Fig. 3: Nuclear flattening primes chromatin for spontaneous differentiation.

a, Representative top views, side views and 3D reconstructions of LaminB1-RFP-tagged hiPS cells subjected to compression (scale bars, 10 µm; images representative of six independent experiments). b, UMAP of scRNA- and scATAC-seq from hiPS cells subjected to compression for timepoints indicated. c, A heatmap of predicted regulons enriched in compressed cells from SCENIC+ analyses of the multiome data. d, A schematic of experimental outline for genome-wide mapping of H3K27ac changes. e,f, Heatmap (e) and metaplot (f) analysis of mean H3K27ac levels at active promoters and predicted active enhancer regions. Note reduction in H3K27ac enrichment at promoters across all conditions and at enhancers in cells compressed in basal medium or exposed to hypertonic shock. g, UpSet plot showing an overlap of enhancers decommissioned in compression and hypertonic shock conditions. h, Venn diagram and Reactome pathway enrichment of compression-specific and shared decommissioned enhancers as defined in g. i, A schematic of the experimental outline for the quantification of the nascent transcriptome. 4sU, 4-thiouridine. j, Quantification of RNA synthesis across conditions from TTseq. Note reduced synthesis across all conditions compared with pluripotency medium condition (n = 3 biological replicates per condition). k, Quantification of total changes in nascent RNA production across conditions relative to the pluripotency medium condition (n = log₂FC of 19,288 genes computed from 3 biological replicates; Tukey’s box plots show 75th, 50th and 25th percentiles). l, A heatmap of z scores from altered nascent RNA levels of relevant transcripts from TTseq quantified by DESeq2. Note increased levels of IEGs specifically in cells compressed in pluripotency medium while key pluripotency and growth factor regulators are repressed. Comp, compression; Hyper, hypertonic; Rec, recovery; pluri, pluripotency.

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