Deciphering mechanisms for olfactory receptor choice in single cells

PROJECT SUMMARY Remarkably, chromosomes avoid entanglement as they are organized into various spatial domains that support diverse functions in the nucleus. Among these functions, 3D organization of the genome is uniquely essential for constraining patterns of gene expression. This is perhaps best demonstrated by the phenomenon of olfactory receptor (OR) choice in the mammalian olfactory epithelium. Olfactory sensory neurons (OSNs) of the olfactory epithelium stochastically choose 1 of ~1400 OR genes to stably express. The chosen OR defines an OSN’s receptive field and is thus a crucial element of OSN identity. Since the discovery of the OR gene pool ~30 years ago, a mechanism for OR choice remains undefined. However, research from our lab has revealed that OR expression depends on differentiation-dependent alterations to nuclear architecture. In olfactory stem cells, heterochromatin detaches from the nuclear periphery leading to inversion of the nucleus. OR genes subsequently organize into multichromosomal compartments near the newly formed heterochromatic core. Lastly a network OR-gene specific enhancers known as Greek Islands (GIs), assemble over a single OR allele, forming an “OR-Enhancer Hub”, to support OR transcription. While all three events are indispensable for OR expression, the formation of the OR-Enhancer Hub is perhaps the most important. 63 GIs have been identified; however, in any cell, only a subset participate in the OR-Enhancer Hub, forming highly specific interactions with the active OR allele. The unique organization of GIs around an OR gene was considered to be the architectural footprint of the single transcriptionally active OR. However, single-cell genomics data from our lab demonstrate that topologically identical structures exist elsewhere in the nucleus. These complexes are defined by the same enhancer constitution as the active hub yet contain transcriptionally silent OR genes. However, additional single- cell experiments suggest these structures might be distinguished by underlying biochemical features. In this proposal we aim to first characterize the chromatin properties that separate active from inactive enhancer hubs, then test possible mechanisms for how these structures might assemble and gain different functional properties. We have devised a single-cell genomic strategy to decipher the chromatin marks and bound transcription factors that differentiate enhancer subtypes, focusing on those that explain GI association to the active OR gene. OR transcription early in development is a known tropic signal for GI recruitment, reinforcing OR choice. Therefore, we will perform analysis of genome structure and functional molecular features in mice mutated to perturb stages of the OR gene expression pathway to identify determinants of OR-Enhancer hub assembly. We anticipate that these experiments will reveal a biological scheme by which the coordinated spatial rearrangement of the genome alongside patterned modifications to chromatin guide gene expression in a unique cell of the nervous system.