Journal: Science (New York, N.Y.)

Article Title: Interpretation of cancer mutations using a multi-scale map of protein systems

doi: 10.1126/science.abf3067

Figure Lengend Snippet: (A) Interactions defining the PIK3CA-actomyosin complex and the context of this system in the NeST hierarchy. (B) Mutation frequencies of genes in this system (shown top seven frequently mutated genes in each cohort). (C) Signal (spectral count fold change versus control) of actomyosin proteins (MYH9, MYH10, MYO1C, LIMA1) in AP-MS experiments with cancer protein baits. Red dots represent signal as interactors of PIK3CA in the CAL-33 cell line; gray dots show corresponding data for other cancer proteins. (D) Per-cell counts of proximity ligation foci when probing for PIK3CA and MYH9 in CAL-33 cells; significantly more foci are observed when probing for both proteins. ***: P <0.001 by one-tailed Student’s T test. (E) CAL-33 cell lines expressing FLAG-PIK3CA (as used in AP/MS experiments) were probed with anti-FLAG and anti-MYH9 antibodies and imaged by DNA-PAINT. Substantial colocalization is observed in small, membrane-proximal puncta. Representative subfields are shown below; scale bar is 500nm. (F) CAL-33 cells were treated with DMSO, blebbistatin (10 μM) and/or alpelisib (0.5 μM) and harvested with indicated antibodies for immunoblotting (N = 3; a representative image is displayed). Signals were normalized relative to total protein and loading control. Error bars indicate mean ± standard error; *: P < 0.05 by one-way ANOVA; images are representative of 3 independent experiments; N = 6 for all measurements. (G) Comparison of the RPPA-measured abundance of indicated phosphorylated/total proteins between cancer cell lines (78) (N = 899) with MYH9 (N = 108) or MYH10 (N = 82) mutations and the wild-type lines. (H) Genomic alterations of PIK3CA and MYH9 in the TCGA-HNSC cohort show a pattern of mutual exclusivity. Wilcoxon rank sum test was used for G and H. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: The different combinations of primary antibodies used were: anti-PIK3CA (Invitrogen, Cat# MA5–17149) diluted at 1/400; anti-FLAG (Cell Signaling Technologies, Cat# 14793S) diluted at 1/800, and anti-MYH9 (ThermoFisher, Cat#PA-29673), diluted at 1/500.

Techniques: Mutagenesis, Ligation, One-tailed Test, Expressing, Western Blot

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Normalized read distribution profiles of H3K36me3 CUT&Tag spanning 5 Kb of gene bodies in WT and Setd2 KO mESCs. IgG was used as the negative control. TSS, transcription start site. TES, transcription end site. Norm. RRPM normalized reference-adjusted reads per million. b Heatmaps of H3K36me3 levels detected by CUT&Tag around gene body regions in WT and Setd2 KO mESCs. 5 Kb windows spanning the TSS to TES of all genes were plotted. Genes were arranged by their enrichments of H3K36me3 in WT cells. Norm. RRPM, normalized reference-adjusted reads per million. c Normalized read distribution profiles of H3K36me3 CUT&Tag spanning 5 Kb of gene bodies in WT and Setd2 KO mESCs. The anti-H3K36me3 antibody used was from Active Motif. TSS, transcription start site. TES, transcription end site. Norm. RRPM normalized reference-adjusted reads per million. d Heatmaps of H3K27ac, H3K27me3 and H3K9me3 CUT&Tag clustering in WT mESCs. Gene expression levels are plotted with column scaled signal scores. H3K27ac and H3K27me3 data are from GSE169049. e Normalized read distribution profiles of H3K36me3 N-ChIP-Rx spanning 5 Kb of gene bodies in WT and Setd2 KO mESCs. IgG was used as the negative control. f As in ( e ), except H3K36me3 X-ChIP-Rx was performed. g IGV tracks showing the enrichment of H3K36me3 by different methods in WT and Setd2 KO mESCs. Three different chromatin loci are shown. Red boxes: promoter regions. Blue boxes: gene body regions. h Schema showing the timing of adding CDK inhibitors (1 μM Flavopiridol, 1 μM THZ1) or high salt (300 mM NaCl) before tagmentation. i Normalized read distribution profiles of Pol II CUT&Tag spanning 5 Kb of gene bodies in WT mESCs. The average read density at all genes determined by NCBI RefSeq was plotted. Nuclei were treated with 1 μM Flavopiridol, 1 μM THZ1 or high salt (300 mM NaCl) for 30 min before tagmentation. j As in ( i ), except H3K36me3 CUT&Tag was performed. k IGV tracks showing the enrichments of Pol II and H3K36me3 by different treatments of CDK inhibitors or a high concentration of salt in WT mESCs. Three different chromatin loci were shown. Red boxes: promoter regions. Blue boxes: gene body regions.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Negative Control, Expressing, Concentration Assay

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Schema showing the calculation of the H3K36me3 index. b Dot plot showing the first screening results. Genes, KO of which changed H3K36me3 indexes less than 15% (gray), decreased or increased the H3K36me3 indexes in blue and red, respectively. c Dot plot showing the second screening results. color scheme as in ( b ). d Protein levels in WT and Smyd5 KO mESCs. Setd2 KO #1 mESCs were used as a positive control for H3K36me3. Cell extracts were analyzed by Western blotting using the specified antibodies. Two independent experiments were performed. Source data are provided as a Source Data file. e The normalized read distribution profiles of H3K36me3 CUT&Tag spanning 5 Kb of gene bodies in WT and Smyd5 KO mESCs. TSS, transcription start site. TES, transcription end site. f Heatmaps of H3K36me3 levels detected by CUT&Tag around gene body regions in WT and Smyd5 KO mESCs. 5 Kb windows spanning the TSS to TES of all genes determined by NCBI RefSeq were plotted. g IGV tracks presenting the enrichment of H3K36me3 by H3K36me3 CUT&Tag in WT and Smyd5 KO mESCs. Red boxes: promoter regions. Blue boxes: gene body regions. h HMT assays of full length SMYD5, SET domain of SUV420H1, and SET domain of SETD2 proteins against nucleosome or octamer substrates. Upper panel: Western blotting. Lower panel: the input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Star indicated the non-specific proteins. i End-point HMT assays of SMYD5 against an equal amount of WT, H3K36M, H4K20M, and H3K36M, H4K20M mutant octamers. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. j HMT assays of full length SMYD5 against different amounts of WT and H3K36M octamer substrates. Upper panel: H3K36me3 western blot. Lower panel, input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Source data are provided as a Source Data file. k End-point HMT assays of SMYD5 against the different amounts of H4K20M and H3K36M/H4K20M octamers. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. Source data are provided as a Source Data file. l ESI-TOF mass spectrometry analysis of Histone H3.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Positive Control, Western Blot, Staining, Mutagenesis, Mass Spectrometry

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Overexpression of Smyd5 does not alter the levels of tested histone marks. O/E, FLAG-Smyd5 overexpression. Two independent experiments were performed. b Knock-in of a FLAG tag at the 5’ end of Smyd5 does not alter the levels of tested histone marks. KI, knock-in of a FLAG tag at the 5’ end of Smyd5 . Two independent experiments were performed. c Normalized read distribution profiles of FLAG CUT&Tag spanning 5 Kb of gene bodies in WT, FLAG-Smyd5 overexpression, and FLAG-tag knock-in Smyd5 mESCs. TSS, transcription start site. TES, transcription end site. O/E, over-expression. KI, FLAG tag knock-in. d Heatmaps of FLAG levels detected by CUT&Tag around gene body regions. 5 Kb windows spanning the TSS to TES of all genes were plotted. e IGV tracks presenting the enrichments of FLAG-tagged proteins by FLAG CUT&Tag. Three different chromatin loci are shown. Red boxes indicated the promoter regions. f Venn diagram illustrating the overlap of FLAG-SMYD5 peaks detected in FLAG-Smyd5 over-expression and FLAG-tag knock-in mESCs. P value was determined by Fisher’s exact statistical test, two-sided. g Correlations between the signals of H3K36me3 and FLAG-SMYD5 at promoters in FLAG-Smyd5 overexpression mESCs. Each dot indicates a single promoter. R, correlation coefficients that were assessed by Pearson product moment correlation. P values were calculated by paired t test, two-sided. h Same as in ( g ), except mESCs with a FLAG tag at the 5’ end of Smyd5 were used. i Correlations between the signals of FLAG-SMYD5 and alternations of H3K36me3 at the promoters in FLAG-Smyd5 over-expression mESCs. Each dot indicates a single promoter. R, correlation coefficients that were assessed by Pearson product moment correlation. P values were calculated by paired t test, two-sided. j Same as in ( i ), except mESCs with a FLAG tag at the 5’ end of Smyd5 were used. k Normalized read density of FLAG-SMYD5 from 3 Kb upstream of the TSS to 3 Kb downstream of the TES in grouped genes with high, medium, and low expression levels in FLAG-Smyd5 over-expression mESCs. Genes were separated into high, medium, and low expression groups based on their expression levels in WT mESCs. High expression, expression levels at top 25%. Low expression, expression levels at bottom 25%. Medium expression, expression levels between high and low expression groups. l Same as in ( k ), except mESCs with a FLAG tag at the 5’ end of Smyd5 were used. m The difference in normalized read densities of H3K36me3 at promoters between parental and Smyd5 KO mESCs relative to alternations of gene expression. Genes that were downregulated in Smyd5 KO cells ( P < 0.05) comparing to parental cells are plotted. R, correlation coefficients that were assessed by Pearson product moment correlation. Confidence interval shows the SEM. n Same as in ( m ), except genes that were upregulated in Smyd5 KO cells ( P < 0.05) comparing to parental cells are plotted. Confidence interval shows the SEM. o Normalized read density of H3K36me3 from 3 Kb upstream of the TSS to 3 Kb downstream of the TES in grouped genes, which were upregulated or downregulated in Smyd5 KO mESCs. Genes were separated into upregulated genes or downregulated genes as defined in ( m ).

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Over Expression, Knock-In, FLAG-tag, Expressing

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Over-expressed SMYD5 interacts with Pol II and H3. SMYD5 was purified by FLAG IP from HEK 293 T over-expressing FLAG-tagged SMYD5. HEK 293 T transfected with empty vectors were used as negative controls. Two independent experiments were performed. b Pol II bound with H3 and overexpressed SMYD5. Pol II was purified by Pol II antibody from HEK 293 T over-expressing FLAG-tagged SMYD5. IgG antibody was used as the negative control. Two independent experiments were performed. c Endogenous SMYD5 interacts with Pol II and H3. SMYD5 was purified by SMYD5 antibody from WT HEK 293 T. IgG antibody was used as the negative control. Two independent experiments were performed. d THZ1 treatment decreased the interaction between SMYD5 and Pol II. SMYD5 was purified by FLAG IP from HEK 293 T overexpressing FLAG-tagged SMYD5. THZ1 treatment was 1 μM for 30 min. Two independent experiments were performed. e Phosphorylation of Pol II CTD increased its interaction with SMYD5 in vitro. GST tagged Pol II CTD was purified and then phosphorylated by the CDK7-Cyclin H complex. Recombinant HIS-tagged SMYD5 was purified and incubated with phosphorylated or unphosphorylated Pol II CTD. GST Seflnose Resin beads were used to pull down Pol II CTD. CTD, Pol II CTD. CTD-p, phosphorylated Pol II CTD. P values were calculated by Hypergeometric distribution test, two-sided, adjusted by BH adjustment for multiple comparisons. Two independent experiments were performed. f HMT assays of full length SMYD5 against octamer substrates in the presence of Pol II CTD or phosphorylated Pol II CTD. Upper panel, H3K36me3. Lower panel, input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Star indicated the non-specific proteins. CTD, Pol II CTD. CTD-p, phosphorylated Pol II CTD. g End-point HMT assays of SMYD5 against an equal amount of H4K20M mutant octamers. Phosphorylated or unphosphorylated Pol II CTD was added to determine the changes of enzymatic activities of SMYD5. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. CTD, Pol II CTD. CTD-p, phosphorylated Pol II CTD. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Purification, Expressing, Transfection, Negative Control, In Vitro, Recombinant, Incubation, Staining, Mutagenesis, Glo Assay

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Schema showing the functional domains and mutations of SMYD5. b Deletion of the C-terminal domain of SMYD5 reduced its interaction with octamers. Recombinant HIS tagged SMYD5 and SMYD5 mutants were incubated with an equal amount of histone core octamers. NI-NTA Seflnose Resin beads were used to pull down SMYD5. Two independent experiments were performed. c Same as in ( b ), except H3/H4 tetramer was used to incubate with WT and mutant SMYD5. Two independent experiments were performed. d Removal of the C-terminal domain repressed the interactions among SMYD5, Pol II, and H3. FLAG-tagged SMYD5 full length (FL) or C-terminal deletion (ΔC) over-expressed HEK 293 T were subjected to FLAG IP. Proteins from input and IP samples were analyzed by Western blotting using the indicated antibodies. HEK 293 T transfected with empty vectors were used as the negative controls. Two independent experiments were performed. e HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates. Upper panel, H3K36me3 was detected by Western blotting. Lower panel, input by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Asterisk indicates non-specific proteins. f End-point HMT assays of full length SMYD5 and mutant SMYD5 against core octamers. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. Each assay was repeated at least three times with similar results. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. g End-point HMT assays of full length and C-terminal deleted SMYD5 against H3K36M mutant octamers. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. h N-terminal domain is important for the interaction between SMYD5 and phosphorylated Pol II CTD. GST tagged Pol II CTD was purified and then phosphorylated by the CDK7-Cyclin H complex. Recombinant HIS-tagged full length and mutant SMYD5 were purified and incubated with phosphorylated Pol II CTD, respectively. Ni-NTA Seflnose Resin beads were used to pull down SMYD5. The input and beads-bound proteins were analyzed by Western blotting using the indicated antibodies. Unphosphorylated Pol II CTD was used as the negative control for the mobility shift of phosphorylated Pol II CTD. CTD, Pol II CTD. CTD-p, phosphorylated Pol II CTD. Two independent experiments were performed. i HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates in the presence of Pol II CTD. Upper panel, H3K36me3 was detected by Western blotting. Lower panel, the input of the reactions was shown by Coomassie Brilliant Blue (CBB) staining. Each assay was repeated at least three times with similar results. Star indicated the non-specific proteins. CTD, Pol II CTD. Two independent experiments were performed. j End-point HMT assays of full length SMYD5 and mutant SMYD5 against octamer substrates in the presence of Pol II CTD. After the reaction, SAM was transferred to SAH which was detected by the MTase-Glo™ assay. N = 3 independent experiments. Data are mean ± SD. P values were calculated by one-way ANOVA. CTD, Pol II CTD. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Functional Assay, Recombinant, Incubation, Mutagenesis, Western Blot, Transfection, Staining, Glo Assay, Purification, Negative Control, Mobility Shift

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Overexpression of Smyd5 in Smyd5 KO mESCs didn’t change the total levels of tested histone marks. Smyd5 KO mESCs were transfected with full length ( FLAG-Smyd5-FL ) or C-terminal deletion ( FLAG-Smyd5-ΔC ) Smyd5 overexpression plasmids. Cell extracts were analyzed by Western blotting using the indicated antibodies. Two independent experiments were performed. Source data are provided as a Source Data file. b Normalized read distribution profiles of FLAG-SMYD5 as detected by FLAG CUT&Tag. c Heatmaps illustrating FLAG-SMYD5 levels detected by FLAG CUT&Tag around gene body regions. d Venn diagram showing the overlap of overexpressed full length and C-terminal deleted SMYD5. P value was determined by Fisher’s exact statistical test, two-sided. KO, Smyd5 KO. e Normalized read distribution profiles of H3K36me3 CUT&Tag signals. f Heatmaps illustrating H3K36me3 levels around gene body regions. g IGV tracks presenting the enrichments of FLAG-SMYD5 and H3K36me3 by CUT&Tag. Three different chromatin loci were shown. Red boxes indicated the promoter regions. Blue boxes indicated gene body regions. h The normalized read distribution profiles of H4K20me3 CUT&Tag signals. i The difference in normalized read densities of H3K36me3 at promoters between Smyd5 KO and Smyd5 reexpression mESCs relative to that of FLAG-SMYD5. R, correlation coefficients that were assessed by Pearson product moment correlation. Confidence interval shows the SEM. Source data are provided as a Source Data file. j Same as in ( h ), except Smyd5 KO mESCs with FLAG-Smyd5-ΔC overexpression were used. Confidence interval shows the SEM. Source data are provided as a Source Data file. k Gene expression heatmap for genes that were restored in FLAG-Smyd5-FL reexpression mESCs. All the changed genes between WT and Smyd5 KO cells that were ‘rescued’ by the WT Smyd5 transgene were plotted. Genes associated with Ras signaling were labeled as red. Genes associated with Wnt signaling were labeled as blue. Two repeats of each sequencing were shown. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Labeling, Sequencing

Journal: Nature Communications

Article Title: SMYD5 catalyzes histone H3 lysine 36 trimethylation at promoters

doi: 10.1038/s41467-022-30940-1

Figure Lengend Snippet: a Smyd5 was elevated in LIHC tumors compared with normal tissues. The expression levels of Smyd5 were generated from the TCGA database through GEPIA analysis . TPM, transcript per million. P values were calculated by one-way ANOVA. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR. b Kaplan–Meier analysis of the overall survival in LIHC cases based on Smyd5 level. Patient data were from the TCGA database through GEPIA analysis . HR, hazard rate ratio. c Schema showing the plasmids used for mouse work. ACT, actin promoter. PB, piggyBac . PBL and PBR, piggyBac repeat termini. U6, U6 promoter. d Smyd5 functions in the tumorigenesis of liver tumors. High expressions of Nras and Ctnnb1 in liver were introduced in mice by HTVI. Smyd5 was knocked down by shRNAs or reexpressed by overexpression of full length (FL) or C-terminal deletion (∆C) Smyd5 . Mice were sacrificed 90–100 days after injection and livers were pictured. Three representative livers in each cohort were shown. e H&E and HA-tag staining of mouse livers. Mouse livers from different cohorts were fixed, paraffin embedded, sectioned, and stained. CTNNB1 and NRAS were HA-tagged and could be stained by HA-tag staining. Representative livers were shown. Scale bars, 2 mm and 0.5 mm in scanned and zoom in figures, respectively. f The liver to body ratio of different cohorts. 13 mice in each group were summarized. Data are mean ± SD. P values were calculated by Student’s t test, one-sided. Source data are provided as a Source Data file. g The expression levels of Smyd5 were analyzed via RT-PCR. The expression level of Smyd5 in normal liver tissues from normal mice was set as 1. The data are represented by the mean ± SD. P values were calculated by Student’s t test, one-sided. Source data are provided as a Source Data file. h Schema showing SMYD5 methylates H3K36me3 at promoters and SETD2 is responsible for H3K36me3 in gene bodies. TSS, transcription stat site. TES, transcription end site.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Abcam, Cat. #ab1791), 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 (Cell Signaling Technology, Cat. #4909, Clone name D5A7, Lot 5), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 (Abcam, Cat. #ab8580, Lot GR3425199-1), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 (Abcam, Cat. #ab8898, Lot GR3291043-1), 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 (Cell signaling Technology, Cat. #9733, Clone name C36B11, Lot 19), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac (Abcam, Cat. #ab4729), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 (Abcam, Cat. #ab239350, Clone name EPR20927-67), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 (Abcam, Cat. #ab9053), 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 (Abcam, Cat. #ab31830, Clone name mAbcam 31830, Lot GR3204774-5), 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU (Abcam, Cat. #ab8955, Clone name IIB5, Lot GR3362257-4), 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 (Sigma, Cat. #F1804, Clone name M2), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 (Abcam, Cat. #ab5095, Lot GR3225147-1), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 (Abcam, Cat. #ab5408, Clone name 4H8, Lot GR3264297-4), 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II (Millipore, Cat. #05-623, Clone name CTD4H8, Lot 3489764), 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Active Motif, Cat. #61101, Lot 28818005), 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 (Abcam, Cat. #ab9050, Lot GR3382010-2), 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag (Sangon Biotech, Cat. #D190101, Lot G112AA0001), 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag (Sangon Biotech, Cat. #D191001, Lot GB15AA0001), 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody (Cell Signaling Technology, Cat. #3724, Clone name C29F4), 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Cat. #111-035-003, Lot 158673), 1:5000 diluted for Western blot.

Techniques: Expressing, Generated, Over Expression, Injection, Staining, Reverse Transcription Polymerase Chain Reaction