EPIFLOW: A HIGH-DIMENSIONAL SPECTRAL FLOW CYTOMETRY PLATFORM FOR SINGLE-CELL EPIGENETIC PROFILING IN THE BRAIN

Understanding cell-type-specific epigenetic dysregulation in neurological disease requires tools capable of capturing chromatin states at single-cell resolution across heterogeneous tissues. We developed EpiFlow, a spectral flow cytometry-based platform enabling simultaneous detection of 15 epigenetic markers at the single-cell level, including histone acetylation (H3K9ac, H3K14ac, H3K27ac, H4K8ac, H4K16ac), methylation (H3K4me3, H3K9me3, H3K27me3, H3K36me2, H3K36me3, H3K79me3, H4K20me2, H4K20me3), DNA modifications (5-mC, 5-hmC), and total histone levels. These markers integrate into functional scores capturing global chromatin architecture and transcriptional states: Chromatin Relaxation, DNA Methylation ratio, Facultative Heterochromatin Activation, Facultative/Constitutive balance, Heterochromatin Index, Enhancer Activity, Initiation & Elongation, and Transcriptional Potential. The platform is robust from yeast to mammals, enabling high-throughput pharmacological screening and complex tissue analysis. In the brain, EpiFlow resolves seven cell types: excitatory and inhibitory neurons, astrocytes, OPCs, oligodendrocytes, microglia, and endothelial cells, providing comprehensive epigenetic coverage of brain cell types.
Applied to epilepsy, EpiFlow revealed cell-type-specific acute responses: inhibitory neurons were more affected than excitatory neurons; endothelial cells showed chromatin relaxation and increased transcriptional potential, suggesting blood-brain barrier stress; microglia displayed enhanced enhancer activity consistent with inflammatory activation; while oligodendrocytes exhibited chromatin compaction, indicating vulnerability to excitotoxicity.
In APP-NLGF Alzheimer's disease mice, we uncovered temporally dynamic alterations: astrocytes showed progressive heterochromatin expansion and increased initiation and elongation indicative of reactive gliosis, while microglia exhibited a biphasic response: early repression followed by late-stage facultative heterochromatin reactivation, marking the inflammatory switch in AD progression.
EpiFlow bridges chromatin regulation and cellular identity, offering unprecedented resolution for understanding cell-type-specific epigenetic dysregulation in brain disease.