Single-cell approaches to probe the function of the unique neuronal epigenome

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Abstract The recent explosion of single cell techniques has revealed striking cellular diversity and complexity in the mammalian nervous system. Neurons are driven to develop their unique features by intrinsic factors, but also must adapt to local environmental features to produce functional circuits. This raises the question: how do neurons maintain or return to stable cellular states in the context of variable inputs? Evidence suggests that this process requires transcriptional regulation by DNA methylation and the methyl-binding protein MeCP2. The levels of 5-methylcytosine (mC) and its oxidized form, 5-hydroxymethylcytosine (hmC), dramatically change in the developing mammalian brain in response to intrinsic cues and environmental input, and the genomic profiles of these marks appear to show unique patterns in different cell-types. MeCP2 modulates the effects of mC and hmC in neurons and is commonly mutated in Rett syndrome. Further, mutations in the TET enzymes responsible for converting mC to hmC have been recently associated with neurological disorders, indicating that hmC may play a key epigenetic role in gene regulation in the brain. Despite this, we have little understanding of why mC and hmC patterns are unique to individual cell-types and how they function in the brain. Recent results from us and others indicate that hmC plays a dual role as a context-specific activator of mC repressed genes and as a stable repressor during neuronal development. Emerging evidence further indicates MeCP2 as an important reader of mC and hmC signals. Meanwhile, developmental studies have shown that a neuron-specific buildup of a unique form of non-CG methylation (mCH), as well as hmC and MeCP2 occurs during the postnatal period, when neurons integrate into circuits and complete their final maturation into specific functional subtypes. This leads to the intriguing hypothesis that mCH, hmC, and MeCP2 are critical for the establishment and maintenance of diverse, specialized neuronal subtypes within microcircuits. However, a critical barrier in understanding mC, hmC, and MeCP2’s function in the brain, and to testing this hypothesis, is the lack of available methods to simultaneously analyze mC, hmC, and gene expression genome-wide in individual cells. Here we propose to develop a novel experimental and computational approach to perform integrative mC, hmC, and gene expression analysis at the single-cell level. We will apply this method to parvalbumin positive interneurons in the visual cortex to determine the patterns of mC and hmC during postnatal neuronal subtype specification and probe how disruption or rescue of MeCP2 in these cells impacts gene regulation at the highest levels of cellular resolution. Together these studies will provide key insights into the function of MeCP2 in the brain, while developing a new technology that can be used to comprehensively assess the unique neuronal methylome and its impact on transcription in normal and disease states.