Lysine-specific demethylase-1 (LSD1) depletion disrupts monogenic and monoallelic odorant receptor (OR) expression in an olfactory neuronal cell line

Abstract

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. Here we show that depletion of LSD1 in an immortalized olfactory-placode-derived cell line (OP6) results 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.

Spatial Determination of Neuronal Diversification in the Olfactory Epithelium

“…ND, No detectable expression of either NQO1 or NCAM2. ͓1͔, ISH performed in the current study; ͓2͔, ISH reported by Serizawa et al (2000); ͓3͔, ISH reported by ; ͓4͔, ISH reported by Grosmaitre et al (2006); ͓5͔, ISH reported Tsuboi et al (2006); ͓6͔, ISH reported by Zhao et al (2013); ͓7͔, ISH reported by Kaluza et al (2004). OR locations identified by association with OR family members are indicated by ͓family͔.…”

“…The localization in normal OE of the amplified OR was determined by ISH ( Fig. 6A) and, when available, compared to published patterns of OR expression (Serizawa et al, 2000;Kaluza et al, 2004;Grosmaitre et al, 2006;Tsuboi et al, 2006;Zhao et al, 2013). We validated both the purity of cells transplanted as well as the sensitivity of our FACS isolation after transplant (Fig.…”

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

“…We have previously shown that only one OR allele (“monoallelic”) of one OR gene (“monogenic”) is expressed at detectable levels in individual OP6 cells, and that many OR genes are expressed in entire OP6 cultures at various passages [ 30 , 31 , 33 ]. Given that OP6 cultures are clonally derived [ 29 ], these observations indicate that OR re-selection has occurred–if OR choice was stable, we would predict that OP6 cultures would be ~homogeneous with respect to OR expression.…”

“…For several years, we and others have used the OP6 cell line as a model for studying OR gene regulation. Based on previous work [ 30 , 31 , 33 ], individual OP6 cells appear to express only one dominant OR allele (monogenic, monoallelic), like mature OSNs in vivo . Therefore, the OP6 cell line provides an opportunity to investigate mechanisms of OR gene selection in a developmentally ~homogeneous (i.e., clonal) cell population.…”

“…When further differentiated using retinoic acid, OP6 cells develop into characteristic bipolar morphology, as well as exhibit chromocenter condensation and developmental gene expression profiles characteristic of a more mature state, substantiating the OP6 founder as an immature OSN [ 25 , 29 – 31 ]. One interesting feature of the OP6 line stems from its immortalization–while expressing only one OR per cell, this choice is not stable during culturing, suggesting that dividing OP6 cells are unable (or less able) to “commit” to an OR [ 30 , 31 , 33 ]. Therefore, the OP6 cell line represents an opportunity to decouple the “OR specification”/“OR selection” process from the “OR commitment” process, a scenario that is difficult to achieve in vivo .…”