277. A LONG NONCODING ERNA FORMS R-LOOPS TO SHAPE EMOTIONAL EXPERIENCE-INDUCED BEHAVIORAL ADAPTATION

Abstract Background Emotional experiences often evoke neural plasticity that supports adaptive changes in behavior, including maladaptive plasticity associated with mood and substance use disorders. These adaptations are supported in part by experience-dependent activation of immediate-early response genes, such as Npas4 (neuronal PAS domain protein 4). However, precise mechanisms controls gene expression in response to emotional experience remain elusive. Aims & Objectives Recent advances have shown that long-noncoding RNAs, which are much more abundant than mRNAs, are crucial players in gene expression. We found that long-noncoding RNAs (lncRNA) form R-loops, three-stranded nucleic acid structure composed of an DNA:RNA hybrid strand and a displaced DNA strand. Despite the potential importance in various transcriptional, translational, and genomic organization, the physiology and function of R-loops in the context of Substance Use Disorder (SUD), and in the brain, is largely unexplored. This project aims to uncover the functions and mechanisms of lncRNAs and its DNA:RNA hybrid structures, R-loops, in gene regulation and SUD-related drug seeking behaviors. Method We utilized a multi-faced approach in mouse brains, including total RNA-sequencing to detect mRNAs and lncRNAs, short hairpin RNA-mediated gene knockdown approach to reduce target gene expression, DNA:RNA hybrid immunoprecipitation approach to identify R-loop structures, and CRISPR-dCas9-RNaseH1 approach to selectively degrade the DNA:RNA hybrid R-loop structure at specific genomic loci. Results Our findings reveal that a conserved long noncoding enhancer RNA (lnc-eRNA), transcribed from an activity-sensitive enhancer, produces DNA:RNA hybrid R-loop structures that support three-dimensional chromatin looping between enhancer and proximal promoter and rapid Npas4 gene induction. Furthermore, in mouse models, Npas4 lnc-eRNA and its R-loop are required for the development of behavioral adaptations produced by chronic psychosocial stress or cocaine exposure. Discussion & Conclusions Understanding how emotional experiences trigger the rapid induction of IEGs, like Npas4, to support or oppose neural circuit adaptations remains an important topic in neuroscience. Our study demonstrate that a highly conserved Npas4 enhancer element produces a non-annotated lnc-eRNA necessary for the rapid induction of Npas4mRNA transcription in NAc and mPFC following an emotional experience. Moreover, the Npas4eRNA appears to regulate Npas4mRNA expression and behavioral adaptations through the formation of R-loops at the Npas4 enhancer and the facilitation of experience-dependent promoter-enhancer 3D looping leading to rapid Npas4mRNA expression. Our findings of Npas4 lnc-eRNA and its R-loops reveal valuable insights into the complex mechanism of IEG induction and emphasize the critical role of Npas4 in regulating behavioral adaptions associated with SUD and chronic stress. Our work highlights the significant contribution of lncRNAs and R-loops in emotional experience-induced gene expression, offering a novel framework for understanding how gene expression shapes neuronal plasticity and behavior.