PROJECT SUMMARY/ABSTRACT There is a sex-based disparity associated with substance abuse disorders, which is evidenced by preclinical and clinical studies. Females are generally more vulnerable to the initiation, escalation and withdrawal effects of substance abuse behavior than males. Although these differences have largely been attributed to hormonal differences, evidence for non-hormonal factors that regulate addiction has been demonstrated by a number of studies. However, the mechanisms underlying sex chromosome influences on substance abuse behavior represent a huge gap in our knowledge base on the epigenetics of substance use disorders. We propose a novel hypothesis that escape from X-chromosome inactivation (XCI) in females contributes to sex associated differences in addiction behavior. We will apply cutting edge technology and uniquely novel approaches and tools we developed recently to comprehensively investigate the impact of XCI escape on sex associated disparities in addiction. XCI is an epigenetic mechanism that occurs in mammalian females and serves to equalize gene expression between the sexes. Females have two X chromosomes (XX), and during XCI, one X chromosome is randomly chosen to be transcriptionally silenced. However, it is known that a number of X linked genes escape XCI and display bi allelic gene expression. The objective of this proposal is to determine the contribution of XCI escape on sex-associated differences in substance abuse disorder. First, we will use novel cutting edge mouse models to characterize cellular mono-allelic (XCI) or bi-allelic (XCI escape) gene expression of specific X-linked genes associated with addiction to opioids and psychostimulants: monoamine oxidase A (Maoa) and GABAA receptor A3 (Gabra3). I pioneered an innovative approach using a gene specific dual bi- cistronic reporter mouse as a tool to enable the visualization of allelic usage of these addiction associated genes in vivo in a model of addiction. Next, we will determine the molecular landscape of XCI in brain tissue and specific neuronal cells during chronic exposure to opioids and psychostimulants, using a highly innovative single cell RNA sequencing technology. To accomplish these goals, I have assembled a talented, multidisciplinary team of research collaborators in addiction, neuroscience, genetic mouse modelling, bioinformatics and biostatistics. This innovative approach to the study and analysis of gene specific XCI escape as an epigenetic mechanism in the context of substance abuse has the potential to open up a new area of research on the epigenetics of addiction. Further, these genetically modified mice can be used to study XCI escape as an epigenetic mechanism in other neurologic disorders. As an early stage investigator, these studies will also advance my long term objective of becoming a future leader in the epigenetics of substance use disorders.