Bridging stochastic and deterministic paradigm of olfactory receptor choice in ants - Project Summary / Abstract Gene families include up to hundreds of highly similar sequences that may perform related but non- redundant functions. Therefore, understanding how the correct gene is activated at the appropriate location and time remains an important question. Odorant receptor (Or) genes present a particularly prominent example of this problem. Previous research in mice (which have >1,000 Ors) and flies (which have 60 Ors) led to an idea that large Or families employ stochastic gene choice, while small Or families follow deterministic specification. The jumping ant Harpegnathos saltator lies in between these extremes, possessing 376 Or genes, most of which are not interspersed throughout the genome, but located within tandem genomic arrays that contain up to 58 genes each. This proposal will test a model where each olfactory sensory neuron (OSN) in ants expresses a single locus, either a single gene or an array, using the same deterministic mechanism as Drosophila melanogaster to choose between a limited number of loci. Single gene loci are expressed deterministically, while loci that contain multiple genes have an added stochastic mechanism by which a single Or promoter is chosen out of the array. To this end, I performed single-nucleus RNA-seq (snRNA-seq) on the H. saltator antennae. Strikingly, although isolated genes were uniquely expressed in non-overlapping sets of OSNs, genes within arrays were co-expressed. In all such cases, they followed a highly stereotypical pattern: given genes A, B, C within an array, either C (the most 3’ gene) was expressed alone in some OSNs, or B and C were co-expressed in another subset of OSNs, or all three genes were expressed concurrently in yet another subset. In addition, the snRNA-seq data revealed extensive antisense transcription in these loci. The antisense RNAs covered genes upstream of the first transcribed gene and appeared to originate from the same promoter. Thus, the antisense RNAs are mutually exclusive with the sense RNAs, suggesting that the antisenses may repress the transcription of genes upstream of the chosen promoter. In aim 1, I will investigate the mechanisms of the co-transcription among clustered genes, determine whether co-transcribed genes are co-translated and whether OSNs that express them target distinct glomeruli in the brain, identify sequence features that enable transcription and provide stability to the antisense RNAs, and test the potential repressive function of the antisense transcripts. Together, this will provide a comprehensive description of the transcriptional mechanisms of the single gene choice in H. saltator Or arrays and uncover how this translates into protein expression. In aim 2, I will determine the transcription factor code associated with each Or gene, collect chromatin interaction and multiome (snRNA-seq paired with ATAC-seq) data to comprehensively identify putative regulatory regions, determine whether one or both alleles are expressed in each cell, and test the function of the candidate enhancers. Together, this will uncover the regulatory architecture of the Or loci, potentially revealing a novel stochastic mechanism of promoter choice within arrays.
