Frequent and biased odorant receptor (OR) re-selection in an olfactory placode-derived cell line

Abstract

We previously characterized a clonal olfactory placode-derived cell line (OP6) as a model system for studying odorant receptor (OR) choice, where individual OP6 cells, similar to olfactory sensory neurons in vivo, transcribe one allele (“monoallelic”) of one OR gene (“monogenic”). The OP6 cell line provides a unique opportunity to investigate intrinsic properties of OR regulation that cannot easily be investigated in vivo. First, whereas OR-expressing cells in vivo are post-mitotic, OP6 cells are immortalized, raising interesting questions about the stability of epigenetic states associated with OR selection/silencing as OP6 cells progress through the cell cycle. Second, OP6 cells have been isolated away from extrinsic developmental cues, and therefore, any long-term OR selection biases are likely to arise from intrinsic epigenetic states that persist in the absence of developmental context. In this study, we investigated OR re-selection frequency and selection biases within clonal OP6 cell populations. We found no evidence of OR stability through the cell cycle: our results were most consistent with OR re-selection events transpiring at least once per cell division, suggesting that chromatin states associated with OR selection in this system might not be maintained in the subsequent generation. In contrast, we found strong evidence for OR selection biases maintained over prolonged culturing across a diverse set of OP6 cell lineages, suggesting the persistence of intrinsic epigenetic states that advantage some OR loci over others. Together, our data suggest that in the absence of instructive cues, intrinsic epigenetic states influencing OR eligibility, but not those determining OR choice, might persist through the cell cycle.

OR Gene Mediated Silencing of an Exogenous Expression Vector

“…The inverse correlation between transgenic OR expression and endogenous OR expression suggests a transcriptional competition between OR alleles. A similar observation is made in the OP6 cell line, as specific OR subsets appear to be biased for frequent selection over other OR genes, possibly due to having inherent competitive advantages (Noble et al, 2018).…”

“…That is, if Olfr1414 (and others) are efficiently silenced because it is a poor competitor, we predict that the endogenous Olfr1414 (and others) will be inefficiently expressed in OP6 cells generally. We have previously published that a subset of ORs appear to be strong or weak competitors OP6 cell populations (Noble et al, 2018;Pathak et al, 2009), and our model predicts that transgenes will correspondingly be inefficiently or efficiently silenced, respectively. One future direction is to conduct parallel RNA-FISH and transgene studies in attempt to observe such a correlation.…”

“…The OP6 and OP27 cell lines have been utilized as a model system for OSN development. Our lab utilizes this system to study OR regulation, since both cell lines express OR genes monallelically and monogenically (Kilinc et al, 2014;Noble et al, 2018;Pathak et al, 2009;Vyas et al, 2017). Like iOSNs in vivo, OP6 and OP27 cells appear to express ORs at low levels (Hanchate et al, 2015;Tan et al, 2017).…”

“…We have used the OP6 cell line to model immature OSNs that are able to select OR genes but appear unable to commit to this choice. Following each cell division event in culture, OP6 cells select a new OR gene to express ("switching"), and there are inherent OR selection biases evident during switching (Noble et al, 2018). The observation that immature OSNs in vivo (Hanchate et al, 2015;Tan et al, 2015), as well as OP6 cells in certain genetic backgrounds (Vyas et al, 2017), appear capable of expressing multiple ORs per cell, albeit at lower pre-commitment levels, suggests that "singularity" might not be an absolute attribute at the outset, and that progressive "iteration" may eventually lead to mutually exclusive OR expression.…”

An Op(timally) Permissive State

“…When OR expression reaches a critical threshold, the UPR pathway detects unfolded OR protein in the ER, which results in a cascade of protein phosphorylation, transcriptional upregulation, and differential translation to facilitate proper OR folding, localization, and signaling . We postulated that the UPR may not be actively functioning in the OP6 cell line since OR selection is not robust or stable in OP cells (Noble et al, 2018) and OR RNA is mostly retained in the nucleus until OP cell differentiation , so it is unlikely that translated OR protein is present in this system to stimulate the UPR pathway. Because of this, we investigated whether OR choice could be stabilized in OP6 cells through chemical induction of the UPR pathway and independent of OR translation.…”