Biology subjects

Sultan, F. A.

Publications and source records attributed to Sultan, F. A..

5 recordsLinked to original sources

bioRxiv2020

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

neuroscience↗

bioRxiv2020

Regulation of dopamine-dependent transcription and cocaine action by Gadd45b

Exposure to drugs of abuse produces robust transcriptional and epigenetic reorganization within brain reward circuits that outlives the direct effects of the drug and may contribute to addiction. DNA methylation is a covalent epigenetic modification that is altered following stimulant exposure and is critical for behavioral and physiological adaptations to drugs of abuse. Although activity-related loss of DNA methylation requires the Gadd45 (Growth arrest and DNA-damage-inducible) gene family, very little is known about how this family regulates the activity of brain reward circuits or behavioral responses to drugs of abuse. Here, we combined genome-wide transcriptional profiling, pharmacological manipulations, electrophysiological measurements, and CRISPR tools with traditional knockout and behavioral approaches in rodent model systems to dissect the role of Gadd45b in dopamine-dependent epigenetic regulation and cocaine reward. We show that acute cocaine administration induces rapid upregulation of Gadd45b mRNA in the rat nucleus accumbens, and that knockout or site-specific CRISPR/Cas9 gene knockdown of Gadd45b blocks cocaine conditioned place preference. In vitro, dopamine treatment in primary striatal neurons increases Gadd45b mRNA expression through a dopamine receptor type 1 (DRD1)-dependent mechanism. Moreover, shRNA-induced Gadd45b knockdown decreases expression of genes involved in psychostimulant addiction, blocks induction of immediate early genes by DRD1 stimulation, and prevents DRD1-mediated changes in DNA methylation. Finally, we demonstrate that Gadd45b knockdown decreases striatal neuron action potential burst duration in vitro, without altering other electrophysiological characteristics. These results suggest that striatal Gadd45b functions as a dopamine-induced gene that is necessary for cocaine reward memory and DRD1-mediated transcriptional activity.

neuroscience↗

bioRxiv2019

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged high-throughput single-nucleus RNA-sequencing to create a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.

neuroscience↗

bioRxiv2018

A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation

Recent developments in CRISPR-based gene editing have provided new avenues to interrogate gene function. However, application of these tools in the central nervous system has been delayed due to difficulties in transgene expression in post-mitotic neurons. Here, we present a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system to drive gene expression in primary neuronal cultures and the adult brain of rodent model systems. We demonstrate robust, modular, and tunable induction of endogenous target genes as well as multiplexed gene regulation necessary for investigation of complex transcriptional programs. CRISPRa targeting unique promoters in the complex multi-transcript gene Brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

neuroscience↗

bioRxiv2018

Enhancer RNAs are necessary and sufficient for activity-dependent neuronal gene transcription

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined ATAC-Seq and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR- based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CBP. Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

neuroscience↗