Glucocorticoid Receptor-Regulated Enhancers Play a Central Role in the Gene Regulatory Networks Underlying Drug Addiction

Abstract

Substance abuse and addiction represent a significant public health problem that impacts multiple dimensions of society, including healthcare, the economy, and the workforce. In 2021, over 100,000 drug overdose deaths were reported in the US, with an alarming increase in fatalities related to opioids and psychostimulants. Understanding the fundamental gene regulatory mechanisms underlying addiction and related behaviors could facilitate more effective treatments. To explore how repeated drug exposure alters gene regulatory networks in the brain, we combined capped small (cs)RNA-seq, which accurately captures nascent-like initiating transcripts from total RNA, with Hi-C and single nuclei (sn)ATAC-seq. We profiled initiating transcripts in two addiction-related brain regions, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), from rats that were never exposed to drugs or were subjected to prolonged abstinence after oxycodone or cocaine intravenous self-administration (IVSA). Interrogating over 100,000 active transcription start regions (TSRs) revealed that most TSRs had hallmarks of bonafide enhancers and highlighted the KLF/SP1, RFX, and AP1 transcription factors families as central to establishing brain-specific gene regulatory programs. Analysis of rats with addiction-like behaviors versus controls identified addiction-associated repression of transcription at regulatory enhancers recognized by nuclear receptor subfamily 3 group C (NR3C) factors, including glucocorticoid receptors. Cell-type deconvolution analysis using snATAC-seq uncovered a potential role of glial cells in driving the gene regulatory programs associated with addiction-related phenotypes. These findings highlight the power of advanced transcriptomics methods to provide insight into how addiction perturbs gene regulatory programs in the brain.

Enhancers associated with unstable RNAs are rare in plants

“…1a, (Duttke et al 2019). csRNA-seq is a simple, scalable and cost-efficient protocol that uses 1-3 ug of total RNA, rather than purified nuclei, as input, and is compatible with any fresh, frozen, fixed, or pathogenic species or tissues (Lim et al 2021;Branche E 2022;Duttke et al 2022b;Lam et al 2023). Recently, csRNA-seq was shown to effectively detect eRNAs in human lymphoblast cells (Yao et al 2022).…”

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats

“… 30 NAcc, 75 samples csRNA-seq data Duttke et al. 75 N/A Annotation of the mRatBN7.2 assembly O’Leary et al. 76 Release 108 Code and computational notebook This paper https://doi.org/10.5281/zenodo.10552887 Experimental models: Organisms/strains Sample metadata for RatCollection This paper https://doi.org/10.5281/zenodo.10552790 Software and algorithms QUAST Mikheenko et al.…”

Cocaine addiction-like behaviors are associated with long-term changes in gene regulation, energy metabolism, and GABAergic inhibition within the amygdala

“…For snATAC-seq library preparations, nuclei were purified from frozen amygdala tissues using an established method 118 .Briefly, frozen amygdala tissue was homogenized using a 2 ml glass dounce with 1 ml cold 1× Homogenization Buffer (HB). The cell suspension was filtered using a 70 μm Flowmi strainer (BAH136800070, Millipore Sigma) and centrifuged at 350g for 5 min at 4°C.…”

“…cDNA was amplified for 12 PCR cycles. For snATAC-seq library preparations, nuclei were purified from frozen amygdala tissues using an established method 118 . Briefly, frozen amygdala tissue was homogenized using a 2 ml glass dounce with 1 ml cold 1x Homogenization Buffer (HB).…”