Isoler-Alcaraz, J. | bioRxiv Science

bioRxiv2026

EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry

The epigenetic landscape of individual cells determines their identity and function, yet current methods for profiling chromatin modifications at single-cell resolution remain low-throughput, costly, or limited in parametric depth. Here we present EpiFlow, a spectral flow cytometry-based platform that enables the simultaneous quantification of 16 epigenetic markers, including histone post-translational modifications, DNA methylation, and hydroxymethylation, at the single-cell level. We demonstrate that EpiFlow is robust across species from yeast to mammals and resolves biologically meaningful epigenetic transitions during the cell cycle, stem cell differentiation, germinal centre B cell maturation, diabetic liver remodelling, and seizure-induced chromatin reprogramming. High-dimensional integration of EpiFlow data enables cell-type classification based solely on epigenetic profiles in liver, brain, blood, and cancer. Furthermore, EpiFlow detects on-target and off-target/indirect effects of epigenetic drugs in a high-throughput-compatible format. Collectively, these results establish EpiFlow as a broadly applicable platform for single-cell epigenetic analysis in basic, pharmaceutical, and translational research.

cell biology↗

bioRxiv2025

Slow RNAPII elongation enhances naive-pluripotency rewiring while preserving replication fork speed

DNA replication and transcription must be intricately coordinated, as both machineries navigate the same chromatin landscape to ensure genome stability and proper cell function. Here, we uncover that a global imbalance between their elongation rates-- specifically, slowed transcriptional elongation alongside rapid replication fork progression--does not elicit replicative stress. Instead, this uncoupling accelerates the acquisition of naive pluripotency during in vitro de-differentiation, revealing an unexpected link between transcription kinetics and cell plasticity. Mechanistically, we show that the transition to naive pluripotency is accompanied by a distinctive alternative splicing program indicative of reduced RNAPII elongation, both in vitro and in vivo. These findings redefine the functional relationship between replication and transcription dynamics and uncover transcriptional velocity as a tunable layer of control over cellular identity transitions. HighlightsO_LIReplication and transcription elongation rates can be uncoupled genome-wide. C_LIO_LISlow transcription elongation accelerates the acquisition of naive pluripotency during in vitro de-differentiation. C_LIO_LIHigh replication fork speed is maintained in slow-transcribing cells during cell state transitions. C_LIO_LIAlternative splicing is distinctly regulated at the naive and primed pluripotency states, both in vitro and in vivo. C_LI

molecular biology↗