Fig. 4: SGF29 phase separation directs a transcriptional program favorable to senescence.
a Principal Component Analysis (PCA) of SGF29 ChIP-seq data in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. b Heatmap showing the chromatin occupancy profiles of SGF29 in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by expression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Peaks identified in hMPCs with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP-SGF29-WT (n = 2890) were used for comparison in all groups. c Metaplots showing the enriched levels of SGF29 occupancies surrounding the TSS regions for protein-coding genes in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. d SA-β-Gal staining of hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by expression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Left, representative images of SA-β-Gal staining. Scale bars, 50 μm. Right, quantitation of the relative percentages of SA-β-Gal-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. *P < 0.05; **P < 0.01 (t-test). e Clonal expansion assay in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Left, representative images of crystal violet staining. Right, quantification of the relative clonal expansion ability. Data are presented as the mean ± SEM. n = 3 biological replicates. *P < 0.05, ***P < 0.001 (t-test). f Immunofluorescence staining of Ki67 in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Left, representative images of Ki67 immunofluorescence staining. Scale bars, 20 μm. Right, quantification of the relative percentages of Ki67-positive cells. Data are presented as the mean ± SEM. n = 3 biological replicates. Over 100 cells were quantified in each replicate. *P < 0.05; **P < 0.01 (t-test). g PCA of transcriptomic profiles in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. h Heatmap showing the relative expression of indicated genes, which were activated in hMPCs expressing EGFP-SGF29-WT (WT) but remained silent in hMPCs expressing EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) mutants, in all groups. Representative Gene Ontology (GO) terms are shown on the right. i Heatmaps showing the relative transcriptional levels and SGF29 occupancies surrounding the TSS regions of 42 genes, which were activated in hMPCs expressing SGF29-WT but remained silent in hMPCs expressing EGFP, or D194A and R207P mutants. j Integrative Genome Viewer tracks of the ChIP-seq and RNA-seq signals at CDKN1A locus in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. k Bar plot showing the ChIP-qPCR detection of the SGF29 enrichment at CDKN1A promoter in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by overexpression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Data are presented as the mean ± SEM. n = 3 biological replicates. *P < 0.05; **P < 0.01 (t-test). l Bar plot showing the qPCR detection of the mRNA levels of CDKN1A in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by expression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively. Data are presented as the mean ± SEM. n = 3 biological replicates. ***P < 0.001 (t-test). m Western blotting detected the protein expression of p21Cip1 in hMPCs (P13) with CRISPR/Cas9-mediated knockdown of endogenous SGF29 followed by expression of EGFP, EGFP-SGF29-WT (WT), EGFP-SGF29-D194A (D194A) and EGFP-SGF29-R207P (R207P) variants, respectively.
