Fig 2: YEATS2 is amplified in NSCLC and is required for cancer cell growth and survival. a YEATS2 gene is frequently amplified in various types of human cancers. Data was obtained from the cBioPortal for Cancer Genomics. b Western blot analysis of YEATS2, GCN5, PCAF, HDAC1, and the indicated histone acetylation in NSCLC cell lines and immortalized “normal” lung fibroblast cell lines. Total H3 and actin are shown as loading control. The arrow indicates the band of YEATS2 protein. Relative H3K9ac, H3K14ac, and H3K27ac levels were quantified (n = 3, mean ± s.e.m.). N.S. not significant; *p < 0.05; **p < 0.01 (Student’s t-test). c Cell proliferation assay of H1299 cells treated with control (shNT) or YEATS2 shRNAs (shY2). Cells (mean ± S.E.M., n = 4) were counted for 4 days after seeding (left panel). Right panel: western blot analysis showing YEATS2 knockdown efficiency. The arrow indicates the band of YEATS2 protein. Error bars represent S.E.M. *p < 0.05; **p < 0.01 (Student’s t-test). d Clonogenic assay of control (shNT), YEATS2 knockdown (shY2), and knockdown H1299 cells rescued with ectopic expression of YEATS2. Empty vector was used as a control. Colonies were stained and photographed 7 days after seeding (right panel). Left panel: western blot analysis of YEATS2 expression level in indicated cells. The arrow indicates the band of YEATS2 protein. e Anchorage-independent growth assay of H1299 cells as in (d). Cells (mean ± S.E.M., n = 4–6) were stained with 0.005% crystal violet blue and photographed 3 weeks after seeding (top panel). Colony numbers (bottom left) and colony diameters (bottom right) were measured and quantified using ImageJ software. Scale bar, 200 µm. N.S. not significant; ***p < 0.001 (Student’s t-test)

Fig 3: YEATS2 is required for ATAC-dependent maintenance of histone H3K9ac on the ribosomal protein genes. a Western blot analysis of YEATS2 and H3 and H4 acetylation in control (shNT) and YEATS2 KD (shY2) cells. H3 and H4 were used as a loading control. The arrow indicates the band of YEATS2 protein. b Average genome-wide H3K9ac occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. c Genome-browser view of the H3K9ac and ZZZ3 ChIP-seq peaks on the indicated ribosomal protein genes in cells as in (b). d qPCR analysis of H3K9ac ChIP of the indicated ribosomal protein genes in cells as in (b). e qPCR analysis of ZZZ3 ChIP of the indicated ribosomal protein genes in cells as in (b). f Average ZZZ3 occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. In d and e, error bars indicate S.E.M. of at least three biological replicates. *p < 0.05; **p < 0.01 (Student’s t-test)

Fig 4: The YEATS domain of YEATS2 recognizes H3K27ac. a Schematic representation of YEATS2 protein structure. The amino acid numbers of the YEATS domains and the full-length protein are shown. HF: histone fold domain. b Western blot analysis of histone peptide pulldowns of GST-YEATS2 YEATS domain and the indicated biotinylated peptides. c Western blot analysis of histone pulldowns of GST-YEATS2 YEATS domain or GST and calf thymus histones. The arrow indicates the band of the GST-YEATS YEATS2 domain. d Western blot analysis of Flag co-IP in 293 T cells transfected with Flag-YEATS2 or vector control. e ITC fitting curves of YEATS2 YEATS titrated with H315–39K27ac, H31–15K18ac, unmodified H31–34K27, H36–15K14ac, and H31–15K9ac peptides. f Overall structure of YEATS2 (aa201–332) bound to the H324–31K27ac peptide in ribbon view. YEATS2 (pale cyan) is shown as ribbons, and the histone H3 peptide (yellow) is depicted as sticks. Purple mesh: Fo–Fc omit map around H324–31K27ac peptide contoured at 1.8 s level. Bottom right, close-up view of the Kac-sandwiching pocket; interplanar distances are labeled in the unit of angstrom. g YEATS2–H3K27ac space-filling-surface view color-coded by electrostatic potential ranging from -10 to 10 kT/e. h Conservation mapping around the H3-binding surface in YEATS2. White and cyan colors indicate low (<0.25) and high (1.0) sequence conservation, respectively. The H3K27ac peptide is shown in yellow stick. i Close-up view of the K27ac-binding pocket of the YEATS2 YEATS domain. The pocket is displayed as semi-transparent surface with key residues shown as green sticks. K27ac is depicted in both yellow stick and space-filling sphere modes. j Hydrogen bonding network between H3K27ac peptide and YEATS2. Hydrogen bonds are shown as pink dashes. Key residues of YEATS2 are depicted as green sticks and labeled black; the H3 peptide is shown as yellow sticks and labeled red. k Western blot analysis of peptide pulldowns of WT YEATS2 YEATS domain or the indicated point mutants with the H3K27ac peptide

Fig 5: Effects of silencing some genes in diagnostic and prognostic panels on ESCC cell phenotypes.a–d Silencing the expression of MMP13 (a), YEATS2 (b), HOXC10 (c) and NECAB2 (d) significantly suppressed KYSE30 and KYSE150 cell proliferation. e–h Silencing the expression of MMP13 (e), YEATS2 (f), HOXC10 (g) and NECAB2 (h) significantly suppressed KYSE30 and KYSE150 cell migration and invasion. Left panel shows representative cell migration and invasion images and right panel shows quantification statistics. Data represent mean ± SEM from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 1.00e-3; ****P < 1.00e-4 and ns not significant of Student’s t test compared with corresponding control