Fig. 6: Chromatin structural changes in HGPS affect PRC2-regulated bivalent genes.

a Transcript-level differential expression (upregulation) was assessed by comparing individual HGPS samples against the group of controls. A two-sided Wald test was used to compute p-values for the differential expression at the transcript level, then uncorrected p-values were aggregated based on their chromatin state using the Lancaster method, considering only genes in SAMMY-seq domains (S4 vs S2). The plot reports the number of transcripts (x axis) and significance (y axis) for each chromatin state (color legend) as defined by ref. ⁴⁹. The horizontal red lines mark the 0.05 p-value threshold. b Average H3K27me3 ChIP-seq signal (y axis) around the Transcriptional Start Site (TSS) of 39 bivalent genes upregulated in HGPS. The x axis reports relative genomic position around the TSS (±5 kb). c Representative genomic tracks (chr2: 118810000-118880000) of H3K27me3 ChIP-seq in control and HGPS samples at indicated passages (p10-19) around the bivalent gene EN1 upregulated in HGPS samples. H3K4me3 ChIP-seq from Roadmap Epigenomics normal skin fibroblasts is reported as well. The (ChIP – input) reads distribution was computed with SPP package and we are limiting the y axis range to zero as a minimum value. d, e Representative images of Proximity Ligation Assay (PLA) experiments in CTRL004 and HGPS167 at late passages (p20). Each fluorescent dot represents the co-localization of Lamin A/C or Progerin and Ezh2 (d) or Bmi1 (e). Nuclei were stained with dapi. Scale bars: 20 μm. The PLA acquisition parameters were the same in control and HGPS cells for each interactor pairs. All data were generated from an average of three independent experiments, the horizontal black line is the median. Each dot shows an independent biological replicate. Exact p-value: 0.0011. f Representative western blot on chromatin fractionation experiments of CTRL004 and HGPS167 at indicated early (left, p10-17) and late (right, p20-27) passages. Equal amounts of each fraction were hybridized with the indicated antibodies. Alpha-tubulin, histone H3, and Lamin A/C were used as loading controls respectively for S1, S2 and S3, S4. g, The graph shows quantifications of Ezh2 and Bmi1 bands of at least two independent biological replicates reporting S4 fraction normalized on S2, the horizontal black line is the median. Each dot shows an independent biological replicate. Exact p-values Ezh2: 0.041; Bmi1: 0.044 h, Cartoon illustrating the proposed model: that expression of progerin in early-passage HGPS fibroblasts interferes with LADs positioning near the nuclear envelope and is accompanied by altered transcriptional profiles at PcG-regulated heterochromatin. Comparisons were done using a two-tail t-test in d, e, g. Statistically significant differences are marked *p < 0.05; **p < 0.01.