west(at)neuro.duke.edu
Epigenomics of Neuronal Development & Maturation
Cellular differentiation requires the precise spatial and temporal orchestration of gene expression programs. Progressive changes in gene transcription during development are driven by epigenetic modifications of genomic DNA and its associated histone proteins, collectively called chromatin, that differentially alter the access of DNA regulatory sequences to the transcriptional machinery. A growing body of evidence shows that chromatin-regulatory proteins can be modulated in neurons by environmental stimuli, raising the intriguing possibility that early life experience may impact brain development by inducing plasticity of the neuronal epigenome. However it is largely unknown when during development neuronal chromatin is subject to epigenetic regulation, where in the genome the key gene regulatory elements are located that mediate neuronal differentiation, and to what extent chromatin structure and state can be modulated by extrinsic stimuli in postmitotic neurons.
We are using state of the art approaches in chromatin profiling such as CUT and RUN, CUT and Tag, HiCAR and Epigenome-editing (among others), to answer some of these questions. Some of the key areas of interest within this space include investigating 1) Polycomb Repressive Complex (PRC2) activity in post-mitotic cerebellar granule neuron maturation, 2) the role of linker histone H1-4 in gene regulation and neuronal maturation, 3) Chromatin remodeling in response to Brain-derived neurotrophic factor (BDNF)-stimulation during neuronal development, 4) The role of chromatin regulators in orchestrating synaptic refinement and plasticity
Frank et al. Nature Neuroscience (2015)
Epigenomic Regulation of Addiction &
Reward-Seeking Behavior
Neurons respond to stimuli in lasting ways, supporting changes at the synapse or the axon by also changing their gene expression. Stimulus-dependent transcription can be influenced by the subtype of neuron, the specific stimulus, or - particularly in the case of addiction - how many times and how recently a neuron has seen a given stimulus.
Addiction is characterized by diverse, lasting changes in the ways different cell types respond to drugs of abuse, as well as naturally rewarding stimuli. We study this process within the Nucleus Accumbens to understand how initial and repeated drug experience can change transcription in diverse cell types, and to functionally test how the underlying mechanisms contribute to altered behavior. To access rare cell types, we use a combination of mouse genetic tools, viruses, and nuclear purification. Epigenetic landscapes and transcriptomes are assayed using next-generation sequencing methods, and specifically manipulated using dCas9-mediated epigenomic editing approaches. Downstream target genes and their protein products are assessed using qRT-PCR, RNAscope FISH, immunostaining, and immunoblotting, and are manipulated using a variety of viral and genetic strategies.
Gallegos et al. Molecular Psychiatry (2022)
Non-Coding Regulatory Element Function in
Activity -Dependent Transcription & Neuronal Maturation
Sensory experience leads to long-term changes in neuronal structure and function, which are important for learning and memory. These long-term changes occur through activity-dependent gene regulation. Activity-inducible genes allow neurons to dynamically respond to stimuli by coupling synaptic activity to the nucleus. This activity-dependent regulation can occur via various gene regulatory elements such as promoters and enhancers. Enhancers are stretches of DNA which, when paired in cis with the promoter, can upregulate gene expression. They are thought to confer cell-type, developmental, and stimulus-specificity to their target genes. Enhancers of activity-dependent genes are only active following neuronal activation, and therefore are thought to confer the stimulus-dependent specificity of these genes. We are currently interested in studying this aspect of enhancers by using and developing CRISPR-based epigenome editing tools to manipulate the activity of enhancers of activity-dependent genes.
Another type of non-coding regulatory element contribution to gene regulation is
Gemberling et al. Nature Methods (2021)