Early life exercise primes the neural epigenome to facilitate gene expression and hippocampal memory consolidation

Abstract

Aerobic exercise promotes physiological and molecular adaptations in neurons to influence brain function and behavior. The most well studied neurobiological consequences of exercise are those which underlie exercise-induced improvements in hippocampal memory, including the expression and regulation of the neurotrophic factor Bdnf. Whether aerobic exercise taking place during early-life periods of postnatal brain maturation has similar impacts on gene expression and its regulation remains to be investigated. Using unbiased next-generation sequencing we characterize gene expression programs and their regulation by specific, memory-associated histone modifications during juvenile-adolescent voluntary exercise (ELE). Traditional transcriptomic and epigenomic sequencing approaches have either used heterogeneous cell populations from whole tissue homogenates or flow cytometry for single cell isolation to distinguish cell types / subtypes. These methods fall short in providing cell-type specificity without compromising sequencing depth or procedure-induced changes to cellular phenotype. In this study, we use simultaneous isolation of translating mRNA and nuclear chromatin from a neuron-enriched cell population to more accurately pair ELE-induced changes in gene expression with epigenetic modifications. We employ a line of transgenic mice expressing the NuTRAP (Nuclear Tagging and Translating Ribosome Affinity Purification) cassette under the Emx1 promoter allowing for brain cell-type specificity. We then developed a technique that combines nuclear isolation using Isolation of Nuclei TAgged in Specific Cell Types (INTACT) with Translating Ribosomal Affinity Purification (TRAP) methods to determine cell type-specific epigenetic modifications influencing gene expression programs from a population of Emx1 expressing hippocampal neurons. Data from RNA-seq and CUT&RUN-seq were coupled to evaluate histone modifications influencing the expression of translating mRNA in neurons after early-life exercise (ELE). We also performed separate INTACT and TRAP isolations for validation of our protocol and demonstrate similar molecular functions and biological processes implicated by gene ontology (GO) analysis. Finally, as prior studies use tissue from opposite brain hemispheres to pair transcriptomic and epigenomic data from the same rodent, we take a bioinformatics approach to compare hemispheric differences in gene expression programs and histone modifications altered by by ELE. Our data reveal transcriptional and epigenetic signatures of ELE exposure and identify novel candidate gene-histone modification interactions for further investigation. Importantly, our novel approach of combined INTACT/TRAP methods from the same cell suspension allows for simultaneous transcriptomic and epigenomic sequencing in a cell-type specific manner.

“SIT” with Emx1‐NuTRAP Mice: Simultaneous INTACT and TRAP for Paired Transcriptomic and Epigenetic Sequencing

“…We have used this method to compare transcriptomic data to epigenomic data from the same set of cells in an experiment to determine how voluntary aerobic exercise in early life might enable memory in adolescence (Raus et al., 2022 ). Fundamentally, using this transgenic mouse and this technique, we compared transcriptomic and epigenomic data from the same set of hippocampal excitatory neurons.…”

“…However, an animal expressing this cassette specifically in hippocampal excitatory neurons had yet to be generated. We study the interaction between epigenetic regulation and transcription in the brain after early life experiences (Raus et al., 2022 ), so we developed a mouse and a method to investigate these connected regulatory networks simultaneously. By crossing an Emx1 ‐Cre mouse (Gorski et al., 2002 ) with the NuTRAP mouse (Roh et al., 2017 ), we generated a transgenic mouse line that labels excitatory neurons in the hippocampus with both nuclear (mCherry and biotin) and ribosomal (GFP) tags.…”

Cell-Type Specific Profiling of Histone Post-Translational Modifications in the Adult Mouse Striatum

“…We found that ICuRuS generated robust and reproducible H3K4me3 and H3K27me3 profiles at sufficient depth to examine cell-type specific chromatin from a single mouse striatum. These findings are an improvement over current methods, such as bulk tissue CnR 7,40 , which obscures cell-type specificity, and single-cell CnR, which requires pooled brain tissue and obscures subject variability 4,5 , a critical barrier given individual differences in stimulus-induced gene expression and susceptibility to psychiatric disease 33,[50][51][52] . Recent studies have applied FACS to isolate MSNs, but we found that the affinity purification strategy used in ICuRuS (and in the original INTACT protocol 9 ) potentially avoids FACS induced ectopic upregulation of activity dependent genes [53][54][55] .…”

“…Bulk tissue analysis reflects the average of cell-type specific changes, which impedes the interpretability of causal relationships between epigenetic modifications, regulation of gene expression and behavior 1-3 . Cell-type specific RNA-seq allows for the deconvolution of bulk tissue RNA-seq data, but cell-type specific epigenomic profiling in brain is only emerging [4][5][6][7] . Fortunately, recent methodological advances facilitate cell-type specific epigenetic profiling in brain [8][9][10][11] .…”