Utilization of epitope tags to improve the isolation of nuclear receptor corepressor SMRT
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Abstract
Endocrine and metabolic diseases such as hyper/hypothyroidism, diabetes and obesity have increased in prevalence around the world in recent decades. Many conditions of this nature stem from the repercussions of improper thyroid hormone action which despite adequate treatment, many patients report persistent symptoms of malaise. Thyroid hormone (TH) acts via the thyroid hormone receptor (TR), a nuclear receptor. TH-related gene expression occurs in the presence of it’s ligand, TH. In the absence of TH, corepressors are recruited to TR to inhibit TH-related gene expression. The silencing mediator of retinoic acid and thyroid receptors (SMRT) binds to TR and forms a corepressor complex that inhibits TH gene expression. The molecular mechanisms of SMRT remain unknown. We aim to determine how the corepressor complex changes across different TH levels by using a novel mouse model developed in our lab. With the specificity of epitope tags found in previous studies, we decided to make an epitope tag to attach to SMRT as a more specific method to detect and isolate SMRT. We used CRISPR/Cas9 to add an epitope tag incorporating two hemagluttin (HA) tags and a BLRP sequence on the C-terminal end of SMRT to generate SMRT-HA tag mice. We studied wild type mice, birA mice, homozygous (HO) SMRT-HA tag mice, and HO SMRT tag x birA mice to determine any differences between genotypes. Our preliminary analysis shows genetic modification of SMRT does not impact phenotype. We found that the epitope tag has no effect on SMRT’s function. Though it has been found to efficiently isolate SMRT, further testing and improvements are needed in order to confirm and isolate SMRT across tissues. Overall, our SMRT-HA tag has potential to not only improve the isolation of SMRT, but also enable further study of its function and molecular mechanisms in every tissue. Further studies are required to improve the efficiency of techniques such as immunoprecipitations and Western blots, along with the efficiency of the tag to make sure that there is limited molecular interference with its function or ability to participate in necessary molecular interactions. Although we have studied SMRT in the context of TH action, the use of this SMRT-HA tag line can be applied across tissues and disease states to widen our knowledge about nuclear receptor action.
Description
2024