Every population has its own unique identity, and behind that is our brain, an organ composed of cellular populations with their own unique DNA methylation identities. However, capturing the true diversity of a population requires the ability to observe each individual in that group and for methylomes that requires innovations in single-cell bisulfite sequencing.
In order to uncover the diverse methylation signatures of neuronal cell types, the labs of Joseph Ecker at Salk Institute for Biological Studies and Eran Mukamel at the University of California, San Diego have developed single-nucleus methylcytosine sequencing (snmC-seq), a new method that involves:
- Nuclei isolation from dissected tissue
- Nuclei sorting using fluorescence-activated cell sorting (FACS), which makes use of the neuronal marker NeuN
- Sodium bisulfite conversion of the isolated nuclei
- Sample pooling and Swift Bioscience’s Accel-NGS Adaptase Module for Single Cell Methyl-Seq Library Preparation
Overall, snmC-seq allows for the large-scale multiplex sequencing of sorted neuronal nuclei that is needed to gain insight into the diverse cell types of the brain. Using their technique, the team generated over 6000 methylomes from the brain’s frontal cortex, which consisted of:
- 3,377 neurons from a young adult (8-week old) mouse with 4.7% of the genome covered in each cell
- 2,784 neurons from a 25-year-old human with 5.7% of the genome covered in each cell
The talented team focused in on non-CG (CH) methylation, which is critical to the brain. They calculated the CH methylation level for each neuron in non-overlapping 100-kb bins and clustered the cells according to their CH methylation profiles, where they annotated the clusters by known cell-type markers. The high dimensionality data from these cellular populations was visualized in two-dimensions using t-distributed stochastic neighbor embedding (t-SNE). However, the team didn’t just stick to CH methylation and looked at the CG methylation profiles, where they also examined transcription factor motif enrichment.
Taken together, their single-nuclei methylome analyses revealed:
- There is more cell subtype methylation diversity in the human brain than the mouse brain
- They identified 21 and 16 subtypes, respectively.
- Inhibitory neurons are more conserved across the two species than excitatory neurons
- A new excitatory neuron subtype in the two species and also a human specific inhibitory neuron subtype
Ecker shares,“We think it’s pretty striking that we can tease apart a brain into individual cells, sequence their methylomes, and identify many new cell types along with their gene regulatory elements, the genetic switches that make these neurons distinct from each other.” Co-senior author Margarita Behrens adds, “Our research shows that we can clearly define neuronal types based on their methylomes. This opens up the possibility of understanding what makes two neurons—that sit in the same brain region and otherwise look similar—behave differently.”
“There are hundreds, if not thousands, of types of brain cells that have different functions and behaviors and it’s important to know what all these types are to understand how the brain works. Our goal is to create a parts list of both mouse and human brains” expands co-first author Chongyuan Luo. While this “parts list” has much to offer basic science it also has great potential for the clinic and leaves Ecker concluding, “If there’s a defect in just one percent of cells, we should be able to see it with this method. Until now, we would have had no chance of picking something up in that small a percentage of cells.”
Single in on all the details of the brains single cells over at Science, August 2017