Navigating the oligodendroglial chromatin landscape using high-throughput single-cell epigenomics

Abstract

The central nervous system (CNS), which includes the brain and spinal cord, is a remarkably complex system composed of billions of cells controlling everything ranging from basic metabolic functions to higher executive processes. This is achieved through the coordinated actions between neurons and glia. Oligodendrocytes, a specific glial population, are the myelinating cells of the CNS and wrap neuronal axons with the myelin sheath to facilitate the action potential and coordinate neuronal circuits. Recent years have shown that these cells, along with their progenitor population – OPCs – are more transcriptionally and functionally diverse than was previously believed. The epigenome acts as a function that converts the conserved genetic code to the diverse phenotypes that are observed, and so studying the epigenome can provide insights into how these states arise. The rise of high-throughput single-cell sequencing technologies has enabled the profiling of complex tissues and unveiled cellular diversity in the epigenome and transcriptome.

This thesis aims to broaden our understanding of gene regulation in the oligodendroglial lineage in both healthy and diseased contexts using a range of single-cell epigenomic methods.

In Paper I we used single-nucleus ATAC-seq to investigate the chromatin landscape of oligodendroglia in experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple sclerosis. We sought to understand the regulatory mechanisms that underlie the immune-like transcriptomic states that oligodendroglia exhibit at peak EAE. We found that healthy oligodendroglia exhibit primed chromatin at a subset of immune genes. We then show that these genes are activated in EAE, through a coordinated action involving the Polycomb Repressive Complex 2, and alterations in the histone landscape.

In Paper II, we developed scCUT&Tag, a new single-cell technology that enables the profiling of histone modifications and transcription factors (TFs). We applied the method to the juvenile mouse brain targeting the histone modifications H3K27me3, H3K27ac, H3K4me3, and H3K36me3, as well as the chromatin-associated proteins OLIG2 and RAD21. We use scCUT&Tag to delineate regulatory principles such as promoter bivalency, H3K4me3 spreading and promoterenhancer interactions.

In Paper III, we used snATAC-seq and nanoCUT&Tag to profile adult human CNS tissue and capture the chromatin accessibility, H3K27me3 and H3K27ac histone landscape in different neural cell types. We unveiled a primed chromatin signature at the development-associated HOX genes in spinal cord-derived oligodendroglia (OLG). Using Micro-C to profile the chromatin architecture, we found that iPSderived human OPCs exhibit a HOX architecture that is compatible both with the primed chromatin state seen in the adult OLGs and with high-grade pontine gliomas. Our results suggest that spinal cord-derived adult OLGs retain epigenetic memory of these genes, which may enable them to promptly transcribe these genes in regenerative contexts but may also make them susceptible to gliomagenesis.

In Paper IV, we developed nanoCTAR (pronounced “nano-star”) a 4-in-1 multimodal single-cell technology. We used nanoCTAR to simultaneously capture accessible chromatin, two histone modifications and the transcriptome in single cells from the developing mouse brain. We showcase nanoCTAR as a versatile, easy-to-implement, cost-effective method for high-complexity single-cell profiling.