ABSTRACT
Olfactory receptor (OR) choice provides an extreme example of allelic competition for transcriptional dominance, where every olfactory neuron stably transcribes one of approximately 2,000 or more OR alleles1,2. OR gene choice is mediated by a multichromosomal enhancer hub that activates transcription at a single OR3,4, followed by OR-translation-dependent feedback that stabilizes this choice5,6. Here, using single-cell genomics, we show formation of many competing hubs with variable enhancer composition, only one of which retains euchromatic features and transcriptional competence. Furthermore, we provide evidence that OR transcription recruits enhancers and reinforces enhancer hub activity locally, whereas OR RNA inhibits transcription of competing ORs over distance, promoting transition to transcriptional singularity. Whereas OR transcription is sufficient to break the symmetry between equipotent enhancer hubs, OR translation stabilizes transcription at the prevailing hub, indicating that there may be sequential non-coding and coding mechanisms that are implemented by OR alleles for transcriptional prevalence. We propose that coding OR mRNAs possess non-coding functions that influence nuclear architecture, enhance their own transcription and inhibit transcription from their competitors, with generalizable implications for probabilistic cell fate decisions.
Subject(s)
Olfactory Receptor Neurons , RNA , Receptors, Odorant , Alleles , Cell Lineage , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Olfactory Receptor Neurons/metabolism , Receptors, Odorant/genetics , Receptors, Odorant/metabolism , Regulatory Sequences, Nucleic Acid/genetics , RNA/genetics , Transcription, Genetic , Genomics , Single-Cell AnalysisABSTRACT
Chromatin immunoprecipitation (ChIP) allows a researcher to determine the genomic occupancy of nuclear proteins, providing insight into the roles of transcription factors, chromatin modifiers, histone modifications, and other factors bound to DNA. Protein-DNA interactions are first fixed in vivo by chemical cross-linking, and then a target protein is captured together with any associated DNA by an antibody mediated pull-down. The co-immunoprecipitated DNA can then be assayed by quantitative PCR or deep sequencing. Here, we demonstrate this technique using murine olfactory sensory neurons (OSNs) purified using fluorescence-activated cell sorting (FACS) and antibodies for the ubiquitous chromatin protein CTCF.
Subject(s)
Olfactory Receptor Neurons , Animals , Mice , Chromatin Immunoprecipitation , Formaldehyde , Antibodies , Chromatin/geneticsABSTRACT
Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.
