This is a resubmission of my renewal application of DA001583-30. In the last review, it received a score of 156 (12.9%) and was not funded. First of all I would like to thank the reviewers for their thorough review and overall very positive assessment of the original application. There were only two points raised in the review, one questioning why not study human gene and the second on physiological relevance of our study on the regulation of MOR receptors. Both are clarified and addressed in the revision. This renewal will continue our study on the regulatory mechanisms underlying the expression of the Mu opioid receptor (MOR) in neurons. Studies during the current award period focused on transcriptional events that provided essential information for our understanding of the genetic basis of how this important drug receptor is regulated. This renewal will focus on regulatory events involving higher-order structures of chromatin (chromatin remodeling) and epigenetic effects. Further, our preliminary studies identified several potentially novel factors that acted beyond transcriptional control, and it is hypothesized that the expression of the functional product of MOR gene, i.e. the mu receptor, is tightly regulated at both transcriptional and post-transcriptional levels. The renewal application is to address this principal hypothesis in three aims.
Aim 1 will continue ongoing studies of MOR gene transcriptional regulation with chromatin as the focus. Firstly, the chromatin structure and nucleosomal arrangement of MOR gene, as well as methylation-induced epigenetic control, will be defined using various biochemical and molecular biology based methods. Secondly, the dynamic behavior of previously identified transcription factors on the MOR promoter will be examined in its endogenous genomic context using primarily chromatin immunoprecipitation.
Aim 2 will extend studies to the post-transcriptional events, primarily translational control of MOR protein expression. Three major regulatory events will be addressed, including the roles of 5'-untranslated regions (UTR), the 3'-UTR and the polyadenylation signal.
Aim 3 will identify external factors involved in the ultimate regulation of MOR protein expression, including vitamin A hormone (retinoic acid) and other signals that possibly modulate translation of MOR mRNA in neurons. Our immediate goal in this renewal application is to determine the mechanism of action of genetic, epigenetic and extracellular signals that act, in combination, to regulate the ultimate expression of appropriate amounts of MOR receptor protein in neurons. Our long-term goal is to be able to delineate signal transduction pathways that affect the manifestation of MOR gene in the context of whole animals, and possibly to contribute to the understanding of genetic and molecular basis of problems related to the use of morphine in humans.
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