Simultaneous DNA Methylation and Chromatin Conformation Analysis Describes Brain Aging at the Single Cell Level
Aging-associated Epigenetic Alterations in the Brain What is Happening at the Single Cell Level?
While increased age represents a risk factor for the development of devasting neurodegenerative diseases (Hou et al.), we still dont understand the epigenetic mechanisms underpinning aging-related deleterious processes in the brain. This lack of knowledge represents a problem, given that we live in a world with an ever-increasing aged population (WHO; October 2024) and lack effective preventive strategies or targeted therapies. Overall, we require data at multiple epigenetic levels DNA methylation, chromatin conformation, chromatin accessibility, and histone modification patterns from single cells resident in multiple brain regions in the hope of advancing. A previous study described alterations to cell-type-specific transcriptomes during brain aging (Jin et al.), and a body of research reported on aging-related epigenetic alterations in brain regions/cell types (Tan et al., Emani et al., Zhang et al., and Chien et al.); however, we lack single-cell insight into aging-related multimodal epigenetic changes across brain regions.
Researchers from the lab of Joseph R. Ecker (Salk Institute) aimed to bridge this knowledge gap by developing a single-cell multiomic brain aging dataset that integrated DNA methylation, chromatin conformation, and spatial transcriptomic data with chromatin accessibility and transcriptome profiles in brain regions affected by neurodegenerative diseases (Zeng et al.). Their new bioRxiv preprint now describes a significant advance in our limited understanding of brain aging, which may provide the impetus to design novel preventive and therapeutic strategies.
Parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or " Paired-Tag " from Epigenome Technologies offers a similar output, generating joint epigenetic and transcriptomic profiles at single-cell resolution. Paired-Tag can detect histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays and avoids the requirement for cell sorting. Applying Paired-Tag technology may enable researchers to take giant leaps forward in our understanding of gene regulatory mechanisms, identify novel therapeutic targets, and improve disease management; what additional insight could Paired-Tag have provided for this study of the aging brain?
An Improved Understanding of Brain Aging via Multimodal Epigenetic Analysis
The authors generated a single-nucleus cell atlas of aging in multiple brain regions (132,551 single-cell methylomes and 72,666 joint chromatin conformation-methylomes), which they integrated with a transcriptomic and chromatin accessibility dataset to yield multimodal maps in nearly forty major cell types. Specifically, they employed single-nucleus methylome sequencing to map DNA methylation and multiomic sequencing (Lee et al.) to simultaneously assess DNA methylation and chromatin conformation in the single cells from major brain regions from C57BL/6 mice of 2, 9, and 18 months of age. The team then integrated a companion single-nucleus ATAC-seq and 10x Multiome datasets generated from the same samples.
The analysis of single-cell DNA methylation data revealed more significant age-related alterations in non-neuronal cells (with glial cells especially susceptible) than in neurons. Unexpectedly, this dataset also revealed the ability of DNA methylation patterns at transposable elements to distinguish cell types and segregate cells into different groups of similar age; this analysis also linked locus-specific DNA demethylation at transposable elements with increased chromatin accessibility and RNA expression , thereby highlighting the cell types affected by the activation of transposable elements. As DNA methylation typically acts to silence transposable elements and maintain genome stability, the data here supports the suggestion that aging-related DNA methylation loss can prompt transposable element activation to accelerate aging (Gorbunova et al. and Morandini et al.) and trigger DNA damage, inflammatory responses, and altered gene expression, which all contribut e to cell dysfunction/age-related decline (De Cecco et al., Simon et al., and Della Valle et al.).
Analysis of single-cell chromatin conformation data revealed a continual and global increase in boundary strength of topologically-associating domains (TADs) and chromatin accessibility at CTCF (CCCTC-binding factor) binding sites on these boundaries during aging as well as an increase in the number of smaller TADs in general (and in excitatory neurons in particular). The strengthened boundaries prompt the formation of more defined and isolated TADs, which may interfere with promoter-enhancer interactions; overall, the authors believe that the described events may represent novel biomarkers for aging in the brain. Previous research had revealed the disruption of 3D genome organization associated with Alzheimer's disease progression (Dileep et al.) and reported the establishment of ultra-long-range intrachromosomal contacts in cerebellar granule neurons during both human and mouse aging (Tan et al.). The data generated here should help us to understand how alterations to the short- and long-range contacts between distant enhancers and promoters impact aging processes in the brain.
The spatial transcriptomic data generated revealed aging-associated variability among the same cell type, which highlighted an important level of brain-region-level heterogeneity that must be considered when unraveling the complexities of brain aging. For example, the study revealed the upregulation of C4b Sekar et al.) -only Finally, the team applied their multimodal epigenetic data to develop a deep-learning model (EpiAgingTransformer ing a comprehensive view of how epigenetic factors orchestrate age-related transcriptional changes.
Simultaneous Single-cell DNA Methylation and Chromatin Conformation in Brain Aging
Overall, this single-cell multiomic has described various aspects of epigenetic aging in the mouse brain, advancing our understanding and offering potential translational applications. The main points highlighted in this study include the vulnerability of non-neuronal cells to changes in DNA methylation, DNA demethylation at cell-type-specific transposable elements, an increase in the number of small TADs in excitatory neurons, and the spatially heterogeneous nature of associated processes across different brain regions during aging.
Moving this research forward may involve epigenetic and transcriptomic profiling at the single-cell level from samples isolated from the aging brain; can Epigenome Technologies help in this endeavor? Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Furthermore, Epigenome Technologies offers a range of other single-cell products and services suitable for various research requirements.
For more on how simultaneous DNA methylation and chromatin conformation analysis describe brain aging at the single-cell level, see bioRxiv, April 2025.