Transcription factor stoichiometry, motif affinity and syntax regulate single-cell chromatin dynamics during fibroblast reprogramming to pluripotency

Ectopic expression of OCT4 , SOX2 , KLF4 and MYC (OSKM) transforms differentiated cells into induced pluripotent stem cells. To refine our mechanistic understanding of reprogramming, especially during the early stages, we profiled chromatin accessibility and gene expression at single-cell resolution across a densely sampled time course of human fibroblast reprogramming. Using neural networks that map DNA sequence to ATAC-seq profiles at base-resolution, we annotated cell-state-specific predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of Tn5-bias corrected TF footprints, linked peaks to putative target genes, and elucidated rewiring of TF- to-gene cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution, connect TF stoichiometry and motif syntax to diversification of cell fate trajectories, and provide new perspectives on the dynamics and role of transient regulatory elements in somatic silencing. ### Competing Interest Statement A.K. is on the SAB of PatchBio Inc., SerImmune., AINovo Inc., TensorBio Inc. and OpenTargets, was a consultant with Illumina Inc. and owns shares in DeepGenomics, Immunai, TensorBio and Freenome Inc. M.A. and L.S. are employees of Illumina. A.B. is an employee of Sanofi.