Scientific Core

1/1 U24 NADIA Scientific Core: Epigenetic/Molecular Core

Principal Investigator: Subhash C. Pandey, Ph.D., University of Illinois Chicago

Abstract:

Alcohol is one of the most widely used addictive drugs in adolescence, and continued use can lead to the development of psychiatric disorders, including alcoholism, in adulthood. Epigenetic processes, such as histone acetylation and DNA methylation mechanisms, have been shown to play a role in neuromaturation by contributing to the stability of gene expression during brain development. The Neurobiology of Adolescent Drinking in Adulthood (NADIA) consortium has revealed that adolescent intermittent ethanol (AIE) exposure produces epigenetic reprogramming in several brain circuits (prefrontal cortex, basal forebrain, hippocampus and amygdala) that persists until adulthood and might be responsible for adult psychopathology. These findings prompt determination of the global epigenome to determine AIE altered specific gene loci within key brain regions. The Epigenetic/Molecular Core is developed to evaluate epigenetic mechanisms in specific brain regions linked to key AIE phenotypes in adulthood. The Epigenetic/Molecular Core will also develop tools for CRISPR/dCas9 approaches for epigenetic editing of target genes to directly link AIE-induced behavioral phenotypes with molecular mechanisms. The objectives of the Core are to provide tools and scientific expertise to test NADIA’s hypotheses that perturbations of epigenetic targets due to AIE may lead to dynamic changes in epigenetic programming leading to transcriptomic changes in key brain areas (prefrontal cortex, amygdala, hippocampus, hypothalamus, and basal forebrain) which are responsible for persistent behavioral and molecular phenotypes in adulthood. These objectives will be achieved with the following proposed work: 1) To examine the status of the epigenome at the whole genome level. We will perform Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) to determine the locations of open and closed chromatin domains in different brain regions studied across NADIA. The core will provide information on global chromatin states during AIE to each component and identify “hub” genes related to the component’s hypotheses for functional studies. 2) To determine gene specific AIE-induced epigenetic modifications (histone acetylation/methylation/DNA methylation) associated with changes in gene expression. 3) To provide gene specific CRISPR/dCas9 tools for NADIA components. The core will develop and test CRISPR/dCas9 technologies to make epigenetic editing (activating or silencing) at specific gene locations and evaluate both functional and behavioral consequences. These studies will provide a better understanding of AIE-mediated changes in gene expression via epigenetic reprogramming across NADIA components. In addition, we will be able to identify mechanisms that may lead to the development of novel treatment strategies for adult psychopathologies resulting from adolescent binge drinking.

Specific Aims:

Adolescent alcohol exposure represents a risk factor for the development of psychiatric disorders including anxiety, depression, cognitive deficits and alcohol use disorder (AUD) later in life (1,2). The Neurobiology of Adolescent Drinking in Adulthood (NADIA) consortium has revealed, during the current funding period, that adolescent intermittent ethanol (AIE) exposure produces epigenetic reprogramming in several brain circuits (prefrontal cortex, basal forebrain, hippocampus and amygdala) that persists into adulthood and might be responsible for adult psychopathology (1,3). These findings prompt investigation of the global epigenome to determine altered specific gene loci within key brain regions in adulthood after AIE. The Epigenetic/Molecular Core will evaluate epigenetic mechanisms in specific brain regions linked to key AIE phenotypes in adulthood. While each individual research project has its own focused hypotheses and goals, all support a shared conceptual molecular-epigenetic framework. This approach will promote integration of ideas, experimental approaches and findings in epigenetic mechanisms of AIE-induced behavioral phenotypes in adulthood. The Epigenetic/Molecular Core will also develop tools for CRISPR/dCas9 approaches for epigenetic editing of target gene to directly link AIE-induced behavioral phenotypes with molecular mechanisms.

The Epigenetic/ Molecular Core of the NADIA will continue to serve as a crucial resource by facilitating the interactive research of each project through investigating alterations in the locations of key epigenetic marks across the genome and identifying domains of open and closed chromatin which, in turn, mediate the changes in gene expression and behavioral phenotypes produced by AIE in adulthood. The objectives of the Core are to provide tools and scientific expertise to test NADIA’s hypotheses that perturbations of epigenetic targets due to AIE may lead to dynamic changes in epigenetic programming leading to transcriptomic changes in key brain areas (prefrontal cortex, amygdala, hippocampus, hypothalamus, and basal forebrain) which are responsible for persistent behavioral and molecular phenotypes in adulthood.

The following Specific Aims will achieve the objectives of the Epigenetic/Molecular Core.

Aim 1: To examine the status of the epigenome at the whole genome level. We will perform Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) to determine the locations of open and closed chromatin domains in different brain regions studied across NADIA. The Core will receive specific brain regions from each NADIA research component and perform ATAC-seq with detailed bioinformatic analyses to assess AIE induced changes in chromatin and identify AIE changes in gene networks. The core will provide information on global chromatin states during AIE to each component and identify gene networks and “hub” genes related to the component’s hypotheses. Epigenetic mechanisms related to each components’ physiological and behavioral hypotheses will then be performed. All data will be fed back to NADIA components.

Aim 2: a) To determine gene specific AIE-induced epigenetic modifications (histone acetylation/methylation/DNA methylation) associated with altered gene expression for already identified candidate genes as well as those identified by ATAC-seq. b) To assess AIE-induced changes in mRNA levels of newly identified target genes using quantitative real-time PCR (RT-qPCR). c) To perform additional epigenetic analysis of genes with or without pharmacological manipulations of control and AIE adult rats for the components. The Core will receive brain regions from each research component and assess gene-specific (promoter, enhancer, and intergenic region) changes in histone acetylation/methylation and DNA methylation using chromatin immunoprecipitation (ChIP) assays for histone modifications [H3K9 acetylation/methylation (H3K9ac, H3K9me2)], H3K27 acetylation/methylation (H3K27ac, H3K27me3) and DNA methylation (MethylMiner™ assay).

Aim 3. To provide gene specific CRISPR/dCas9 tools for components. The core will develop and test CRISPR/dCas9 technologies to make epigenomic modifications (activating or silencing) at specific gene locations and evaluate both functional and behavioral consequences. The core will use a broad suite of dCas9- effectors fusion proteins that will target several different histone marks (H3K9ac, H3K27ac, H3K27me3, H3K9me2 using CRISPR/dCas9-p300, CRISPR/dCas9-KRAB, and CRISPR/dCas9-Lsd1, respectively) and DNA methylation regulators (CRISPR/dCas9-Tet1 or CRISPR/dCas9-Dnmt3b). Important neuronal and glial genes that have already been identified by each component will be tested first along with novel genes emerging from ATAC-seq. This will likely reveal novel AIE mechanisms that are altering neurocircuits and their resulting behavioral phenotypes.

Through an examination of AIE-mediated changes in gene expression via epigenetic reprogramming across NADIA components and neurocircuits, we will be able to identify mechanisms that may lead to the development of new treatment strategies for adult psychopathologies resulting from adolescent binge drinking.