱导的重编程成功可以通过Sox 2的共表达大大增强，Sox 2是多能性重编程以及胚胎和神经干细胞维持所必需的TF。在本提案中，我们的目标是通过分析响应于Sox 2的星形胶质细胞转录组的变化来深入了解Sox 2介导的增强星形胶质细胞向神经元谱系转换的分子机制。此外，我们计划确定结合伴侣的Sox 2在星形胶质细胞的免疫沉淀，然后通过质谱和染色质免疫沉淀研究其结合到假定的Sox 2结合位点的启动子区域中确定的Sox 2在星形胶质细胞中的靶基因。鉴于建议的程序性作用，染色质重塑在发育过渡期，我们最终将评估如何brahma相关因子（BAF）复杂的星形胶质细胞和其他细胞之间的不同，以及其组成是否随成熟而变化。在此数据的基础上，我们将研究是否修改BAF复合物组成的星形胶质细胞对胚胎干细胞或神经干细胞特异性的状态，促进直接谱系重新编程的星形胶质细胞成神经元的原神经基因。

Our studies during the last funding period have demonstrated that immature astroglia can be completely and stably lineage reprogrammed into functional neurons by a single neurogenic transcription factor (TF). In contrast, in more mature astroglia single neurogenic TFs fail to induce reprogramming. However, proneural gene-iduced reprogramming success can be greatly enhanced by co-expression of Sox2, a TF essential for pluripotency reprogramming as well as embryonic and neural stem cell maintenance. In the present proposal we aim at obtaining insights into the molecular mechanisms underlying the Sox2-mediated enhancement of astroglia-to-neuron lineage conversion by analyzing changes in the astrocyte transcriptome in response to Sox2. Furthermore we plan to identify binding partners of Sox2 in astrocytes by immunoprecipitation followed by mass spectrometry and to study their binding to putative Sox2 binding sites in promoter regions of identified Sox2 target genes in astrocytes by chromatin immunoprecipitation. Given the suggested programmatic role of chromatin remodelers during developmental transitions we finally will assess how the brahma-associated factor (BAF) complex differs between astrocytes and other cells and whether its composition changes with maturation. On basis of this data we will examine whether modifying the BAF complex composition in astrocytes towards either an embryonic stem cell- or neural precursorspecific state facilitates direct lineage reprogramming of astrocytes into neurons by proneural genes.