Summary The genome is partitioned into topologically associated domains (TADs) and genomic compartments of shared chromatin valance. This architecture is constrained by the DNA polymer, which precludes genic interactions between chromosomes. Here, we report a dramatic divergence from this pattern of nuclear organization that occurs in mouse olfactory sensory neurons (OSNs). In situ HiC on FAC-sorted OSNs and their progenitors shows that olfactory receptor (OR) gene clusters from 18 chromosomes make specific and robust interchromosomal contacts that increase with differentiation. These contacts are orchestrated by intergenic OR enhancers, the Greek Islands, which first contribute to the formation of OR compartments and then form a multi-chromosomal super-enhancer that associates with the single active OR. Greek Island-bound transcription factor Lhx2 and adaptor protein Ldb1 regulate the assembly and maintenance of OR compartments, Greek Island hubs, and OR transcription, providing mechanistic insight and functional support for the role of trans interactions in gene expression.

Graphical Abstract Highlights d A conserved antisense promoter is located within each of the Pcdha alternate exons d Antisense lncRNA transcription leads to DNA demethylation of promoters and CBSs d CTCF/cohesin drive the assembly of Pcdha enhancer/ promoter complex via loop extrusion d Coupling lncRNA transcription to DNA demethylation ensures stochastic promoter choice SUMMARYStochastic activation of clustered Protocadherin (Pcdh) a, b, and g genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdha gene choice involves the activation of an antisense promoter located in the first exon of each Pcdha alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdha gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdha promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdha promoter choice.

The eukaryotic genome is partitioned into topologically associated domains (TADs) that assemble into compartments of shared chromatin valance. This architecture is influenced by the physical constraints imposed by the DNA polymer, which restricts DNA interactions predominantly to genomic segments from the same chromosome. Here, we report a dramatic divergence from this pattern of nuclear organization that occurs during the differentiation and specification of mouse olfactory sensory neurons (OSNs). In situ HiC on FAC-sorted OSNs shows that olfactory receptor (OR) genes from numerous chromosomes make frequent, extensive, and highly specific interchromosomal contacts that strengthen with differentiation. Moreover, in terminally differentiated OSNs, >30 intergenic enhancers generate a multi-chromosomal hub that associates only with the single active OR from a pool of ~1400 genes. Our data reveal that interchromosomal interactions can form with remarkable stereotypy between like neurons, generating a regulatory landscape for stochastic, monogenic, and monoallelic gene expression.Mouse ORs are encoded by a family of ~1400 genes that are organized in 69 heterochromatic genomic clusters distributed across most chromosomes. Every mature OSN (mOSN) expresses one OR gene from one allele in a seemingly stochastic fashion 1-3 . Previous work suggested that repressive and activating interchromosomal interactions contribute to the singular OR expression 4-6 . However, these interactions have only been analyzed with the use of biased and low-throughput approaches (3C, 4C, capture HiC, and DNA FISH), which have either limited genomic resolution or restricted genomic coverage. Thus, it remains unknown how prevalent and specific these interactions are, and how they form in relationship to OSN differentiation and OR expression. Moreover, in situ HiC 7 , which reduces the occurrence of non-specific ligation events observed in dilution HiC, revealed that interchromosomal associations between non-repetitive, genic regions are extremely infrequent 8,9 , and only emerge upon depletion of cohesin complexes 10,11 . Thus, to explore the landscape of interchromosomal interactions in a biological system that likely depends on them, and to provide a conclusive answer into whether interchromosomal contacts actually occur with biologically meaningful frequency and specificity, we performed in situ HiC in distinct cell populations of the main olfactory epithelium (MOE).

Neural type–specific expression of clustered Protocadherin (Pcdh) proteins is essential for the establishment of connectivity patterns during brain development. In mammals, deterministic expression of the same Pcdh isoform promotes minimal overlap of tiled projections of serotonergic neuron axons throughout the brain, while stochastic expression of Pcdh genes allows for convergence of tightly packed, overlapping olfactory sensory neuron axons into targeted structures. How can the same gene locus generate opposite transcriptional programs that orchestrate distinct spatial arrangements of axonal patterns? Here, we reveal that cell type–specific Pcdh expression and axonal behavior depend on the activity of cohesin and its unloader, WAPL (wings apart–like protein homolog). While cohesin erases genomic-distance biases in Pcdh choice, WAPL functions as a rheostat of cohesin processivity that determines Pcdh isoform diversity.

Singular olfactory receptor (OR) gene expression 1,2 coincides with the formation of a multichromosomal enhancer hub that associates with the only transcribed OR allele in each cell 3,4 . This hub consists of converging transcriptional enhancers 3 , or "Greek Islands", defined by stereotypic binding of Lhx2 and Ebf on a shared, composite DNA motif 5 . How this multi-chromosomal hub, or any other genomic compartment, assembles is unknown, and so is the significance of compartmentalization in transcription. Here, we report that LIM domain binding protein 1 (Ldb1), which is recruited by Lhx2 and Ebf to Greek Islands, promotes robust and specific trans interactions between these enhancers. In addition to disrupting Greek Island hubs, Ldb1 deletion also causes significant downregulation of OR transcription. Thus, our data provide insight to the formation of genomic compartments, confirm the