Epigenetic Regulation of Olfactory Receptor Genes and Dedifferentiation of an Olfactory Placode Cell Line
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).