3D chromatin organisation in direct neuronal reprogramming – re-shaping the nucleus to mold new neurons

转录因子如何重新连接3D基因组结构以及这种调控如何影响细胞命运的决定是染色质生物学的基本问题之一。由单一的神经源性因子将神经胶质细胞重编程为神经元提供了一个很好的系统来研究这一现象，并确定在神经元直接重编程中发生的染色质结构的整体变化。为了解决这个引人入胜的项目，染色质结构和高分辨率Hi-C方面的专家Boyan Bonev和胶质细胞到神经元重编程的先驱Magdalena Götz建议结合他们的独特专业知识，剖析神经元重编程过程中3D核结构、染色质可获得性和基因表达之间的关系。我们的第一个目标是利用高分辨率的高分辨Hi-C来确定星形胶质细胞和诱导神经元中全基因组的3D基因组组织。我们还将利用星形胶质细胞的体外模型，这些细胞对重新编程更具抵抗力，以确定染色质压缩和长期相互作用的全球变化是否导致这种可塑性的缺乏。然后，我们将探索转录变化(RNA-seq数据已经生成)如何与染色质循环的变化相对应，并通过Hi-Chip研究重编程因子(Neurogenin2)的直接结合在多大程度上参与重新塑造染色质结构。此外，使用基因组工程将Ngn2拴在特定的基因座上，并删除Ngn2结合的增强子，我们将确定Ngn2结合是否必要和/或足以导致染色质环状。在提案的最后部分，我们将研究YY1是否对神经元重新编程过程中细胞类型特异性调控相互作用的形成起重要作用，并使用芯片-MS和位点特异性蛋白质组学方法来确定在胶质细胞到神经元重新编程过程中与染色质重新连接相关的其他因素。综上所述，该项目不仅将对单个转铁蛋白如何重组3D染色质结构产生前所未有的见解，还将有助于改进这一过程，以完善诱导神经元。

How transcription factors can rewire 3D genome architecture and how such regulation affects cell fate decisions is one of the fundamental questions in chromatin biology. Reprogramming of glial cells into neurons by a single neurogenic factor provides an excellent system to study this phenomenon and to determine the global changes in chromatin architecture which occur in direct neuronal reprogramming. To tackle this fascinating project Boyan Bonev, an expert in chromatin architecture and high resolution Hi-C and Magdalena Götz, who pioneered the glia-to-neuron reprogramming, propose to combine their unique expertise and dissect the relationship between 3D nuclear architecture, chromatin accessibility and gene expression during neuronal reprogramming. We first aim to determine the 3D genome organization genome-wide in astrocytes and induced neurons using high-resolution Hi-C. We will also exploit an in vitro model of astrocytes that are more resistant to reprogramming to determine if global changes in chromatin compaction and long-range interactions underlie this lack of plasticity. We will then explore how transcriptional changes (RNA-seq data have been generated already) correspond to changes in chromatin looping and examine by Hi-ChIP to which extent direct binding of the reprogramming factor (Neurogenin2) is involved in re-shaping chromatin architecture. Furthermore, using genome engineering to tether Ngn2 to specific loci and delete Ngn2-bound enhancers, we will determine if Ngn2 binding is necessary and/or sufficient to cause chromatin looping. In the last part of the proposal, we will examine if YY1 is important for formation of cell type specific regulatory interactions during neuronal reprogramming and use ChIP-MS and locus-specific proteomics approach to identify other factors relevant for rewiring the chromatin during glia-to-neuron reprogramming. Taken together, this project will not only yield unprecedented insights into how a single TF can reorganize 3D chromatin architecture, but also help to improve this process to perfect the induced neurons.