Direct Single Cell Methylome Profiling in Memory Circuits: Rapid and Massive DNA demethylation induced by Serotonin and Nitric Oxide

So called persistent cell memory and experience induced plasticity is the result of dynamic interactions between multiple transcriptional and site-specific epigenetic modifications in a cell’s genome that do not involve changes in the DNA sequence. Many of these epigenetic events are cell-specific and currently unknown. Here, we (i) developed an unbiased single-neuron assay of genome-wide methylation events at the entire genomic scale complemented by RNA-seq analysis from the same cell, and (ii) identified target genes involved in the control of unique neuronal identities and learning processes within one of the simplest memory-forming circuit of the mollusc Aplysia, a powerful model in neuroscience. To characterize single-neuron DNA methylation profiles, we employed a combination of a novel methylated genome enrichment strategy and massive parallel sequencing using next generation sequencing platform, SOLiD. In parallel, we designed sequencing libraries for directional RNA-seq. As a result, we achieved >40x coverage of the cellular genome in each of the six neuronal cell types tested. First, our data suggest that >40% of neuronal transcripts (including ncRNAs) are differentially expressed among tested cell revealing novel neuron-specific genes. Second, although DNA methylation has so far been considered a very slow and static process, our findings unexpectedly revealed very rapid (<2hrs) and massive DNA demethylation in postmitotic Aplysia neurons in response to one application of Serotonin (5-HT) and Nitric Oxide (NO). 5-HT & NO are known as the facilitatory transmitters and inductor of synaptic reconfiguration and long-term plasticity within the memory-forming circuits. Third, we propose that the process releases transcriptional repression (e.g. gene silencing via DNA methylation of promoter regions) leading to coordinated activation of multiple genes underlying learning and memory. The examples of methylated targets we have identified are the promoter regions of the CREB 2 and Ubiquitin Hydrolase - these two “master genes” are essential for the initiation of coordinated gene expression cascades as a function of learning and memory formation. Fourth, we also identified that DNA methylation occurs outside of CpG islands including gene body regions (rather than promoters), expanding the scope of regulatory mechanisms and the need for genome-wide correlation of methylome and transcriptional outputs from the same cell at different functional states that leads to cell fate specification including the establishment of unique neuronal identities and plasticity in memory circuits..