Journal: Science Advances

Article Title: FeaSion decodes the regulatory landscape and functional diversity of RNA polymerase II CTD phosphorylation

doi: 10.1126/sciadv.adz2345

Figure Lengend Snippet: ( A ) Stacked bar plot showing the kinases (left) and phosphatases (right) identified in RNAPII ChIP-MS, CRISPR screening and RNAPII mut ChIP-MS. ( B ) Pie plots showing the family of kinases identified in CRISPR screening. ( C ) Heatmap showing the NormZ of CRISPR screening for CLK1, CLK4, and YES1. ( D ) Western blot analyses of the RNAPII, pY1, pS2, pT4, pS5, and pS7 in CLK1, CLK4, or YES1 KO cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( E ) Metagene profiles and boxplots (top) and log 2 (fold change) metagene heatmaps (bottom) showing pS7 and RNAPII ChIP-seq density in control, CLK1 KO, and CLK4 KO cells, as well as in untreated and TG003-treated (20 μM, 4 hours) cells (top). Statistical significance was assessed by a two-sided Wilcoxon test. *** P < 0.001. The value on the box plot indicating the fold change. ( F ) Similar to (E), but for pY1 and RNAPII ChIP-seq in control and YES1 KO cells, as well as in untreated and CHR6953755-treated (10 μM, 24 hours) cells. ( G ) Schematic workflow of in vitro phosphorylation assay. ( H ) In vitro phosphorylation activity of CLK1 (left), CLK4 (middle), and YES1 (right) on CTD. The CTD was detected using HA tag, and CLK1, CLK4, and YES1 were detected using Flag tag. ( I ) Pie chart (top) and metagene heatmap (bottom) showing the distribution of the top 500 CLK1 (left) CLK4 (middle) and YES1 (right) ChIP-seq binding sites and their densities. ( J ) Bar plot showing the gene ontology enrichment result of CLK1 (top), CLK4 (middle) and YES1 (bottom) binding genes. ( K ) Tracks of ING5 of CLK1 (left), DLX2 of CLK4 (middle), and IMPACT of YES1 (right) ChIP-seq signals and the changes of pS7 or pY1 and RNAPII after their knockout. miRNA, microRNA; snoRNA, small nucleolar RNA. TES, transcription end site.

Article Snippet: Then, RNAPII inhibitor α-amanitin (Sigma-Aldrich, #129741) that was used to degrade RNAPII , ABL1 inhibitor Dasatinib (MedChemExpress, #HY-10181) that was used to inhibit pY1 , CDK inhibitor DRB (Sigma-Aldrich, #D1916) that was used to inhibit pS2 , PLK1 and PLK3 inhibitor GW843682X (MedChemExpress, #HY-11003) that was used to inhibit pT4 , CDK7 inhibitor THZ1 (Sigma-Aldrich, #5.32372) that was used to inhibit pS5 and pS7 , CLK1 and CLK4 inhibitor TG003 (MedChemExpress, #HY-15338) , SRI-29329 (MedChemExpress, #HY-123600) , SRC family inhibitor PP2 (MedChemExpress, #HY-13805) , SU6656 (MedChemExpress, #HY-B0789) , and YES1 inhibitor CH6953755 (MedChemExpress, #HY-135299) ( ) were diluted in culture medium and added to cells with indicated concentration and time.

Techniques: CRISPR, Western Blot, Control, ChIP-sequencing, In Vitro, Phospho-proteomics, Activity Assay, FLAG-tag, Binding Assay, Knock-Out

Journal: Science Advances

Article Title: FeaSion decodes the regulatory landscape and functional diversity of RNA polymerase II CTD phosphorylation

doi: 10.1126/sciadv.adz2345

Figure Lengend Snippet: ( A ) Stacked bar plot showing the kinases (left) and phosphatases (right) identified in RNAPII ChIP-MS, CRISPR screening and RNAPII mut ChIP-MS. ( B ) Pie plots showing the family of kinases identified in CRISPR screening. ( C ) Heatmap showing the NormZ of CRISPR screening for CLK1, CLK4, and YES1. ( D ) Western blot analyses of the RNAPII, pY1, pS2, pT4, pS5, and pS7 in CLK1, CLK4, or YES1 KO cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. ( E ) Metagene profiles and boxplots (top) and log 2 (fold change) metagene heatmaps (bottom) showing pS7 and RNAPII ChIP-seq density in control, CLK1 KO, and CLK4 KO cells, as well as in untreated and TG003-treated (20 μM, 4 hours) cells (top). Statistical significance was assessed by a two-sided Wilcoxon test. *** P < 0.001. The value on the box plot indicating the fold change. ( F ) Similar to (E), but for pY1 and RNAPII ChIP-seq in control and YES1 KO cells, as well as in untreated and CHR6953755-treated (10 μM, 24 hours) cells. ( G ) Schematic workflow of in vitro phosphorylation assay. ( H ) In vitro phosphorylation activity of CLK1 (left), CLK4 (middle), and YES1 (right) on CTD. The CTD was detected using HA tag, and CLK1, CLK4, and YES1 were detected using Flag tag. ( I ) Pie chart (top) and metagene heatmap (bottom) showing the distribution of the top 500 CLK1 (left) CLK4 (middle) and YES1 (right) ChIP-seq binding sites and their densities. ( J ) Bar plot showing the gene ontology enrichment result of CLK1 (top), CLK4 (middle) and YES1 (bottom) binding genes. ( K ) Tracks of ING5 of CLK1 (left), DLX2 of CLK4 (middle), and IMPACT of YES1 (right) ChIP-seq signals and the changes of pS7 or pY1 and RNAPII after their knockout. miRNA, microRNA; snoRNA, small nucleolar RNA. TES, transcription end site.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 30 min and then incubated overnight at 4°C with the RPB1 NTD antibody (Cell Signaling Technology, #14958), phospho-RPB1 CTD (Ser 2 ) antibody (Cell Signaling Technology, #13499), phospho-RPB1 CTD (Ser 5 ) antibody (Cell Signaling Technology, #13523), phospho-RPB1 CTD (Tyr 1 ) antibody (Merck Millipore, #MABE350), phospho-RPB1 CTD (Thr 4 ) antibody (Cell Signaling Technology, #26319), phospho-RPB1 CTD (Ser 7 ) antibody (Cell Signaling Technology, #13780), CLK1 antibody (Santa Cruz Biotechnology, #sc-515897), CLK4 antibody (Proteintech, #31205-1-AP), YES1 antibody (Proteintech, #20243-1-AP), α-tubulin antibody (Proteintech, #66031-1-Ig), GFP antibody (Proteintech, #50430-2-AP), HA antibody (Proteintech, #51064-2-AP), or glyceraldehyde-3-phosphate dehydrogenase antibody (Proteintech, #10494-1-AP) to the target protein.

Techniques: CRISPR, Western Blot, Control, ChIP-sequencing, In Vitro, Phospho-proteomics, Activity Assay, FLAG-tag, Binding Assay, Knock-Out