DoseH-seq: A single-cell multiome platform to decode gene-dosage logic driving developmental reversion and cell fate reprogramming

Cell identity is governed by graded transcription factor (TF) activity, yet current single-cell tools do not resolve how TF dosage shapes both gene expression and chromatin accessibility. We present DoseH-seq, a dosage-resolved single-nucleus RNA+ATAC multiome assay built on standard 10x Genomics workflows. DoseH-seq integrates sample hashing with quantitative tracking of continuous lentiviral overexpression and knockdown across multiplexed conditions and time points. We validate DoseH-seq in both single and multiperturb designs, including dual overexpression and overexpression/knockdown experiments, across different cell types and conditions. We apply DoseH-seq to resolve the dose and context-dependent roles of NFIX, a somatic TF enriched at regulatory elements more active in youthful cells, in fibroblasts, pluripotent stem cells (PSCs) and during reprogramming. In fibroblasts, increased NFIX opens regulatory elements whose motifs compete with myofibroblast identity TFs, consistent with counteracting mesenchymal drift and a restricted developmental reversion. During reprogramming, high NFIX overexpression activates AP-1 and stabilizes the somatic state. Conversely, transitory moderate-level NFIX overexpression, when Yamanaka factors are limiting, synergistically opens chromatin transiently to dismantle the somatic network, with potential analogous roles in oncogenic identity remodelling. During NFIX-induced PSC differentiation, transient reprogramming elements bearing NFI and degenerate pluripotency TF motifs (including OCT4) are re-engaged, consistent with developmental roles, mechanistically linking reprogramming with differentiation. Our data reveal graded dosage effects in somatic and pluripotency TF interactions, highlighting DoseH-seq as a generalisable perturbation-multiomics platform for resolving gene-dosage interactions governing cell identity and cell-state transitions.