Epigenomic Regulation of a Large, Neuron-specific Chromatin Domain

脑细胞的基因组被组织成数千个“拓扑相关域”（TADs）， 线性基因组在数百个酶中自我折叠。更深入地了解电子监管 和功能将推进有关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，脑细胞中控制神经元结构和功能的调节机制， 仍然完全未被探索。在这个建议中，我们将研究一个子集的神经元的维护， 严重依赖于集合域分叉1（Setdb 1/Eset/Kmt 1 e）大型、百万碱基规模的“超级TAD”， 编码组蛋白H3-赖氨酸9甲基转移酶。这包括1.2兆字节跨度拓扑 在成簇的原钙粘蛋白（cPcdh）基因座的相关结构域，包括>70 Pcdh和非Pcdh 神经元连接的重要基因。 我们建议在体内解剖控制神经元3D基因组的调控层，包括SETDB 1- 敏感的神经元superTADs。目标1将检验SETDB 1屏蔽神经元基因组的假设 多功能染色质组织者CCCTC结合因子（CTCF）的过度结合。为此目的， 我们将通过原位Hi-C分析，绘制成年大脑中海马能投射神经元的3D基因组， 小脑皮质的皮质和抑制性投射神经元，比较野生型与Setdb 1和Ctcf 缺乏神经元目的#2将探索cPcdh基因的单细胞随机约束，包括潜在的 Setdb 1和Ctcf消融后以及环结合非编码区（epi）基因组编辑后的改变 在本地超级计算机中的序列。此外，我们将研究神经元- 特定删除Setdb 1和Ctcf。我们将评估自上而下的吻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 全细胞膜片钳记录的微型兴奋性（mEPSC）突触后电流在多个 发育时间点，连同树突棘表征。总之，这些实验 这里提出的将提供深入的见解监管机制管理的维护和 成熟神经元中大兆碱基规模的高阶染色质结构的功能。这包括 染色体连接体在神经细胞核内塑造大脑连接体的有趣作用， 调节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.