Robust, scalable, and informative clustering for diverse biological networks.

Clustering molecular data into informative groups is a primary step in extracting robust conclusions from big data. However, due to foundational issues in how they are defined and detected, such clusters are not always reliable, leading to unstable conclusions. We compare popular clustering algorithms across thousands of synthetic and real biological datasets, including a new consensus clustering algorithm-SpeakEasy2: Champagne. These tests identify trends in performance, show no single method is universally optimal, and allow us to examine factors behind variation in performance. Multiple metrics indicate SpeakEasy2 generally provides robust, scalable, and informative clusters for a range of applications.

Gadd45 in Neuronal Development, Function, and Injury.

The growth arrest and DNA damage-inducible (Gadd) 45 proteins have been associated with numerous cellular mechanisms including cell cycle control, DNA damage sensation and repair, genotoxic stress, neoplasia, and molecular epigenetics. The genes were originally identified in in vitro screens of irradiation- and interleukin-induced transcription and have since been implicated in a host of normal and aberrant central nervous system processes. These include early and postnatal development, injury, cancer, memory, aging, and neurodegenerative and psychiatric disease states. The proteins act through a variety of molecular signaling cascades including the MAPK cascade, cell cycle control mechanisms, histone regulation, and epigenetic DNA demethylation. In this review, we provide a comprehensive discussion of the literature implicating each of the three members of the Gadd45 family in these processes.

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 activity within the nucleus accumbens 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.

Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems.

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 Assay for Transposase-Accessible Chromatin using Sequencing (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 CREB-binding protein (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.

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

Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. Here, we leveraged single-nucleus RNA-sequencing to generate 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 an immediate early gene expression program that is up-regulated following cocaine experience in vivo and dopamine receptor activation in vitro. Multiplexed induction of this gene program with a large-scale CRISPR-dCas9 activation strategy initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Together, these results define the transcriptional response to cocaine with cellular precision and demonstrate that drug-responsive gene programs can potentiate both physiological and behavioral adaptations to drugs of abuse.

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 human synapsin promoter 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.

A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. 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.

A Novel Dual Lentiviral CRISPR-based Transcriptional Activation System for Gene Expression Regulation in Neurons.

Robust and efficient gene expression control enables the study of a gene's function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we describe a protocol to generate high-titer lentiviruses with neuron-optimized CRISPR-activation constructs (dual lentiviruses consisting of a gene-specific single guide RNA and the CRISPR-activator) for use in primary neurons in vitro or in the adult brain in vivo. This protocol enables modular, scalable, and multiplexable gene regulation in the nervous system and does not require a transgenic model organism.

The role of the Gadd45 family in the nervous system: a focus on neurodevelopment, neuronal injury, and cognitive neuroepigenetics.

The growth arrest and DNA damage-inducible (Gadd)45 proteins have been associated with numerous cellular mechanisms including cell-cycle control, DNA damage sensation and repair, genotoxic stress, neoplasia, and molecular epigenetics. The genes were originally identified in in vitro screens of irradiation- and interleukin-induced transcription and have since been implicated in a host of normal and aberrant central nervous system processes. These include early and postnatal development, injury, cancer, memory, aging, and neurodegenerative and psychiatric disease states. The proteins act through a variety of molecular signaling cascades including the MAPK cascade, cell-cycle control mechanisms, histone regulation, and epigenetic DNA demethylation. In this review, we provide a comprehensive discussion of the literature implicating each of the three members of the Gadd45 family in these processes.

Dissection of Different Areas from Mouse Hippocampus.

The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate gyrus, CA3 and CA1 (here, we do not distinguish the "hippocampus proper" which consists only of CA1, CA3 and smaller CA2 and CA4 areas, from the "hippocampal formation," composed of these in addition to the dentate gyrus and subiculum). Distinct cell types give rise to unique axonal fiber pathways in the dentate gyrus, CA3 and CA1 subregions; accordingly, these areas may exhibit differential molecular profiles in response to a number of behavioral paradigms and pharmacological and genetic treatments. It is therefore in the interest of the investigator to dissect a specific subregion from the whole hippocampus. Here we outline a protocol for subregion-specific dissection from the adult mouse.

Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

Genetic deletion of Gadd45b, a regulator of active DNA demethylation, enhances long-term memory and synaptic plasticity.

Dynamic epigenetic mechanisms including histone and DNA modifications regulate animal behavior and memory. While numerous enzymes regulating these mechanisms have been linked to memory formation, the regulation of active DNA demethylation (i.e., cytosine-5 demethylation) has only recently been investigated. New discoveries aim toward the Growth arrest and DNA damage-inducible 45 (Gadd45) family, particularly Gadd45b, in activity-dependent demethylation in the adult CNS. This study found memory-associated expression of gadd45b in the hippocampus and characterized the behavioral phenotype of gadd45b(-/-) mice. Results indicate normal baseline behaviors and initial learning but enhanced persisting memory in mutants in tasks of motor performance, aversive conditioning and spatial navigation. Furthermore, we showed facilitation of hippocampal long-term potentiation in mutants. These results implicate Gadd45b as a learning-induced gene and a regulator of memory formation and are consistent with its potential role in active DNA demethylation in memory.

Epigenetic mechanisms in memory and synaptic function.

Although the term 'epigenetics' was coined nearly seventy years ago, its critical function in memory processing by the adult CNS has only recently been appreciated. The hypothesis that epigenetic mechanisms regulate memory and behavior was motivated by the need for stable molecular processes that evade turnover of the neuronal proteome. In this article, we discuss evidence that supports a role for neural epigenetic modifications in the formation, consolidation and storage of memory. In addition, we will review the evidence that epigenetic mechanisms regulate synaptic plasticity, a cellular correlate of memory. We will also examine how the concerted action of multiple epigenetic mechanisms with varying spatiotemporal profiles influence selective gene expression in response to behavioral experience. Finally, we will suggest key areas for future research that will help elucidate the complex, vital and still mysterious, role of epigenetic mechanisms in neural function and behavior.