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)
Vyas, Rutesh Niranjanbhai (Author), (Robert P. Lane) (Thesis advisor)
Function of the mammalian olfactory system depends on specialized olfactory sensory neurons (OSNs) that each express only one allele (“monoallelic”) of one odorant receptor (OR) gene (“monogenic”). The lysine-specific demethylase-1 (LSD1) protein removes activating H3K4 or silencing H3K9 methylation marks in a variety of developmental contexts, and is thought to be important for proper OR regulation. Most of the focus in the field has been on a potential “activating” function for LSD1; e.g., in the demethylation of H3K9 associated with the expressed OR allele. In this thesis, we show that depletion of LSD1 in an immortalized olfactory-placode-derived cell line (OP6) results in a significant increase in multigenic and multiallelic OR transcription per cell, while not seemingly disrupting the ability of these cells to activate new OR genes during clonal expansion. These results are consistent with LSD1 having a role in silencing additional OR alleles, as opposed to being required for the activation of OR alleles, within the OP6 cellular context. We propose a model for initial OR gene selection, whereby management of both H3K9 methylation (by enzymes that add this mark at ORs; e.g. G9a and GLP) and H3K4 methylation (by enzymes that remove this mark at ORs; e.g. LSD1) are required for reduction of the number of competing OR candidate genes for activation. This model predicts that disruption of either enzyme (G9a to add H3K9 methylation or LSD1 to remove H3K4 methylation) might shift the equilibrium towards increased H3K4 /decreased H3K9, thereby increasing the probability of multiple OR activation events per cell. To further test this hypothesis, we perturbed G9a expression using the CRISPR-Cas9 technique in order to test whether perturbation of G9a contributed to an equilibrium shift towards decreased H3K9 methylation (and increased probability of multiple OR activation events per cell, as with the LSD1 perturbation). We made several observations in this experiment that suggest a much broader developmental role for G9a in the OSN lineage. We observed that CRISPR-treated OP6 cells transform/de-differentiate into stem-cell like colonies that exhibit global disruption of heterochromatin, expression of stem-like markers, and ultimately, differentiation and organization of progeny cells into developmental structures. In this thesis, I will review the field of olfactory system development (Chapter 1), discuss published results indicating a role for the LSD1 protein in regulating OR expression during olfactory sensory neuronal (OSN) development (Chapter 2), discuss unpublished results suggesting a role for the G9a protein in regulating OSN development (Chapter 3), and finish with a discussion of questions raised by this work and future experimental directions inspired by preliminary results (Chapter 4)., Old URL: https://wesscholar.wesleyan.edu/etd_diss/103, In Copyright – Non-Commercial Use Permitted (InC-NC)