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通过单一的神经源性转录因子（TF）完全和稳定地谱系重编程为功能神经元。相比之下，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，前源基因诱导的重编程成功可以通过Sox2的共表达大大增强，Sox2是多能性重编程以及胚胎和神经干细胞维持所必需的TF。在目前的提案中，我们旨在通过分析星形胶质细胞转录组响应Sox2的变化，深入了解Sox2介导的星形胶质细胞向神经元谱系转换增强的分子机制。此外，我们计划通过免疫沉淀法和质谱法确定Sox2在星形胶质细胞中的结合伙伴，并通过染色质免疫沉淀法研究它们与星形胶质细胞中Sox2靶基因启动子区域中推测的Sox2结合位点的结合。鉴于染色质重塑者在发育转变过程中的程序性作用，我们最终将评估星形胶质细胞和其他细胞之间brahma相关因子（BAF）复合物的差异，以及其成分是否随着成熟而变化。基于这些数据，我们将研究将星形胶质细胞中的BAF复合物组成修改为胚胎干细胞或神经前体细胞特异性状态是否有助于星形胶质细胞通过前体细胞基因直接谱系重编程为神经元。

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.