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 by environmental stimuli, raising the intriguing possibility that early life experience may impact development by inducing plasticity of the epigenome. Neurons are extremely long-lived cells that, as part of their fundamental function in information processing, undergo robust and dynamic changes in their gene expression repertoires long after they have left the cell cycle and committed to a postmitotic identity. Thus neurons are ideal for studying functions of the dynamic epigenome beyond its role in establishing cell identity.

We have used the differentiation of cerebellar granule neurons (CGNs) in the postnatal brains of male and female mice as a model system for identifying molecular mechanisms of neuronal maturation. The huge abundance and postnatal development of CGNs makes them an ideal cell type for biochemical studies of the molecular mechanisms that guide neuronal differentiation. CGNs account for >99% of all neurons and >85% of all cells in the cerebellar cortex, lowering the barriers to studying a specific cell type developing in vivo. CGNs derive from committed granule neuron precursors (GNPs). Upon leaving the cell cycle these progenitors develop first into immature neurons and then into mature neurons, which can be distinguished by their unique gene expression programs. By comparing multiple stages of CGN differentiation we can identify chromatin mechanisms of gene transcription that operate during differentiation and maturation both in proliferating and in postmitotic cells of the neuronal lineage.

We are using state of the art approaches in chromatin profiling including ATAC-seq, CUT&RUN, CUT&Tag, and the low-input chromatin conformation protocol HiCAR to build a foundational understanding of the regulatory logic of dynamic chromatin states that underlie stages of neuronal maturation.  We can also track the morphological development and circuit integration of newly born CGNs in vivo using in vivo electroporation of granule neuron progenitors to obtain sparse labeling and cell-autonomous genetic manipulation of these neurons.   

Chromatin-regulatory proteins are among the most common classes of genes mutated in autism spectrum disorder (ASD) and intellectual disability (ID). It is well established that cellular differentiation requires the precise orchestration of gene expression programs, which is mediated by dynamic changes to chromatin state and structure. However, it remains unclear why de novo mutations in a subset of chromatin regulators give rise to the specific neurological disruptions that underlie ASD/ID.  Synaptic proteins are the other major class of gene products that show de novo mutations in ASD/ID, and disruption of neuronal connectivity is thought to be a major underlying disease pathology in ASD. Our overarching hypothesis is that there are convergent roles played by multiple ASD/ID-associated chromatin regulators that mediate the orchestrated activation of genes underlying synapse development in postmitotic neurons. Our focus on neurons in the postnatal brain is relevant to therapeutics because by the time ASD/ID is diagnosed in early childhood, there is little that can be done to change fetal development, whereas gene therapy has the potential to correct ongoing deficiencies in neuronal function.

We focus in detail on the biochemical and cellular mechanisms by which ASD/ID-associated mutations in specific chromatin regulators result in impaired neuronal development.

One project examines functions of the lysine demethylase Kdm6b, which remodels the distribution of the repressive histone modification H3K27me3 in developing neurons. We have shown that disruption of the lysine demethylase function of KDM6B impairs synaptic gene expression and synapse development. This is notable given evidence of ASD/ID-associated mutations in KDM6B, including point mutations in the lysine demethylase region.

A second project investigates the biochemical and cellular mechanisms by which ID-associated mutations in the variant linker histone H1.4 impair neuronal development. Histone H1 proteins bind at the entry and exit sites of nucleosomes and contribute to chromatin compaction and 3D architecture. Frame-shift mutations that disrupt the C-terminal of H1.4 cause Rahman Syndrome, a genetic form of ID. We have shown that expression of mutant H1.4 expression in neurons disrupts synaptic gene expression and are working to establish the mechanism.

In the brain, synaptic activity provides a salient stimulus for the adaptation of neuronal function through the induction of stimulus-dependent gene transcription. Distal transcriptional enhancers play both permissive and instructive roles in this process. In their permissive role, the pattern of active enhancers predetermines which genes can be induced in any given kind of neuron, such that distinct programs of activity-regulated gene expression are induced in different types of neurons, even in response to a common stimulus. A functionally important corollary is that different types of neurons adapt in distinct ways to changes in neuronal firing. At the instructive level, enhancers serve as docking sites for activity-inducible transcription factors, thus conferring temporal regulatory activity upon a given gene. 

We have been studying the importance of enhancers in the context of transcriptional responses by drugs of abuse that contribute to the development of addiction. Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome, especially at enhancers, may underlie the persistence of cocaine-induced addictive-like behaviors.

We are testing this hypothesis by using a dCas9/CRISPR based, functional genomics strategy to modify the regulatory state of the enhancer elements of two foundational activity-regulated genes: the neurotrophin Bdnf and the synaptic plasticity gene Arc. We hypothesize that regulating the epigenome at these enhancers will titrate the quantity of activity-induced gene expression to set thresholds for the induction of reward-driven motivated behaviors.

We seek to discover mechanisms that link genome regulation with neuronal and behavioral plasticity. To achieve this goal, we deploy a broad range of experimental approaches including, but not limited to, chromatin sequencing, single molecule fluorescence in situ hybridization, bioinformatic modeling, mouse genetics, and behavior. We also take advantage of novel leading-edge methods that can be applied in new ways to advance understanding of our system. For example we have combined mice carrying cell-type specific transgenes with single nuclear RNA sequencing to identify gene regulatory programs within rare interneuron populations that reside in physiologically relevant neural circuits of behaving mice.

Ongoing projects in the lab are pioneering the use of multiple new technologies in neurons including the following:

1) To understand the three-dimensional organization of genomic DNA in neurons, we are using a novel low-input chromatin sequencing method called HiCAR that was developed by our colleague Yarui Diao in the department of Cell Biology. Because HiCAR is more efficient than standard HiC, we have been able to assess the dynamics of genome architecture changes in neurons across developmental stages, in response to drug treatments that disrupt epigenome features, and in genetic models of neurodevelopmental disorders. See data in project on chromatin regulation of postmitotic neuronal maturation.

2) To experimentally edit the epigenome and then determine the consequences for neuronal and network function in vivo, we are using dCas9-CRISPR based mouse strains engineered by our colleague Charlie Gersbach in the department of Biomedical Engineering. We are also developing new dCas9 fusions to expand the ways that we can locally modify transcription along the genome. See Gemberling et al. (2021) Nat Methods 18: 965-74.  

3) To study the function of chromatin regulators in the context of the human genome, we are expanding our experimental systems to include NGN2-induced human iPSC-derived neurons. The ease of generating large numbers of relatively homogenous neurons from genetically manipulatable iPSCs makes these cells ideal for studying the functional consequences of ASD/ID-associated gene mutations using sequencing assays and molecular genetic screens. We are also using these cells to study human-specific gene regulatory elements.