the impermanence of OR selection and destabilization of OSN identity in OP cells
Noble, Joyce Carol (Joyce Carol Noble) (author), (Robert Lane) (Thesis advisor), Wesleyan University Molecular Biology & Biochemistry (Degree grantor)
The olfactory system is necessary for environmental perception in animals, where olfactory sensory neurons (OSNs) can detect volatile molecules in the air. For OSNs, terminal maturation is temporally coordinated with, and dependent on, the expression and function of olfactory receptors (ORs) that directly bind to odorants. In mouse, an OR is stochastically selected in a monogenic and monoallelic fashion from 1000 potential ORs, granting each neuron its own unique identity. Though the exact mechanism underlying OR expression regulation is not fully understood, research in this field has provided evidence that it is likely a coordinated process of cis and trans factors and nuclear organization that ensures singular OR expression.
I utilized an immature olfactory neuron cell line clonally derived from the developing olfactory placode (OP6) as a model system to study OR regulation and OSN development. I investigated OR re-selection frequencies and selection biases within OP6 colonies and cell populations. I found that OR re-selection occurs as frequently as at least once every cell cycle, and although OR selection is not stable, expression biases persist even with prolonged culturing. Overall, these findings suggest that though the chromatin environment to maintain OR selection is not stable in mitotic OP cells, epigenetic states that dictate OR selection probability are persistent.
I also investigated if a candidate chromatin modifier, G9a, may be involved in OSN lineage maintenance. Histone methyltransferase G9a is involved in the expression of olfactory-specific genes, and acts as a barrier in the reprogramming of neuronal precursor cells. To investigate the role of H3K9 methylation in OSN development, I developed a G9a drug inhibition protocol on OP cell populations. Surprisingly, drug-treated OP6 cells appeared to lose neuronal characteristics, exhibiting embryoid body-like colony formation, which was not evident in untreated OP6 cells or in treated GD25 (fibroblast) cells. Several stem cell markers were also upregulated within these OP6 colonies relative to untreated cells. Interestingly, initial colony formation and maintenance appears to be dependent on the cell cycle-promoting large T-antigen. These results suggest that epigenetic and mitotic barriers may prevent lineage regression towards stem-like states and provides evidence for G9a's direct involvement in neuronal identity maintenance in OP cells.
Lastly, I investigated the function of several known factors of robust OR expression - transcription factor Lhx2, locus control regions, and the unfolded protein response - in the OP cell where selection, but not commitment, is occurring. I observe little evidence of Lhx2 functioning on OR expression as ChIP reveals no enrichment of Lhx2 at OR promoters or LCRs. LCRs do not contain canonical chromatin states such as H3 acetylation and DNase hypersensitivity that normally would classify them as functional. LCRs also do not aggregate to interact in trans. However, preliminary data suggests that they may interact in cis. Finally, I provided evidence that ER stress alone is not sufficient to upregulate the downstream factors associated with the OR-specific UPR pathway, a necessary event in vivo for OR commitment. This data suggests that these factors (Lhx2, LCR) or pathways (UPR) that appear to be a requirement for commitment are likely not necessary for OR expression in the pre-commitment neuron.
The de-repression of previously selected ORs in the mitotic olfactory neuron and the destabilization of neuronal identity through G9a perturbation imply a unique characteristic of cell state malleability in immature olfactory neuronal cells. These studies provide evidence to suggest that olfactory neurons may have a greater developmental plasticity than other cell types, a hypothesis that is consistent with the need to meet the highly regenerative capacity of this system., In Copyright – Non-Commercial Use Permitted (InC-NC)