Figure 4: The histone chaperone complex HUCA is required for chromatin incorporation of H3.3cs1.
From: Histone H3.3 and its proteolytically processed form drive a cellular senescence programme
(a) Confocal microscopy of Flag-tagged H3.3FL and H3.3cs1. DAPI, H3K9me3 (red), Flag (green) staining and merged (red and green) images shown. (b) Confirmation of ASF1a and UBN1 knockdown in IMR90 ER::RASV12 cells by reverse transcription-quantitative PCR. Values are normalized to expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH); mean±s.d. (n=2 biological replicates for ASF1a and n=3 technical replicates for UBN1). (c) Chromatin fraction from growing and senescent IMR90 ER::RASV12 cells expressing either shLuc, shASF1a or shUBN1 probed with αH3cs1. αH-RAS immunoblot shows inducible expression of RASV12. Core histones stained with Amido Black for loading. (d) Immunoblot of the chromatin fraction of IMR90 cells expressing the indicated histones with αFlag. (e) Representative SA-β-Gal staining images of IMR90 expressing the indicated histones ( × 10 images shown); quantification shown on the right; mean±s.d. (n=2 biological replicates with three technical replicates). (f–i) Quantification of EdU incorporation, SAHF-positive cells, CCNA and p16 levels in IMR90 cells expressing the indicated histones. Values are normalized to expression of GAPDH; mean±s.d. (n=3 technical replicates); P values for e–i as indicated; two-tailed unpaired Student’s t-test. For p16, expression is relative to pBabe control. NS, not significant.
