STEMIN transcription factor drives selective chromatin remodelling for gene activation within a relaxed chromatin during reprogramming in the moss Physcomitrium patens

Land plants exhibit remarkable cellular plasticity, readily reprogramming differentiated cells into stem cells in response to internal and external stimuli. While chromatin remodelling is crucial for cellular reprogramming, its interplay with gene expression during reprogramming into stem cells remains elusive. In the moss Physcomitrium patens, wounding induces reprogramming of leaf cells facing wounded cells to change into chloronema apical stem cells through the activation of the AP2/ERF transcription factor STEMIN. In this study, we employed combined single-nuclei RNA and ATAC-sequencing to explore the interplay between gene expression and chromatin dynamics during STEMIN-mediated reprogramming. Profiling 20,883 single-nuclei from gametophores, protonemata, and cut leaves, we identified eleven distinct cell types including reprogramming leaf cells. Our analysis revealed that reprogramming leaf cells exhibit a partly relaxed chromatin landscape and STEMIN transcription factors selectively enhance accessibility at specific genomic loci essential for stem cell formation. Thus, our results indicate that wounding initiates a broad chromatin relaxation, creating a permissive environment and specific transcription factors act to refine this permissive state by specifically relaxing chromatin regions critical for reprogramming. Overall design: Wild type (Wt) Physcomitrium patens (Hedw.) Mitt. and the derived triple deletion mutant ?stemin1?stemin2?stemin3#6-48-1 (ste)(Gu et al., 2020) were grown from disrupted gametophyte tissue on solid BCDAT medium (Nishiyama et al., 2000) at 25°C under continuous white light for 1 week to propagate protonemata or 22 days to generate gametophore enriched samples. Nuclei for the gametophore samples (T00) were isolated directly from tissue harvested from BCDAT medium-grown plates after 22 days of cultivation. For time-course experiments, 22-day-old tissue was collected in liquid BCDAT medium, subjected to mechanical disruption using a Polytron 1200E homogenizer and then allowed to recover in liquid BCDAT medium on an illuminated shaker. Nuclei were isolated and pooled as follows: nulcei isolated 3, 4.5, and 6 hours after disuption were pooled for the 3-6 h timepoint (T03); nuclei isolated 10, 12, and 14 hours after disruption were pooled for the 10-14 h timepoint; nuclei isolated 24 and 36 hours after disruption and nuclei isolated from 1-week-old protonemata were pooled for the 24-36 h +protonemata time point (T24).