Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: (A) Phosphorylation of RNA Pol II at Ser 2 and Ser 5 are differentially affected by Fcp1 CTD phosphatase. Yeast strains grown at 30°C (OD600 = 0.8) were further incubated at 37°C (even lanes) or 30°C (odd lanes) for 55 min. Whole-cell extracts were prepared from each strain. Extract protein (80 μg) was assayed by immunoblotting with 8WG16 (CTD, recognizing the nonphosphorylated CTD of Rpb1), H14 and H5 (CTD-S5-P and CTD-S2-P, phosphorylated CTD on Ser 5 and Ser 2 position, respectively), B3 (phosphorylated CTD at either Ser 5 or Ser 2), G2 (recognizing Rpb1 outside of CTD) and polyclonal antibodies against Fcp1, Ceg1, and Cet1. Yeast strains used are YMK16α, shuffled with plasmids expressing wild-type FCP1 (pFK1; lanes 1,2), fcp1-1 (pFK4; lanes 3,4) and fcp1-2 (pFK7; lanes 5,6). (B) Specificity of CTD antibodies. Biotinylated peptides (100 ng or 1 μg) containing four CTD repeats with the indicated phosphorylations were coupled to the wells of streptavidin-coated 96-well plates. The indicated antibodies were used to probe the peptides and binding was detected by indirect chemiluminescence.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Phospho-proteomics, Incubation, Western Blot, Expressing, Binding Assay

Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: Ser 2 and Ser 5 phosphorylations on the ADH1 gene are differentially affected by the Fcp1 CTD phosphatase during transcription. Chromatin immunoprecipitation was performed with YSB763 shuffled with the same Fcp1 plasmids used in Figure ​Figure11 (pFK1, pFK4, and pFK7). To monitor the presence of each protein along the ADH1 gene, chromatin was immunoprecipitated with various antibodies and PCR amplified with primer pairs recognizing promoter (P) and coding (C) regions (see schematic at bottom). Each PCR reaction contained a second primer pair that amplifies a region of chromosome V devoid of ORFs, thus providing an internal control for background (*). Each panel shows a different immunoprecipitation with respective antibodies as follows: α-CTD-S5-P (H14), α-CTD-S2-P (H5), α-CTD (8WG16), α-Rpb3HA (12CA5, recognizing an HA epitope on RNA polymerase subunit Rpb3), and α-Cet1 (a polyclonal antibody recognizing the triphosphatase subunit of capping enzyme). Input shows the signal from the chromatin before immunoprecipitation. Signals were quantitated by PhosphorImager and normalized as described previously (Komarnitsky et al. 2000). The signals for the promoter with wild-type chromatin solution were assigned as 1, except for the CTD-S2-P immunoprecipitation, in which the signal from the coding region was taken as 1. A zero indicates that the signal was <0.005. The primer pairs used are (P) ADH1−235 and ADH1−13, (C) ADH1844 and ADH11013, (−) Intergenic V −1 and Intergenic V −2.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Control

Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: Ser 2 and Ser 5 phosphorylations on the PMA1 gene are differentially affected by the Fcp1 CTD phosphatase during transcription. The same chromatin immunoprecipitates shown in A were used for PCR amplification with primer sets that amplify DNA throughout the PMA1 gene (see schematic diagram at bottom). Promoter; PMA1-370 and PMA1-90, Coding region 1 (CD1): PMA1168 and PMA1376; Coding region 2 (CD2): PMA1584 and PMA1807; Coding region 3 (CD3): PMA11010 and PMA11250; Coding region 4 (CD4): PMA12018 and PMA12290.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Amplification

Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: The TFIIH kinase Kin28 is not required for CTD Ser 2 phosphorylation of elongating polymerase. Chromatin IP/PCR was carried out with kin28-16 mutants combined with FCP1, fcp1-1, or fcp1-2 (YMK223, YMK224, and YMK225, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Immunoprecipitating antibodies are indicated to the left of the autoradiographs and PMA1 primer pairs are as in Figure ​Figure33.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Phospho-proteomics, Chromatin Immunoprecipitation, Transformation Assay

Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: The Srb10 kinase component of RNA Pol II holoenzyme is not required for CTD Ser 2 phosphorylation of elongating polymerase. Chromatin IP/PCR was carried out with Srb10(D290A) combined with FCP1, fcp1-1, or fcp1-2 (YMK162, YMK164, YMK166, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Srb10 is the kinase associated with RNA Pol II holoenzyme. Srb10 (D290A) is catalytically inactive but successfully incorporated into the holoenzyme. Immunoprecipitating antibodies and PMA1 primer pairs are as in previous figures.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Phospho-proteomics, Chromatin Immunoprecipitation, Transformation Assay

Journal:

Article Title: Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain

doi: 10.1101/gad.935901

Figure Lengend Snippet: The CTDK-I kinase Ctk1 is required for CTD Ser 2 phosphorylation in vivo. Chromatin IP/PCR was carried out with a ctk1 deletion mutant combined with FCP1, fcp1-1, or fcp1-2 (YSB762 shuffled with pFK1, pFK4, and pFK7, respectively). Strains were also transformed with pY3AtURA to provide an HA epitope-tagged Rpb1 subunit. Ctk1 is the catalytic kinase subunit of CTDK-I. Immunoprecipitating antibodies and PMA1 primers are as in previous figures.

Article Snippet: CTD phosphorylation occurs predominantly at Ser 2 and Ser 5, and these modifications represent an important mechanism for regulating Pol II activity ( West and Corden 1995 ; Bensaude et al. 1999 ).

Techniques: Phospho-proteomics, In Vivo, Chromatin Immunoprecipitation, Mutagenesis, Transformation Assay

Journal: Science Advances

Article Title: Structure and noncanonical Cdk8 activation mechanism within an Argonaute-containing Mediator kinase module

doi: 10.1126/sciadv.abd4484

Figure Lengend Snippet: ( A ) Structural organization of Med12. The first and second helices (orange ribbon) in Med12N are labeled as H1 and H2, respectively. Five HEAT domains (Med12HEAT) are shown in transparent surface. ( B ) Domain organization of Med12. The N- and C-terminal regions of Med12 (Med12N and Med12C) that form interactions with Cdk8/CycC and Med13, respectively, are indicated. Colors are as in (A). ( C ) Interactions of Med12 with Cdk8, CycC, and Med13. Cdk8, CycC, and Med13 are shown in colored surface representations. ( D ) The Med12 N-terminal region (residues 1 to 105) associates with Cdk8/CycC. GST-Med12 fragments in Escherichia coli lysates as indicated were immobilized on glutathione Sepharose beads and incubated with yeast cell lysate (CycC-TAP/Med12Δ/Med13Δ) containing Cdk8/CycC. ( E ) Kinase activity of yeast Cdk8/CycC stimulated by GST-Med12-(1–105). Phosphorylation of GST-CTD-6xHis was detected by the antibody that recognizes phosphorylated Ser 5 of CTD. For GST-Med12-(1–105), 250 ng (+) or 1 μg (++) of protein was used in the reactions. ( F ) Immunoprecipitation (IP) assay. Deletion of the C-terminal region (residues 1346 to 1427) of Med12 caused loss of Med13 from CKM.

Article Snippet: The antibody that recognizes phosphorylated Ser 5 of CTD (GenScript, A10634) was used to detect CTD phosphorylation.

Techniques: Labeling, Incubation, Activity Assay, Phospho-proteomics, Immunoprecipitation

Journal:

Article Title: BRCA1 Can Modulate RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Levels

doi: 10.1128/MCB.24.16.6947-6956.2004

Figure Lengend Snippet: BRCA1 inhibits CTD phosphorylation in HCC1937 transfected cells. HCC1937 cells transiently transfected with the wild type or mutant BRCA1-C derivatives (A) or full-length BRCA1 (B) were analyzed by immunoblotting using antibodies raised against the serine 5-phosphorylated form of the CTD (upper panels) and the N-terminal region of Rpb1 (lower panels). IIo and IIa, the hyper- and the hypophosphorylated forms of RNA polII, respectively. Two, 3, and 4 μg of BRCA1-C constructs and 4, 6, and 8 μg of full-length BRCA1 constructs were used.

Article Snippet: A total of 100 μg of proteins from nuclear extracts was run by SDS-PAGE and revealed by immunoblotting with antibodies raised against serine 5-phosphorylated CTD (H14; BabCO) and Rpb1 (N20; Santa Cruz Biotechnology).

Techniques: Transfection, Mutagenesis, Western Blot, Construct

Journal: The Journal of Cell Biology

Article Title: Role of TIF1α as a modulator of embryonic transcription in the mouse zygote

doi: 10.1083/jcb.200603146

Figure Lengend Snippet: Ablation of TIF1α leads to aberrant localization of RNA polII, SNF2H, and BRG-1 but does not affect HP1β localization or histone H3 acetylation. (a) Zygotes were microinjected with the antibodies anti-Flag or anti-TIF1α or were not injected and were cultured for 7 h, until the late zygote stage. The embryos were then analyzed with the indicated antibodies using a 60× oil objective under confocal microscopy. For each antibody, embryos from the three groups were processed in parallel and were analyzed using the same confocal laser power. Shown are representative pronuclei of at least 10 zygotes analyzed for each experimental group and for each antibody used. The same results were observed in both female and male pronuclei. (b) Pattern of BrUTP incorporation upon ablation of TIF1α. BrUTP incorporation was analyzed by indirect immunofluorescence in zygotes after microinjection of antibodies. After injection of BrUTP and the indicated antibodies, embryos were cultured for 7 h, fixed, and analyzed using a BrdU antibody. All the samples were processed in parallel and analyzed under a 60× oil objective using the same confocal parameters. (left) Representative pronuclei of six (noninjected [ni] and Flag) and nine (TIF1α) zygotes. For quantification, the area of the pronuclei was first delimited and extracted using Volocity, and the area displaying BrUTP incorporation within each pronucleus was quantified using the same software. The graph shows the mean ± SD of at least six replicates for each group of embryos. *, P = 0.0001, t test. (c) BRG-1 was localized in the cytoplasm and was barely detected in the pronuclei upon TIF1α ablation. Zygotes were microinjected as in panel a and processed for immunostaining with a BRG-1 antibody in parallel. Shown are representatives of at least 10 zygotes.

Article Snippet: The antibodies used in this work are as follows: TIF1α (Santa Cruz Biotechnology, Inc.), KAP1 (TIF1β; Abcam), RNA polII (recognizing the CTD phosphorylated in Ser5; CTD4H8; Upstate Biotechnology), BRG-1 (Santa Cruz Biotechnology, Inc.), hSNF2H (provided by P. Varga-Weisz, The Babraham Institute, Cambridge, UK; ), tubulin (Sigma-Aldrich), HP1β (IGBMC), HP1γ (IGBMC), acetylated histone H4 (Upstate Biotechnology), acetylated K14 histone H3 (provided by B. Turner, University of Birmingham, Birmingham, UK), and acetylated K18 histone H3 (Abcam).

Techniques: Injection, Cell Culture, Confocal Microscopy, Immunofluorescence, Microinjection, Software, Immunostaining

Journal: Genes & Diseases

Article Title: Role of post-translational modification of the Y box binding protein 1 in human cancers

doi: 10.1016/j.gendis.2015.05.001

Figure Lengend Snippet: Known and confirmed post-translational modifications in human YBX1.

Article Snippet: S136, T271 , CTD , Phosphorylation , – , Kettenbach et al 2011.

Techniques: Phospho-proteomics

Journal: Genes & Diseases

Article Title: Role of post-translational modification of the Y box binding protein 1 in human cancers

doi: 10.1016/j.gendis.2015.05.001

Figure Lengend Snippet: Predicted but unconfirmed post-translational modifications in human YBX1.

Article Snippet: S136, T271 , CTD , Phosphorylation , – , Kettenbach et al 2011.

Techniques: Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: Schematic representation. ( A ) Domain organization of Spt6 and its variants used in this study. Residues for the N’ terminus, core, tSH2 domain and different constructs of Spt6 are indicated. The core consists of five domains: HtH, helix-turn-helix domain; YqgF, looks similar to the RuvC catalytical domain; HhH, helix-hairpin-helix domain; DLD, death-like domain; S1 domain. Pictograms correspond to the flexible N’ terminus, core, and tSH2 domain. ( B ) Cartoon representation of histones and DNA used for nucleosome reconstitution, binding and assembly assay. ( C ) Peptide sequences of Pol II Rpb1 linker and CTD used in binding studies. The residues of the peptides in red indicate phosphorylations

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: N-terminal region of Spt6 disrupts binding with Pol II and interacts with tSH2 domain and Spt6 core when added in trans. ( A ) Fluorescence anisotropy titration of phosphorylated Rpb1 CTD and Rpb1 linker peptides with Spt6 and ΔN-Spt6. Left panel – Rpb1 CTD binding with Spt6 lacking the flexible N-terminal region (cyan) K D ∼ 6.7 ± 1.56 μM; Spt6 (violet) K D ∼ n.d., not detected. Right panel – Rpb1 linker binding with Spt6 lacking the flexible N-terminal region (cyan) K D ∼ 11 ± 2.2 nM, Spt6 (violet) K D ∼ 290 ± 70 nM. Data represent means ± SD of technical triplicates. Normalized fluorescence anisotropy is plotted as a function of protein concentration. The data were normalized for visualization purposes and the experimental isotherms were fitted to a single-site saturation with non-specific binding model. ( B ) Fluorescence anisotropy titration shows that (left panel) N’ terminus binds to wild-type tSH2 (purple) K D ∼ 0.79 ± 0.20 μM, tSH2 variant with K1355A, K1435A double amino-acid substitutions (blue) K D ∼ 0.67 ± 0.39 μM, tSH2 variant with R1282H substitution (grey) K D ∼ n.d., not detected. ΔN-Spt6-ΔC associates with its N’ terminus (added in trans) with a K D ∼ 1.5 ± 0.7 μM (black, right panel). Points represent the mean ± SD of technical triplicates. The data were normalized for visualization purposes and the experimental isotherms were fitted to a single-site saturation with non-specific binding model

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Binding Assay, Fluorescence, Titration, Protein Concentration, Variant Assay

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: Cryo-EM structure of Spt6 and comparison with other structures of Spt6. ( A ) Graphical representation of construct used for Cryo-EM studies. ( B ) Local resolution estimation, shading from red to blue indicates local resolution according to colour gradient. Domains are identified. ( C ) Resolution estimated using gold-standard FSC. Resolution is given for FSC 0.143. Flexibile regions (the N-terminal part of the central helix and associated regions) were masked out. ( D ) Electron density map variability of reconstituted ΔN-Spt6. Overlay of two cryo-EM density maps highlighting extreme positions of the central helix in violet and light green. The remaining parts of Spt6 are virtually identical and are shown in grey. For detailed comparison, see . ( E ) Overlay of unbound and Pol II-bound Spt6 structures. The cryo-EM structure of Spt6 bound to Pol II (violet, PDB ID: 6TED, ) and the crystal structure of the free form of Spt6 (light green, PDB ID: 3PSF, ). The largest conformational changes are observed for the highlighted central helix, which correspond to the variability of the cryo-EM density maps shown in (D).

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Cryo-EM Sample Prep, Comparison, Construct

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: SAXS analysis of Spt6. Experimental SAXS profile of Spt6-ΔC ( A , top), Spt6 ( B , top), ΔN-Spt6 ( C , top) and theoretical scattering (red trace) calculated for model structures shown in bottom panels. ( D ) Structure of the Pol II–DSIF–PAF–SPT6 complex showing mutual orientation between the domains of SPT6 and Pol II. Pol II, pink; Rpb4, magenta; Rpb7, navy blue; Spt4, light green; Spt5, dark green; Spt6, grey; Spt6-tSH2, orange. Template and nontemplate DNA strands shown in blue and cyan, respectively; RNA, red. Color coding of Spt4, Spt5, DNA and RNA analogous as in for easier orientation

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Fragment-based Differential Targeting of PPI Stabilizer Interfaces

doi: 10.1021/acs.jmedchem.9b01942

Figure Lengend Snippet: Identification of fragments binding to the interface of 14-3-3σ with p53. (A) Fragment AZ-001. (B) Crystal structure of AZ-001 (yellow sticks) in complex with 14-3-3σ (white surface) and p53pT387 (orange sticks), PDB 6S40. The final 2 F o – F c electron density maps are shown as blue mesh. (C) Detailed view of the binding pocket of AZ-001. The most prominent interaction is a salt-bridge (dotted black line) between the amidine of AZ-001 and carboxyl group of E14 of 14-3-3σ. (D) Fragment AZ-002. (E) Crystal structure of AZ-002 (yellow sticks) in complex with 14-3-3σ (white surface) and p53pT387 (orange sticks), PDB 6RWI. The final 2 F o – F c electron density maps are shown as blue mesh. (F) Detailed view of the binding pocket of AZ-002. Also with AZ-002, the most prominent interaction is a salt-bridge (dotted black line) between the amidine of AZ-002 and carboxyl group of E14 of 14-3-3σ.

Article Snippet: CTD Thr387 phosphorylated 32-mer peptide of p53 (AnaSpec) was immobilized onto a CMD200 M sensor chip (Xantec Bioanalytics) by using standard NHS/EDC amine-coupling chemistry.

Techniques: Binding Assay