Rose Marie Akiki, Sierra Simmerson, Caila Worley, Rebecca G Cornbrooks, Kosuke Magami, Alain Greige, Kristen K Snyder, Daniel J. Wood, Mary Claire Herrington, Philip Mace, Kyle Blidy, Nobuya Koike, Stefao Berto, Christopher W. Cowan, Makoto Taniguchi

Emotional experiences often drive behavioral responses that enable organisms adapt to their environment. These experiences trigger the expression of specific gene, initiating transcriptional programs that underlie cellular, synaptic, and behavioral plasticity. This gene expression is tightly regulated and plays critical role in adaptive responses to emotional stimuli. Conversely, dysregulation of these transcriptional program is implicated in maladaptive behaviors associated with neuropsychiatric disorders, including mood and substance use disorder (SUD). We recently demonstrated that long non-coding RNAs (lncRNAs) form DNA:RNA hybrid structure, R-loops, across the genome. Genes associated with these R-loops are identified in the glutamatergic and dopaminergic signaling pathways, calcium signaling pathways, and synaptic plasticity-related genes. We discovered that neuronal activity induces the formation of R-loop at the enhancer region of immediate early gene, Npas4, a gene that we previously identified as a key regulator of stress-induced anhedonia-like behavior and drug reward-associated contextual memory. This R-loop is essential for linking distal enhancer to the proximal promoter, facilitating Npas4 gene expression, and regulating cocaine reward-associated contextual memory. Together, these data identify R-loops as novel regulatory mechanism that translates emotional experience into gene expression and behavioral plasticity. In this presentation, we will discuss the functional mechanisms of R-loops in gene expression and behavior in response to emotional experiences.

Department of Neuroscience, Medical University of South Carolina

Funding support: NIH_T32 DA07288, R01 DA032708, R01 MH129521, P20 GM140964, P20 GM148302, P50 DA046373, and UL1 TR001450.

Brad Balderson1 , Narayan Pokhrel2 , Arnav Gurha2 , Yanning Zuo2 , Benjamin Johnson2 , Olivier George2 , Abraham A. Palmer2,3 , Graham McVicker1 , Francesca Telese2

The nucleus accumbens (NAc) is a key subcortical brain structure that regulates reward and is involved in addiction. However, the molecular basis of individual differences in addiction phenotypes are not well characterized. To dissect the molecular basis of oxycodone addiction, we used an intravenous self administration assay (IVSA) to measure oxycodone addiction-like behaviours in outbred Heterogeneous Stock (HS) rats. NAc tissues were collected from 85 HS rats after 5 weeks of abstinence from oxycodone. We obtained whole genome sequencing, and also 10X multi-ome from 500,000 single-cell nuclei. Latent factor analysis on gene expression and chromatin accessibility across 14 NAc cell types identified a molecular pattern of changes significantly associated with oxycodone in-take (factor of oxycodone, FOXY). A major driver of FOXY was the abundance of Drd3-expressing medium spiny neurons (MSNs), with higher abundance associated with increased oxycodone in-take. We also identified gene expression and chromatin accessibility changes in Grm8-expressing MSNs and Chat expressing interneurons as a major component of FOXY. These cell types exclusively express the opioid receptor Oprm1 in the NAc. To evaluate if FOXY was genetically driven, we called cis-acting eQTLs and caQTLs across all cell types, and trained Predixscan models to predict gene expression and chromatin accessibility for identified eGenes and caPeaks in each cell type. These cis-variant predicted expressions indicated 18% of the variance in FOXY could be explained by genetic differences between individuals, and this variance was marginally predictive of oxycodone-intake (R=0.18, p=0.14). Overall, we identified a pattern of molecular and cell abundance changes in the NAc associated with oxycodone in-take, and at least 18% of the variance in this pattern was explained by genetic differences between individuals.

1 Salk Institute for Biological Studies, Integrative Biology Laboratory, La Jolla, CA 2 Department of Psychiatry, University of California San Diego, La Jolla, CA, USA 3 Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA

Ana Pombo

Midbrain dopamine neurons (DNs) are the first responders to the acute exposure of cocaine and are central to the development of drug addiction. Following a single cocaine injection, DNs undergo synaptic and transcriptional changes that are resolved within days but preserve a long-term cellular memory of the exposure by a mechanism that remains unknown. To investigate whether persistent alterations in 3D genome organization are involved in the cellular memory of an acute drug exposure, we applied Genome Architecture Mapping (GAM), single nucleus transcriptomics (snRNA-seq) and chromatin accessibility mapping (snATAC-seq) in the mouse midbrain 24 hours or 14 days after a single injection of cocaine or saline control. We found that DNs retain extensive chromatin reorganization 14 days after a single exposure across multiple genomic scales, including topologically associating domains (TADs), chromatin condensation and looping interactions, which affect genes previously associated with chronic cocaine addiction, synaptic plasticity and metabolic adaptation.To assess whether altered 3D genome structure 14 days post exposure has consequences for the transcriptional responses to cocaine, we re-exposed animals to a second injection of cocaine after 14 days of abstinence. Transcriptional responses were more extensive and often did not match the direction or intensity of changes seen upon the first exposure. Importantly, transcriptional responses following re-injection occurred predominantly at genes with reorganized chromatin architecture. Together, these findings identify 3D genome remodelling as a key mechanism underlying cellular memory of cocaine exposure, providing new insight into the earliest molecular events in addiction and the plasticity of the genome.

Max Delbrück Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Epigenetic Regulation and Chromatin Architecture Group, Berlin, Germany.

Department of Biology and Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD, USA.

Gabriella M. Silva¹, Joseph A. Picone¹, Annalise Hassan¹, Natalie L. Truby¹, R. Kijoon Kim¹, Corinne Smith¹, Shelbey S. Strandberg¹, Jessica L. Bell¹, Xiaohong Cui¹, Theingi Aung¹, Peter J. Hamilton¹

Zfp189, which encodes a Krüppel-associated box zinc finger protein (KZFP) transcription factor (TF), differentially accumulates in rodent cortical and limbic brain regions in response to stress- or drug-use experience. Here, we aimed to illuminate the brain cell-type specific molecular mechanisms through which this and other KZFP TFs produce cocaine-related brain changes, with emphasis on investigating transposable elements (TEs), since KZFPs like ZFP189 are known regulators of TEs. To investigate this, we quantified TE transcripts in existing single nuclei and bulk RNA-sequencing datasets of rodents exposed to cocaine. TE transcripts often go un-analyzed in transcriptomic data due to multi-mapping reads. We identified dynamic TE transcript expression across cocaine exposure, especially in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes. To directly regulate brain TEs, we created novel synthetic ZFP189 TFs and synthetic KZFP-interacting TRIM28, each capable of exerting distinct forms of transcriptional control at KZFP-regulated genes, including TEs. These KZFP TFs were virally delivered to the NAc of mice, including conditional delivery to MSNs, and the consequences on cocaine-related behaviors, including intravenous self-administration, and transcriptional response, by bulk and snRNAseq, were characterized. We discover that normal KZFP function in brain is critical to produce cocaine-induced NAc transcription and an escalation of cocaine-taking behaviors, and this can be impeded with synthetic KZFP TFs. Our synthetic KZFPs release TEs that form cis-regulatory contacts with down-regulated, predominantly immune-related genes. Collectively this work points to the KZFP-mediated transcriptional repression of brain TEs as an important mechanistic step in cocaine-induced gene expression and behavioral changes.

¹Department of Neuroscience and Anatomy; Virginia Commonwealth University School of Medicine

Funding from NIH grants: R01DA058089, R01DA058958