Some of life’s most important behaviors are guided by the sense of smell. Detecting and interpreting odor information influences food-seeking, predator avoidance, sociality, competition, mating rituals, and more, shaping how organisms interact with their environment. In vertebrates, odors are detected by olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE). OSNs rely on olfactory receptors (ORs) to recognize odorants and trigger neural activation. The OR gene pool is typically vast, containing between 200-4000 olfactory receptor genes across mammals, yet mature OSNs stably express only one gene from one allele. Data from mice show that ORs are anatomically restricted to designated sections of the MOE, but within these zones, OR expression appears mosaic and random. Since the discovery of the OR gene pool 30 years ago, deciphering how OSNs choose which OR they are going to express remains a central question.
While multiple differentiation-dependent alterations to the OSN nucleus are required for OR expression, the most notable contribution comes from the organization of OR-gene specific enhancers, called Greek Islands (GIs), around the chosen allele. GIs use the transcription factors Lhx2 and Ebf1, as well as the coactivator Ldb1, to form a nucleoprotein complex known as the Greek Island Hub (GIH) to associate with the active OR gene and support its transcription. Bulk Hi-C data show that GIs form strong, specific, and singular associations with the active OR gene, suggesting a possible role for the GIH in singular OR choice. However, single-cell Hi-C analysis shows that multiple GIHs exist in every OSN with no clear differences between them, complicating the contribution of the GIH. Furthermore, ectopic OR gene activation is sufficient to drive association of an OR locus with a GIH and bias choice, suggesting a role for OR transcription itself in supporting its own stable expression.
To clarify the genomic transformations that result in the formation of multiple GIHs, I performed combined scRNA-seq and scATAC-seq in the MOE. I determined that a selective inactivation event was taking place during the INP3-to-iOSN transition, where OSNs would silence a large fraction of the GI pool. GI inactivation takes place during a phase preceding OR choice, where OR expression is polygenic but skewed towards one OR. My single-cell Hi-C analysis verifies the presence of multiple GIHs per cell, with similar GI-GI interaction properties, but I also observe that the single active GIH contains much more specific GI-OR gene interactions than those in inactive GIHs. These architectural differences are supported by Liquid Hi-C and H3K27ac HiChIP analysis where I observe that the active GIH is more highly acetylated than inactive GIHs and possesses more euchromatic physical properties. Taken together these data show that while most GIs were initially euchromatic during the polygenic phase of OR expression, once choice has taken place, GIHs possess distinct OR interaction properties, chromatin marks, and physical features that are determined by their association with the active OR gene. I believe that these data are best explained by a winner-takes-all event, where GIHs containing transcribed OR genes during the polygenic phase are in competition for choice.
Once one OR begins to win, it recruits resources to maintain its expression which consequently results in the silencing of other GIHs. Ectopic induction of OR gene transcription is sufficient to bias choice and silence other ORs by impeding their specific association with a GIH. I find that this does not depend on the coding properties of OR protein, as the transcription of non-coding OR mRNAs still results in OR gene silencing. I describe this competition as a symmetry breaking process, where asymmetrical reorganization of transcriptional resources to a single GIH is mediated by non-coding properties of a single highly expressed OR mRNA, culminating in the stable expression of that allele alone for the remainder of a cell’s lifetime.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/7zh7-cp88 |
| Date | January 2024 |
| Creators | Pourmorady, Ariel David |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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