YEATS2 is upregulated in head and neck cancer.

(A) Computational strategy used to find epigenetic factors with poor patient prognosis overexpressed in HNC. (B) Heatmap depicting the TCGA mRNA expression of top 10 epigenetic factors shortlisted using strategy mentioned in (A). (C) RT-qPCR result showing mRNA expression of YEATS2 in HNC samples (n= 8). (D) Kaplan-Meier curve of YEATS2 showing association with overall head and neck cancer patient survival. (E-F) Gene expression profile of YEATS2 in publicly available HNC microarray datasets, GSE30784 (E) and GSE9844 (F). (G) Immunoblot showing protein levels of YEATS2 in HNC samples. (H) Volcano plot of differentially expressed genes in RNA-seq analysis of shControl vs. shYEATS2 in BICR10 cells. (I) Results of overrepresentation analysis of genes significantly downregulated in shControl vs. shYEATS2 RNA-seq data. Error bars, min to max; two-tailed t test, ∗∗∗p < 0.001.

Results for differential gene expression analysis for 10 shortlisted epigenetic factors.

YEATS2 drives EMT in head and neck cancer cells.

(A) Immunoblot showing the expression levels of various EMT factors upon YEATS2 knockdown in BICR10. (B-C) Results of invasion assay with quantification (below), after knockdown (B) or overexpression (C) of YEATS2 in BICR10 (Scale bar, 200 μm). (D-E) Wound healing assay performed after knockdown (D) or overexpression (E) of YEATS2 in BICR10 (Scale bar, 275 μm). (F-G) Results of 3D invasion assay showing change in invasive potential of BICR10 cells in collagen matrix after silencing (F) or overexpression (G) of YEATS2 (quantification shown below). (Scale bar: 4X, 200 μm; 10X, 50 μm) Error bars, mean ± SEM; two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, n= 3 biological replicates.

Regulation of EMT by YEATS2 is SP1-dependent in HNC.

(A) Luciferase assay results showing the difference in relative luciferase activity of the two YEATS2 promoter deletion constructs in BICR10. (B) Plot showing decrease in mRNA expression of YEATS2 on SP1-knockdown in BICR10 cells. (C) Immunoblot showing the reduced expression level of YEATS2 upon SP1-knockdown in BICR10. (D) Plot depicting SP1 binding on YEATS2 promoter in SP1-ChIP assay in BICR10. (E) Plot showing difference in relative luciferase activity of YEATS2 Luc-508 in shControl vs. shSP1 BICR10 cells. (F) Schematic showing mutation of SP1-binding site sequence in YEATS2 Luc-508 construct (above), and relative luciferase activity of wild-type (WT) vs. mutant (Mut) YEATS2 Luc-508 in BICR10 (below). (G) Immunoblot depicting the decreased Twist1 levels on SP1 knockdown and its subsequent rescue of expression upon YEATS2 overexpression in BICR10 (* indicates YEATS2 band). (H) Invasion assay images (with quantification on right) showing decrease and rescue of the percentage of invaded cells in shSP1 BICR10 cells, and shSP1 cells with YEATS2 overexpression, respectively. Scale bar, 200 μm. Error bars, mean ± SEM; two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3 biological replicates.

YEATS2 and GCDH regulate histone crotonylation in HNC.

(A) GSEA plot showing enrichment of LYSINE_DEGRADATION_PATHWAY in TCGA samples stratified as YEATS2_high as compared to YEATS2_low samples. (B) Schematic depicting the canonical lysine degradation pathway highlighting the step that leads to crotonyl-CoA production via GCDH. (C) RT-qPCR result showing enhanced mRNA expression of GCDH in tumor vs. normal samples (n= 8). (D) Immunoblot showing enhanced levels of H3K27Cr in tumor vs. normal samples. (E) Representative IHC images showing the colocalization of nuclear GCDH with H3K27Cr (Scale bar, 100 μm). Cells with nuclear GCDH-H3K27Cr localization indicated by red arrows, whereas non-nuclear staining indicated by black arrows. (F and H) Immunoblot depicting the decrease in H3K27Cr levels on (F) YEATS2- and (H) GCDH-knockdown in BICR10 cells. (G and I) Immunoflorescence images depicting the decrease in H3K27Cr levels on (G) YEATS2- and (I) GCDH-knockdown in BICR10 cells (Scale bar, 5 μm). Error bars, mean ± SEM; two-tailed t test, ∗p < 0.05, ∗∗∗p < 0.001. n = 3 biological replicates.

YEATS2 regulates expression of EMT-related SPARC in HNC.

(A) Venn diagram showing SPARC gene obtained after integration of RNA-seq data (genes downregulated in shControl vs. shYEATS2), YEATS2 ChIP-seq data and, HALLMARK_EPITHELIAL_TO_MESENCHYMAL gene signature. (B) Integrative genome viewer (IGV) plot showing decrease in SPARC expression in shControl vs. shYEATS2 RNA-seq data. (C) YEATS2-ChIP-qPCR results showing decreased binding of YEATS2 on SPARC promoter in shYEATS2 BICR10 cells. (D) qRT-PCR results showing decreased expression of SPARC on YEATS2-knockdown in BICR10. (E) Immunoblot showing decreased expression of SPARC in conditioned media derived from shControl and shYEATS2 BICR10 cells. (F) p300-ChIP-qPCR results showing decreased binding of YEATS2 on SPARC promoter in shYEATS2 BICR10 cells. (G-H) Immunoblot depicting the overexpression of Flag-tagged SPARC in shYEATS2 BICR10 cells (G), and Invasion assay images (with quantification on right) showing decrease and rescue of the percentage of invaded cells in shControl vs. shSP1 BICR10 cells, and shSP1 cells with YEATS2 overexpression, respectively. Scale bar, 200 μm. ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3 biological replicates.

Supplementation of sodium crotonate increases SPARC levels in YEATS2-dependent manner in HNC cells.

(A) H3K27Cr-ChIP-qPCR results showing decrease in H3K27Cr enrichment on SPARC promoter on YEATS2 knockdown. (B) H3K27Ac-ChIP-qPCR results showing non-significant change in H3K27Ac enrichment on SPARC promoter on YEATS2 knockdown. (C) H3K27Cr-ChIP-qPCR results showing increase in H3K27Cr enrichment on SPARC promoter on treating BICR10 cells with 2.5 mM sodium crotonate (NaCr). (D-E) RT-qPCR (D) and immunoblot (E) showing enhanced SPARC expression in untreated BICR10 cells vs. BICR10 cells treated with 2.5 mM NaCr. (F) Invasion assay images (with quantification below) showing increased invasion on treating BICR10 cells with 2.5 mM NaCr (Scale bar, 200 μm). (G-H) Immunoblot showing inability of cells to rescue SPARC expression on YEATS2-knockdown (G) and, H3K27Cr-ChIP-qPCR data depicting the lack of significant difference in H3K27Cr levels between shYEATS2 vs. shYEATS2+ 2.5 mM NaCr cells (H). Error bars, mean ± SEM; two-tailed t test, ns-non-significant, ∗∗p < 0.01, ∗∗∗p < 0.001.

GCDH expression is SP1-dependent and regulates H3K27Cr-mediated SPARC expression with YEATS2 synergistically.

(A) qRT-PCR results showing decrease in SPARC expression on GCDH-knockdown in BICR10 cells (B) Immunoblot showing the reduced expression of SPARC upon GCDH-knockdown in SCC9 cells. (C) Decrease in H3K27Cr levels on SPARC promoter in shGCDH BICR10 cells. (D) Immunoblot showing the reduced expression of GCDH on SP1-knockdown in BICR10 cells. (E) Plot showing SP1 binding on GCDH promoter in SP1-ChIP assay in BICR10. (F) IF images showing reduced nuclear localization in shYEATS2 BICR10 cells. Scale bar, μm. (G) Immunoblot depicting decreased SPARC expression on SP1 knockdown and its subsequent rescue upon YEATS2 and GCDH overexpression in shSP1 BICR10 cells. Error bars, mean ± SEM; two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3 biological replicates.

YEATS2 regulates gene expression by maintaining H3K27Cr levels globally.

(A) Differential ChIP-seq profile of H3K27Cr in shControl vs. shYEATS2. (B) Overrepresentation analysis showing pathways enriched among genes with decreased H3K27Cr enrichment in H3K27Cr ChIP-seq data. (C) Overlap of genes with reduced H3K27Cr on their promoter and downregulated genes in RNA-seq. (D-E) IGV plot showing representative examples of genes common in (C).

Schematic showing YEATS- and GCDH-mediated regulation of EMT through SPARC upregulation in head and neck cancer.

Clinical characteristics of patients.

Sequences of primers used in this study.

Sequences of shRNA used in this study.