Epigenomic Regulation of a Large, Neuron-specific Chromatin Domain

脑细胞的基因组被组织成数千个与拓扑相关的结构域(TADS)，其中 线形基因组在数百个千碱基之间自行折叠。对TAD监管有更深入的了解 和功能将促进关于神经精神疾病的表观基因组和遗传风险架构的知识 疾病。然而，到目前为止，调节脑细胞中TAD结构和功能的机制 仍然完全没有被探索过。在这个方案中，我们将研究非常子集的神经元维持 严重依赖集-域-分支1(SetDB1/Eset/Kmt1e)大型、兆基规模的“超TAD”， 编码组蛋白H3-赖氨酸9甲基转移酶。这包括拓扑跨度为1.2兆的数据库 聚集的原钙粘附素(CPcdh)基因座上的相关结构域，包括&>70 Pcdh和非Pcdh 对神经元连接很重要的基因。 我们建议在体内解剖控制神经元3D基因组的调节层，包括SETDB1- 敏感的神经元超TADs。Aim#1将检验SETDB1保护神经元基因组的假设 由多功能染色质组织者CCCTC结合因子(CTCF)过度结合。为此， 我们将通过原位Hi-C分析绘制成人大脑谷氨酸能投射神经元的3D基因组图 大脑皮层和小脑皮质抑制性投射神经元：野生型与Setdb1和CTCF的比较 神经元缺陷。目标2将探索cPcdh基因的单细胞随机约束，包括潜在的 SetDB1和CTCF消融后的改变以及环结合非编码的基因组编辑(Epi)后的改变 本地SuperTAD内的序列。此外，我们将研究神经元-之后的功能连接- 具体删除SetDB1和CTCF。我们将评估自上而下旋转内侧突触驱动的变化 青春期和成年期之间的额叶-视皮层投射神经元，具有投射特异性 微兴奋性突触后电流的全细胞膜片钳记录 发育时间点，以及树突棘的特征。总而言之，这些实验 在这里提出的建议将提供对管理维护和 大型高阶染色质结构在成熟神经元中的功能。这包括一个 神经核内的染色体连接体塑造大脑连接体的有趣作用，通过 调控cPcdh基因的表达。

The genome of brain cells is organized into thousands of `topologically associated domains' (TADs), with the linear genome folded upon itself across hundreds of kilobases. A deeper understanding of TAD regulation and function will advance knowledge about epigenomic and genetic risk architectures of neuropsychiatric disease. However, to date regulatory mechanisms governing TAD structure and function in brain cells remain completely unexplored. In this proposal, we will study neuronal maintenance of a subset of very large, mega-base scale `superTADs' that critically depend on Set-domain-bifurcated 1 (Setdb1/Eset/Kmt1e), encoding a histone H3-lysine 9 methyltransferase. This includes the 1.2 megabase-spanning topologically associated domain at the clustered Protocadherin (cPcdh) locus, encompassing >70 Pcdh and non-Pcdh genes important for neuronal connectivity. We propose to dissect, in vivo, the regulatory layers governing the neuronal 3D genome, including SETDB1- sensitive neuronal superTADs. Aim #1 will test the hypothesis that SETDB1 shields neuronal genomes from excess binding by the multifunctional chromatin organizer CCCTC binding factor (CTCF). To this end, we will map, by in situ Hi-C assays, the 3D genomes of glutamatergic projection neurons in adult cerebral cortex and inhibitory projection neurons of cerebellar cortex, comparing wildtype with Setdb1 and Ctcf deficient neurons. Aim #2 will explore single cell-stochastic constraint of cPcdh genes, including potential alterations after Setdb1 and Ctcf ablation and after (epi)genomic editing of loop-bound non-coding sequences within the local superTAD. Furthermore, we will study of functional connectivity after neuron- specific deletion of Setdb1 and Ctcf. We will assess changes in synaptic drive onto top-down rostromedial frontal-to-visual cortex projection neurons between adolescence and adulthood, with projection-specific whole-cell patch clamp recordings of miniature excitatory (mEPSC) postsynaptic currents at multiple developmental time points, together with dendritic spine characterization. Taken together, the experiments proposed here will provide deep insights into regulatory mechanisms governing the maintenance and function of large megabase-scale higher order chromatin structures in mature neurons. This includes an intriguing role of the chromosomal connectome inside neuronal nuclei shaping the brain's connectome, by regulating expression of the cPcdh genes.