Title: Spatially determined olfactory receptor choice is regulated by Nfi-dependent heterochromatin silencing and genomic compartmentalization
Description: Pattern formation during development is guided by tightly controlled gene regulatory networks that lead to reproducible cell fate outcomes. However, stochastic choices are often employed to further diversify cell fates. These two mechanisms are closely interlinked in the mouse olfactory system, where stochastic expression of one of one out of >1,000 olfactory receptor (OR) genes is restricted to anatomical segments, or “zones”, organized along the dorsoventral axis of the olfactory epithelium (OE). Despite recent progress in understanding the processes underlying OR choice, the mechanism by which the dorsoventral position of an olfactory sensory neuron (OSN) dictates its OR repertoire has remained elusive and is the focus of this thesis. To gain insight into a possible mechanism I compared the transcriptomes, chromatin landscape, and nuclear architecture of cells isolated from ventral and dorsal zonal segments of the OE. I determined the developmental window in which cells become restricted in their zonal OR repertoire and found this coincided with both the deposition of heterochromatic histone marks H3K9me3 and H3K79me3 on OR genes and their coalescence into a multi-chromosomal compartment.

Comparing heterochromatin levels and OR compartment composition in OSNs from different zones, I determined in each case OR genes with more dorsal indexes have higher levels of H3K9me3/H3K79me3 and thus become silenced, while OR genes with more ventral indexes have no heterochromatin and consequently are excluded from OR compartments. Thus, ORs that are “competent” for activation are relatively more accessible, while still being recruited into the OR compartment where they can interact with the proximally positioned enhancer hub. I found that this mechanism is regulated by Nfi family transcription factors that are expressed in a ventral (high) to dorsal (low) gradient in the OE. Deletion of Nfi A, B and X transforms the heterochromatin and OR compartmentalization in ventral OSNs to a more dorsal state, and shifts their preferred OR repertoire towards more dorsal ORs. This result implicates Nfi proteins as key regulators of zonal OR expression. Finally, I probed the nuclear architecture in single cells to look for the source of stochastic choice within zonal segments.

I found high variability in inter-chromosomal OR compartment and enhancer hub composition between individual OSNs that stemmed from the unpredictable and variable positioning of chromosomes in the interphase nucleus. Overall, this thesis provides evidence for a mechanism of zonal OR choice that combines deterministic restrictions imposed by a gradient of Nfi with random inter-chromosomal contacts.