Date of Degree
odorant receptors, epigenetic marks, trafficking, G protein-coupled receptors, assay
Odorant receptors (ORs) are expressed by mature olfactory sensory neurons (OSNs), in a monogenic and monoallelic fashion (singular gene choice). It has been repeatedly published that the singular expression of ORs is a consequence of epigenetic regulation. The model invokes the depositing of epigenetic marks (off marks) that reflect transcriptional inactivity on all OR genes in immature neurons followed by the activation of one OR allele in maturing neurons. OR expression has been exclusively studied via transgenic animal models, due to the lack of cell line system that can undergo singular gene choice. Here, we used chromatin immunoprecipitation-quantitative polymerase chain reaction assay (ChIP-qPCR) with antibodies against histone modifications representing epigenetic off and on marks in mouse embryonic stem cells (mESCs) to show that the M71 OR promoter, where M71 is thoroughly studied OR gene in ours and other labs, contains only off marks in mESCs. We established a high-throughput approach that scored for the expression of CRE protein and for the conversion of fluorescence (TdTomatoàGFP fluorescent shift) as a reporter in case of M71 OR activation. To attempt M71 OR expression, we administered thousands chemical compounds to the reporter mESCs, yet none led to the expression of our CRE reporter revealing the powerful repression of the M71gene in mESCs. To further increase the opportunity for M71 expression, we transfected into these reporter cells an M71-IRES-CRE transgene with OR gene choice enhancers, but we observed no expression of the reporter visa vie random integration. In addition, a set ofepigenetic chemical compounds did not induce expression of stable CRE protein.
We hypothesizethat the results might be due to negative regulatory sequences used in OR genes that act as suppressor elements in any cell type including mESCs, such as the OR enhancers, OR promoter, or coding sequence. Based on this reasoning, we utilize our mESC-transgenic approach to test for the presence of “negative” DNA sequences that are specific for the OR genes: the enhancer element, the 461bp M71 OR promoter, and the 1kb M71 OR coding sequence.
ORs are G protein-coupled receptors (GPCRs) and like many GPCRs cannot express at the cell surface outside of their normal cell type. The second part of my thesis focuses on how GPCRs traffic to the cell membrane in the heterologous cell line OP6. To explore this peculiarity, we performed a structure-function study between the β2-adrenergic receptor (β2AR), which traffics well to the plasma membrane and M71 OR, which does not. Using OP6 cells, wedetermined that the N-terminus and the first transmembrane domain of β2AR (β2AR NtàTM1) trafficked to the cell surface on its own, similarly to the full length of the receptor, while the M71 NtàTM1 still did not. We eventually deleted the Nt from the β2AR NtàTM1protein, which initially appeared to abolish plasma membrane trafficking. A detailed analysis of this mutant revealed that it had in fact trafficked to the plasma membrane, but in “C-terminus out/N-terminus in orientation” rather than the conventionally expected “N-terminus out/C-terminus in” orientation. A re-analysis of the M71 NtàTM1 “non-trafficking” mutant revealed that it too was in a C-terminus out/N-terminus in orientation.
Parvanova, Irena G., "Getting at the Surface: A Promoter and Coding Sequence Characterization of an Odorant Receptor" (2019). CUNY Academic Works.