A Multimodal Single-Cell Epigenomic and 3D Genome Atlas of the Human Basal Ganglia

The basal ganglia are a group of forebrain nuclei critical for motor control and reward processing, and their dysfunction contributes to neurological and neuropsychiatric disorders. Here, we present the first multimodal single-cell epigenomic atlas of the human basal ganglia across major subregions and cell types. We jointly profiled DNA methylation and 3D chromatin conformation in 197,003 nuclei from eight basal ganglia subregions using multi-omic sequencing (snm3C-seq), and integrated these data with existing DNA methylation and chromatin conformation sequencing datasets to build a unified atlas of 261,331 cells spanning 31 subclasses and 59 groups. This atlas reveals extensive cell-type- and region-specific differential methylation, enriched for distinct transcription factor motifs, and validated by MERFISH spatial transcriptomics, which uncovered epigenetic gradients linked to transcriptional output. Compared to neuronal cells, non-neuronal cells exhibit distinct 3D genome organization including smaller chromatin compartments, increased long-range inter-compartment contacts, shorter loops, and stronger CG hypomethylation in A compartments. We further identified genes that display compartment switches, are strongly correlated with compartment scores, and exhibit differential domain boundaries and chromatin looping across basal ganglia cell types. We identified multiple medium spiny neuron subtypes defined by distinct hypomethylated signature genes, with 3D genome embeddings emphasizing dorsal, ventral, and hybrid populations. By integrating chromatin accessibility and histone modification profiles, we reconstructed cell-type-resolved enhancer-promoter links and gene regulatory networks, providing a comprehensive epigenomic framework for interpreting genetic risk loci and regulatory architecture in the human basal ganglia.