Reprogramming KZFP function to understand drug-specific transcription and behavior - PROJECT SUMMARY In this revised R01, we propose to use novel synthetic transcription factors (TFs) delivered to the mouse nucleus accumbens (NAc), a key brain reward region, to uncover and manipulate the cell-type-specific brain molecular processes that distinguish stimulant versus opioid addiction. This will enable us to generate knowledge critical for the design of the next generation of drug-specific, anti-addiction medications. An existing body of literature suggests that, in response to use of addictive drugs, the function of key TFs within the cells that comprise brain reward regions drive transcriptional adaptations and lasting changes in drug use behaviors. Preliminary and published data point to NAc ZFP189, a member of the KRAB-domain containing zinc-finger (KZFP) TF family, as highly sensitive to cocaine use, and causal in worsening stimulant- but not opioid-related behaviors. This suggests that the NAc ZFP189 TF is uniquely sensitive to, and contributes to, the brain adaptations that worsen stimulant addiction. Here, we have reprogrammed ZFP189 to create synthetic TFs, each possessing functional moieties that exert distinct forms of transcriptional control at in vivo target genes. This enables us to uncover how the stimulant versus opioid use experience differentially primes the epigenetic status of NAc cells to facilitate the function of drug-specific TFs. In Aim 1 we will virally deliver synthetic ZFP189 TFs to the NAc of mice, dose with repeated stimulant or opioid treatments, and perform multiome single nuclei ATAC and RNA sequencing. This will reveal the biological mechanisms through which stimulant versus opioid exposure enables drug-specific TF function, the transcriptional adaptations that facilitate specific drug addictions, and the NAc cell-types in which this occurs. In Aim 2, we will virally deliver synthetic ZFP189 TFs to mouse NAc and investigate their contribution to stimulant versus opioid conditioned and drug self- administration reward-related behaviors. This will reveal how drug-specific transcriptional neuroadaptations may differentially drive a worsening of stimulant- versus opioid-related behaviors. Lastly, in Aim 3, we will introduce novel synthetic TF co-factors, capable of re-programming the in vivo gene-regulatory function of the entire KZFP TF family, to interrogate how distinct KZFP members are recruited to worsen stimulant- versus opioid addiction. To accomplish this, we will virally deliver variants of the KZFP co-factor, TRIM28, to simultaneously dysregulate members of the KZFP TF family within the NAc. We will perform self-administration for stimulants and opioids and subject tissues to single nuclei RNA sequencing. This will uncover the collective contribution of NAc KZFPs to the worsening of stimulant and opioid drug behaviors and reveal the KZFPs, and their regulated genes, that most drive these damaging effects. Together, this research will provide new approaches to identify the TF functions at the core of specific drug addictions and yield refined gene candidates as targets for future drug-specific medications.
