In a groundbreaking advancement at the intersection of epigenetics and spatial biology, researchers have unveiled a novel method for simultaneous, spatially resolved profiling of the DNA methylome and transcriptome within complex tissue architectures. This pioneering technique, dubbed spatial-DMT, enables scientists to decipher the intricate interplay between epigenetic modifications and gene expression in situ, providing an unprecedented window into cellular identity and regulation within the mammalian brain. Published recently in Nature, this study spotlights the spatial heterogeneity of non-CpG methylation — particularly mCH (methylated cytosine in non-CG context), with emphasis on mCA — across the developing mouse brain, offering profound insights into how epigenetic landscapes shape neuronal function and differentiation.
DNA methylation, the addition of methyl groups to cytosine bases in DNA, is a key epigenetic mark known to regulate gene transcription. While CpG (cytosine-guanine dinucleotide) methylation has been extensively studied, non-CpG methylation such as mCA is emerging as a unique hallmark of neuronal tissues. The spatial distribution and regulatory roles of these modifications, however, remained elusive due to technological limitations in jointly mapping methylation and transcription within the native tissue context. Spatial-DMT overcomes this barrier by integrating DNA methylome sequencing with RNA sequencing on the same tissue sections, offering a high-resolution map of epigenetic and transcriptomic signatures, matched precisely to anatomical regions.
Leveraging spatial-DMT, the investigators profiled a postnatal day 21 (P21) mouse brain section encompassing critical neuroanatomical areas—the dentate gyrus (DG), cornu ammonis (CA) sectors CA1/2 and CA3, and cerebral cortex. Initial global methylation analyses revealed a notable disparity: mCA and mCG (methylated CG) levels were distinctly lower in hippocampal subregions such as DG and CA compared to the cortex. This uneven distribution hints at region-specific epigenetic regulation, aligning with the diverse functional specializations of these brain areas.
Clustering analyses of both DNA methylation and transcriptomic data independently delineated spatially distinct domains that coincided remarkably with known histological landmarks. This congruence was further emphasized through integrated weighted nearest neighbor (WNN) analysis that combined methylation and RNA profiles. Each cluster corresponded to discrete anatomical subregions, underscoring how epigenetic patterns and gene expression converge to define tissue microenvironments.
To unravel the specific influence of mCG and mCA on gene regulation, the team focused on signature genes for each cluster and correlated differential DNA methylation with changes in gene expression. For instance, the transcription factor Prox1, essential for the maintenance of granule cell identity in the DG, showed strong positive association with both mCG and mCA levels. Similarly, Bcl11b, pivotal in neuronal progenitor differentiation within the hippocampus, exhibited regulation by both methylation contexts, suggesting a coordinated epigenetic control mechanism.
Intriguingly, Ntrk3, a receptor tyrosine kinase indispensable for nervous system function, demonstrated a distinct pattern: its expression in CA1/2 and DG tightly correlated with mCG, but not with mCA methylation. This finding highlights that not all genes are governed identically by different methylation contexts, reflecting gene-specific regulatory modes. Moreover, Satb1, a transcription factor involved in cortical neuron differentiation, also displayed a strong correlation exclusively with mCG methylation in the cortex, further supporting functional divergence in epigenetic control.
The study also revealed complexity in gene repression mechanisms. The gene Cux1, a regulator of neuronal development, showed a negative correlation between expression and hypermethylation at both CG and CA sites in CA3, indicating that methylation serves as a repressive signal. Yet, in CA1/2, only CA methylation appeared linked to transcriptional silencing, independent of CpG methylation. Such locus- and region-specific epigenetic dynamics reflect a nuanced, layered regulatory network guiding neuronal specification.
Beyond these gene-centric analyses, spatial-DMT illuminated cell-type-specific epigenetic and transcriptomic heterogeneity across neuronal and glial populations. Neurons widely expressed genes like Syt1 and Rbfox3 throughout cortical layers, while markers such as Cux2, Cux1, and Satb2 were enriched in upper cortical layers. Conversely, Bcl11b was predominantly found in deeper layers. Glial cells including oligodendrocytes and astrocytes showed distinct regional enrichments, with oligodendrocyte markers localizing to the corpus callosum and hippocampus, reflecting their specialized roles in these areas.
The power of spatial-DMT was further underscored by integration with single-cell RNA sequencing (scRNA-seq) references. Annotated spatial clusters matched well with established cell types, including oligodendrocytes, DG granule neurons, and telencephalic excitatory neurons. This cross-modal validation confirmed the robustness of spatial-DMT in resolving complex tissue architecture at cellular resolution. Mapping of cell types also reflected expected neuroanatomical distributions, such as laminar-specific localization of excitatory neuron subtypes in the cortex and discrete hippocampal subregion specificity.
This dual profiling technique addresses longstanding challenges in neuroscience and epigenetics by providing direct spatial context to both gene expression and DNA methylation. Unlike conventional single-cell approaches, which may dissociate cells and thus lose positional information, spatial-DMT preserves tissue morphology, enabling comprehensive analyses of how epigenetic landscapes influence transcriptional states within intact brain circuits.
The implications of these findings are broad and transformative. The demonstration of differential roles of mCG and mCA in regulating key developmental and functional genes reshapes our understanding of epigenetic modulation in neural tissue. Importantly, the predominance of negative correlations between DNA methylation and gene expression across contexts affirms the generally repressive role of cytosine methylation, while also revealing gene- and region-specific exceptions that merit further investigation.
Looking forward, spatial-DMT opens new avenues for exploring epigenetic regulation in diverse biological contexts beyond neuroscience, including development, disease pathology, and regenerative medicine. By enabling simultaneous, spatially resolved epigenomic and transcriptomic profiling, researchers can better decipher cellular identities, lineage relationships, and the molecular underpinnings of complex tissue ecosystems.
Ultimately, this breakthrough embodies a formidable leap in spatial multi-omics, promising to unravel the epigenetic codes that orchestrate cellular function and tissue organization with molecular precision in their native environments. The marriage of spatial epigenetics and transcriptomics heralds a new era of integrated molecular cartography with vast potential to advance our understanding of brain biology and beyond.
Subject of Research:
Spatial profiling of DNA methylome and transcriptome to investigate region- and cell-type-specific epigenetic regulation in the mouse brain.
Article Title:
Spatial joint profiling of DNA methylome and transcriptome in tissues.
Article References:
Lee, C.N., Fu, H., Cardilla, A. et al. Spatial joint profiling of DNA methylome and transcriptome in tissues. Nature (2025). https://doi.org/10.1038/s41586-025-09478-x
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