Cell-state specific 3D genome architecture in heterogeneous cell populations of the brain.

在发育和环境反应中，基因表达的准确水平是通过非编码调节区与其靶基因之间的特定三维(3D)接触来调节的。调控元件与其靶基因的空间关系的缺陷与疾病有关，对于理解复杂的神经疾病越来越重要，这些疾病往往与染色质因子的放松调控有关。然而，研究神经系统4D基因组结构的努力一直局限于使用大量组织、体外分化的细胞或从大脑分离的神经元。因此，剖析伴随着特定神经元激活过程的4D核组改变，以及它们与神经元激活期间和昼夜节律过程中的动态平衡反应之间的关系仍然是一个主要的挑战。在这项建议中，我们旨在了解4D基因组和大脑特定细胞中基因表达之间的关系，以及它在与Shank3突变相关的神经发育障碍中的放松调控，这些突变是自闭症谱系障碍(ASD)的危险因素。除了突触作用外，Shank3还穿梭到细胞核，在那里它调节基因表达，以响应神经元的活动。首先，我们将确定野生型和突变型Shank3小鼠海马区特化锥体神经元的3D基因组拓扑结构，以研究Shank3基因组靶点和作用机制。其次，我们将开发一种新的技术来剖析3D折叠模式和核因子细胞水平之间的定量关系，这是更精细地剖析脑等异质组织中3D基因组折叠机制的关键一步。

Accurate levels of gene expression during development and in environmental responses are mediated through specific three-dimensional (3D) contacts between non-coding regulatory regions and their target genes. Defects in the spatial relationship of regulatory elements to their target genes are linked to disease, and are increasingly important to understand complex neurological disorders, often associated with deregulation of chromatin factors. However, efforts to study 4D genome architecture in the nervous system have been limited to the use of bulk tissues, cells differentiated in vitro or neurons dissociated from the brain. Thus, it remains a major challenge to dissect the 4D nucleome alterations that accompany activation processes in specific neurons, and how they relate with homeostatic responses during neuronal activation and in circadian processes. In this proposal, we aim to understand the relationship between the 4D genome and gene expression in specific cells of the brain, and its deregulation in neurodevelopmental disorders associated with Shank3 mutations, which are risk factors for Autism Spectrum Disorders (ASD). Further to its synaptic roles, Shank3 shuttles to the nucleus where it modulates gene expression in response to neuronal activity. First, we will determine 3D genome topologies in specialized pyramidal neurons in the hippocampus of wildtype and mutant Shank3 mice, to investigate Shank3 genomic targets and mechanisms of action. Second, we will develop a novel technology to dissect quantitative relationships between 3D folding patterns and cellular levels of nuclear factors, an essential step towards finer dissection of 3D genome folding mechanisms in heterogeneous tissues such as the brain.