Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain

Abstract

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation–methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular–spatial and regulatory genome diversity of the mouse brain.

Single-cell analysis of chromatin accessibility in the adult mouse brain

“…It is possible that additional mouse brain cCREs remain to be discovered because many cell types defined by scRNA-seq or other molecular modalities are not currently represented in the snATAC-based cell clusters. Furthermore, the current catalogue was at the resolution of cell subclasses, and may not reflect subtle differences between cell types, subtypes and states defined in the companion single-cell transcriptomics or single-cell methylome studies 5,6,41 .…”

“…9h). Third, the cell-type-specific chromatin accessibility profiles of these cCREs strongly correlate with DNA hypomethylation 41 (Fig. 2f, Methods and Extended Data Fig.…”

“…10f). The top proximal-distal cCRE pairs and positive pairs showed enrichment signals using the chromatin conformation data from the companion study 41 (Methods and Extended Data Fig. 10g,h).…”

“…Rows represent subclasses, and columns are representative cCREs sampled from each module. Right, heat map showing the snDNA-methylation signals from the snmC-seq 41 analysis at the genomic locations of the corresponding cCREs for the same subclasses. Bottom, heat maps similar to those above but for only the 460,000 cCREs with no overlaps with the ENCODE rDHSs.…”

“…We cross-referenced the dataset from the companion study 41 , in which a comprehensive chromatin conformation/methylome joint profile throughout the adult mouse brain is described, and most of the subclass annotations (244 subclasses of 275 subclasses in our data) are shared between these two datasets.…”