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.