The ability to detect odorants relies on the generation of thousands of different olfactory receptor (OR) neurons during the development of the olfactory epithelium (OE), the primary sensory organ of the mammalian olfactory system. The identity of each neuron is determined by the expression of a single OR gene from over ~1500 genes scattered across the genome. The mechanism of this choice is an intriguing and unsolved problem in the field of gene regulation. This thesis presents a large-scale investigation of the regulatory architecture of distal enhancer elements that regulate ORs and yields new insight into the mechanism of OR choice.
In the first part of the dissertation I identify 35 possible cis-regulatory elements for ORs and I characterize their function in the olfactory epithelium. These sequences exhibit canonical chromatin hallmarks of enhancers, as well as enrichment for the repressive histone modification H3K79me3. Transgenic analysis of these elements in zebrafish and mouse shows broad expression in olfactory neurons. Genetic deletion of one element, Lipsi on chromosome 2, demonstrates its requirement for the expression of proximal olfactory receptors in the mouse olfactory epithelium.
In the second chapter I describe experiments probing the three-dimensional nuclear organization of olfactory receptor enhancers. Intriguingly, these elements form long-range interactions in the nuclei of olfactory neurons which may be functional for OR expression. Multiple OR enhancers from different chromosomes interact frequently with a transcribed olfactory receptor gene. Global analysis of enhancer-enhancer interactions show that interchromosomal interactions are extensive in olfactory neurons and are specific to OR enhancer sequences. Genetic disruption of these long-range enhancer interactions disrupts the singular expression of OR genes in OSNs. These observations support a model whereby the assembly of an enhanceosome consisting of enhancers from different chromosomes drives singular OR expression.
The third section of the dissertation explores the transcription factors that bind these enhancers and mediate their function in OR gene expression. Ebf4 binds an O/E-like motif on enhancers which is necessary and sufficient for enhancer activity. BPTF, part of the NURF chromatin remodeling complex, binds enhancers, is required for OR gene expression, and facilitates enhancer-enhancer interactions in olfactory neuron nuclei.