Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Computational strategy used to find epigenetic factors with poor patient prognosis overexpressed in head and neck cancer (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 (quantification on right) in nuclear protein lysates extracted from HNC tissues (n=23). ( H ) Volcano plot of differentially expressed genes in RNA-seq analysis of shControl vs. sh YEATS2 in BICR10 cells. ( I ) Results of overrepresentation analysis of genes significantly downregulated in shControl vs. sh YEATS2 RNA-seq data. Error bars, min to max, mean ± SEM for ( G ); two-tailed t-test, ∗∗∗p<0.001, N-Normal, T-Tumor. Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot displayed in . Figure 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Microarray, Western Blot, RNA Sequencing, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A–B ) RT-qPCR result showing mRNA expression of ( A ) RUVBL1 and ( B ) MRGBP in head and neck cancer (HNC) samples. ( C ) Scatter plot showing positive correlation of YEATS2 expression with the expression of genes included in the hallmark EPITHELIAL_MESENCHYMAL_TRANSITION gene signature, in HNC TCGA data. ( D ) Expression of YEATS2 in different grades of TCGA HNC samples, showing a significant increase in expression from grade 1 to subsequent grades. ( E ) Gene set enrichment analysis (GSEA) plot showing significant enrichment of metastasis-associated gene sets in TCGA samples stratified as YEATS2_high as compared to YEATS2_low samples. ( F ) Immunoblot showing protein levels of YEATS2 in nuclear lysates extracted from HNC samples (YEATS2 band indicated by *). ( G ) Results of overrepresentation analysis of genes significantly upregulated in shControl vs. sh YEATS2 RNA-seq data. Error bars, min to max; two-tailed t-test, ns- non-significant, ∗p<0.05, N-Normal, T-Tumor. Figure 1—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 1—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 1—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, RNA Sequencing, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( 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: ×4 , 200 μm; ×10 , 50 μm) Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, n=3 biological replicates. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 2. Original files for western blot displayed in . Figure 2—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Western Blot, Expressing, Knockdown, Invasion Assay, Over Expression, Wound Healing Assay, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Immunoblot showing the expression levels of various EMT factors upon YEATS2 knockdown in SCC9. ( B and D ) Results of invasion assay after knockdown ( B ) or overexpression ( D ) of YEATS2 in SCC9 (Scale bar, 200 μm). ( C ) Immunoblot showing the expression level of Twist1 upon YEATS2 overexpression in SCC9. ( E–F ) Wound healing assay results performed after knockdown ( E ) or overexpression ( F ) of YEATS2 in SCC9 (Scale bar, 275 μm). ( G–H ) Results of 3D invasion assay showing change in invasive potential of SCC9 cells in collagen matrix after silencing (G, quantification below) or overexpression (H, quantification on right) of YEATS2. (Scale bar: ×4 , 200 μm; ×10 , 50 μm) Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, n=3 biological replicates. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 2—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Western Blot, Expressing, Knockdown, Invasion Assay, Over Expression, Wound Healing Assay, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( 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-qPCR assay in BICR10. ( E ) Plot showing difference in relative luciferase activity of YEATS2 Luc-508 in shControl vs. sh SP1 BICR10 cells (n=2). ( 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 endogenous YEATS2 band). ( H ) Invasion assay images (with quantification on right) showing decrease and rescue of the percentage of invaded cells in sh SP1 BICR10 cells, and sh SP1 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. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot displayed in . Figure 3—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Luciferase, Activity Assay, Construct, Expressing, Knockdown, Western Blot, Binding Assay, ChIP-qPCR, Mutagenesis, Sequencing, Over Expression, Invasion Assay, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Luciferase assay results showing the difference in relative luciferase activity of the two YEATS2 promoter deletion constructs in SCC9. ( B–C ) Expression levels of SP1 ( B ) and KLF5 ( C ) in TCGA HNC gene expression dataset. ( D ) Plot showing decrease in mRNA expression of YEATS2 on SP1-knockdown in SCC9 cells. ( E ) Immunoblot showing the reduced expression level of YEATS2 upon SP1-knockdown in SCC9. ( F ) Plot depicting SP1 binding on YEATS2 promoter in SP1-ChIP-qPCR assay in SCC9 cells. ( G ) Relative luciferase activity of wild-type (WT) vs. mutant (Mut) YEATS2 Luc-508 in SCC9 (n=2). ( H ) Invasion assay images (with quantification on right) showing decrease and rescue of the percentage of invaded cells in sh SP1 SCC9 cells, and sh SP1 cells with YEATS2 overexpression, respectively. Scale bar, 200 μm. Error bars, mean ± SEM; two-tailed t-test, one-way ANOVA for ( G ), ns- non-significant, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, n=3 biological replicates. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 3—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 3—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Luciferase, Activity Assay, Construct, Expressing, Gene Expression, Knockdown, Western Blot, Binding Assay, ChIP-qPCR, Mutagenesis, Invasion Assay, Over Expression, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) plot showing enrichment of KEGG LYSINE_DEGRADATION_PATHWAY in TCGA samples stratified as YEATS2_high as compared to YEATS2_low samples (NES- net enrichment score, NOM p- nominal p-value). ( 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 (quantification on right) in nuclear lysates extracted from HNC tumor vs. normal samples (n=23). ( E ) Representative IHC images showing the levels of ECHS1, GCDH, YEATS2, H3K27cr, and Twist1 in HNC normal vs. tumor tissue samples (n=8) (Scale bar, 100 μm). ( F ) Quantification of IHC experiment performed in ( E ). ( G–H ) Immunoblot depicting the decrease in H3K27cr levels on ( G ) YEATS2- and ( H ) GCDH-knockdown in BICR10 cells. ( I–J ) Immunofluorescence images depicting the decrease in H3K27cr levels on ( I ) YEATS2- and ( J ) GCDH-knockdown in BICR10 cells (Scale bar, 10 μm). Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001. n=3 biological replicates. N- normal, T- tumor. Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 4—source data 2. Original files for western blot displayed in . Figure 4—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Immunofluorescence, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A–B ) Plots showing an increase or decrease in expression of GCDH ( A ) and ECHS1 ( B ) genes, respectively, in HNC TCGA data. ( C ) Scatter plot showing positive correlation between YEATS2 and GCDH expression levels in HNC TCGA data. ( D ) Immunoblot showing enhanced levels of H3K27cr in nuclear lysates extracted from HNC tumor ( T ) vs. normal ( N ) samples. ∗∗p<0.01, ∗∗∗p<0.001. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 4—figure supplement 1—source data 2. Original files for western blot displayed in .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: Representative IHC images showing the levels of ECHS1, GCDH, YEATS2, H3K27cr, and Twist1 in head and neck cancer (HNC) normal ( N ) vs. tumor ( T ) tissue samples (Scale bar, 100 μm).

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques:

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A–B ) Immunoblot depicting the decrease in H3K27cr levels on ( A ) YEATS2- and ( B ) GCDH-knockdown in SCC9 cells (YEATS2 band indicated by *). ( C ) Immunofluorescence images (quantification on right) depicting the increase in H3K27cr levels on YEATS2 overexpression (Scale bar, 10 μm). Figure 4—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 4—figure supplement 4—source data 2. Original files for western blot displayed in . Figure 4—figure supplement 4—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Western Blot, Knockdown, Immunofluorescence, Over Expression

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Pie chart showing the genomic distribution of YEATS2 peaks in BICR10 cells. ( B ) Integrative genome viewer (IGV) plot showing the enrichment of YEATS2 on the promoter of SPARC in YEATS2 ChIP-seq data. Arrows indicate the regions probed for YEATS2 binding on SPARC in YEATS2-ChIP-qPCR assay; region corresponding to F1R1 showed significant change in YEATS2 occupancy in sh YEATS2 BICR10 cells. ( C ) Increased expression of SPARC gene in TCGA HNC tumor vs. normal data. ( D ) YEATS2-ChIP-qPCR results showing decreased binding of YEATS2 on SPARC promoter in sh YEATS2 SCC9 cells. ( E ) RT-qPCR results showing decreased expression of SPARC on YEATS2-knockdown in SCC9. ( F ) Immunoblot showing decreased expression of SPARC on YEATS2-knockdown in SCC9. ( G ) EP300 expression levels in Normal vs. Tumor HNC samples from TCGA. Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, n=3 biological replicates, N-normal, T-tumor. Figure 5—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 5—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: ChIP-sequencing, Binding Assay, ChIP-qPCR, Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Venn diagram showing SPARC gene obtained after integration of RNA-seq data (genes downregulated in shControl vs. sh YEATS2 ), YEATS2 ChIP-seq data and, hallmark EPITHELIAL_MESENCHYMAL_TRANSITION gene signature. ( B ) Integrative genome viewer (IGV) plot showing decrease in SPARC expression in shControl vs. sh YEATS2 RNA-seq data. ( C ) YEATS2-ChIP-qPCR results showing decreased binding of YEATS2 on SPARC promoter in sh YEATS2 BICR10 cells. ( D ) RT-qPCR 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 sh YEATS2 BICR10 cells. ( F ) p300-ChIP-qPCR results showing decreased binding of p300 on SPARC promoter in sh YEATS2 BICR10 cells. ( G ) Immunoblot showing decreased SPARC levels on p300 knockdown in conditioned media from shControl and sh EP300 BICR10 cells. ( H ) Immunoblot showing co-immunoprecipitation of YEATS2 by p300 in YEATS2-overexpressed BICR10 cells (endogenous YEATS2 band is highlighted by *). ( I–J ) Immunoblot depicting the overexpression of Flag-tagged SPARC in sh YEATS2 BICR10 cells ( I ), and invasion assay images ( J ) (with quantification on right) showing decrease and rescue of the percentage of invaded cells in shControl vs. sh YEATS2 BICR10 cells, and sh YEATS2 cells with SPARC 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. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot displayed in . Figure 5—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: RNA Sequencing, ChIP-sequencing, Expressing, ChIP-qPCR, Binding Assay, Quantitative RT-PCR, Knockdown, Western Blot, Derivative Assay, Immunoprecipitation, Over Expression, Invasion Assay, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( 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 (n=2). ( C ) RNA Pol II ChIP-qPCR showing decrease in Pol II occupancy on SPARC after YEATS2 downregulation in BICR10. ( D ) H3K27cr-ChIP-qPCR results showing increase in H3K27cr enrichment on SPARC promoter on treating BICR10 cells with 2.5 mM sodium crotonate (NaCr). ( E–F ) RT-qPCR ( E ) and immunoblot ( F ) showing enhanced SPARC expression in untreated BICR10 cells vs. BICR10 cells treated with 2.5 mM NaCr. ( G ) Invasion assay images (with quantification on right) showing increased invasion on treating BICR10 cells with 2.5 mM NaCr (Scale bar, 200 μm). ( H ) H3K27cr-ChIP-qPCR data depicting the lack of significant difference in H3K27cr levels between sh YEATS2 vs. sh YEATS2 + 2.5 mM NaCr cells. ( I ) Immunoblot showing inability of NaCr treatment to rescue SPARC expression in YEATS2-knockdown BICR10 cells. ( J ) H3K27cr ChIP-qPCR showing decrease in H3K27cr levels in BICR10 shControl vs. sh EP300 cells. ( K–L ) p300 ( K ) and H3K27cr ChIP-qPCR ( L ) showing decrease and subsequent rescue in p300 binding and H3K27cr enrichment on SP1 knockdown and YEATS2 overexpression, respectively. Error bars, mean ± SEM; two-tailed t-test, ns- non-significant, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 6—source data 2. Original files for western blot displayed in . Figure 6—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: ChIP-qPCR, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Invasion Assay, Binding Assay, Over Expression, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) H3K27cr-ChIP-qPCR results showing decrease in H3K27Cr enrichment on SPARC promoter on YEATS2 knockdown in SCC9 cells. ( B ) H3K27ac-ChIP-qPCR results showing non-significant change in H3K27ac enrichment on SPARC promoter on YEATS2 knockdown in SCC9. ( C ) H3K27cr-ChIP-qPCR results showing increase in H3K27cr enrichment on SPARC promoter on treating SCC9 cells with 2.5 mM sodium crotonate (NaCr). ( D–E ) RT-qPCR ( D ) and immunoblot ( E ) showing enhanced SPARC expression in untreated SCC9 cells vs. SCC9 cells treated with 2.5 mM NaCr. ( F ) Invasion assay images (with quantification on right) showing increased invasion on treating SCC9 cells with 2.5 mM NaCr (Scale bar, 200 μm). Error bars, mean ± SEM; two-tailed t-test, ns- non-significant, ∗p<0.05, ∗∗p<0.01. Figure 6—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 6—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 6—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: ChIP-qPCR, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Invasion Assay, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) RT-qPCR 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 sh GCDH BICR10 cells (n=2). ( 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 ) Invasion assay images (quantification shown below) showing decrease in invasion of BICR10 cells on GCDH knockdown (Scale bar, 200 μm). ( G ) IF images (quantification shown below) showing reduced nuclear localization of GCDH in sh YEATS2 BICR10 cells. Scale bar, 5 μm. ( H ) Immunoblot depicting decreased SPARC expression on SP1 knockdown and its subsequent rescue upon dual YEATS2 and GCDH overexpression in sh SP1 BICR10 cells. Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001, n=3 biological replicates. Figure 7—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 7—source data 2. Original files for western blot displayed in . Figure 7—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Binding Assay, Invasion Assay, Over Expression, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) RT-qPCR results showing decrease in SPARC expression on GCDH-knockdown in SCC9 cells. ( B ) Co-IP Immunoblot showing lack of interaction between YEATS2 and GCDH in HEK293T cells. ( C ) Scatter plot showing positive correlation between GCDH and SP1 expression levels in HNC TCGA data. ( D ) Immunoblot showing the reduced expression of GCDH on SP1-knockdown in SCC9 cells. ( E ) Plot showing SP1 binding on GCDH promoter in SP1-ChIP assay in SCC9. ( F ) Invasion assay images showing an increase in invasion of BICR10 cells on GCDH overexpression (Scale bar, 200 μm). Error bars, mean ± SEM; two-tailed t-test, ∗p<0.05, ∗∗p<0.01, n=3 biological replicates. Figure 7—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 7—figure supplement 1—source data 2. Original files for western blot displayed in . Figure 7—figure supplement 1—source data 3. Raw data file for .

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Invasion Assay, Over Expression, Two Tailed Test

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: ( A ) Differential ChIP-seq profile of H3K27cr in shControl vs. sh YEATS2 . ( 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 ) Integrative genome viewer (IGV) plot showing representative examples of genes ( CREB3L2 and ERG ) common in ( C ).

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: ChIP-sequencing, RNA Sequencing

Journal: eLife

Article Title: Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer

doi: 10.7554/eLife.103321

Figure Lengend Snippet: Schematic showing YEATS2- and glutaryl-CoA dehydrogenase (GCDH)-mediated regulation of epithelial-to-mesenchymal transition (EMT) through H3K27cr-dependent SPARC upregulation in head and neck cancer.

Article Snippet: Antibodies used: YEATS2 (Proteintech, 24717–1-AP), SP1 (CST, 9389 S), SPARC (CST, 5420 S), GCDH (Sigma, HPA043252), N-Cadherin (Abcam, ab19348), Vimentin (Abcam, ab137321), Twist1 (CST, 46702 S), GAPDH (CST, 5174 S), GFP (Affinity Biosciences, T0006), Flag-tag (Novus, NBP1-06712SS), H3K27cr (PTM Bio, PTM-545RM), Histone H3 (Active Motif, 61475), p300 (CST, 54062 S).

Techniques: