Journal: bioRxiv

Article Title: A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

doi: 10.1101/2020.01.08.899328

Figure Lengend Snippet: (A) SCC FAK-WT cells were transfected with siControl and siAmbra1 (siGENOME pool). After 48 h whole cell and nuclear lysates were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Lamin A/C and anti-GAPDH were used as a control for the purity of the nuclear lysates as well as a loading control. (B) SCC FAK-WT cells were transfected with siControl and siAmbra1. After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-Brg1, anti-Akap8, anti-Cdk8, anti-Cdk9, anti-p-Atf2 T71 and anti-Atf2. Anti-Histone H4 served as a marker for chromatin as well as a loading control. (C) The graph shows relative chromatin protein levels normalised to Histone H4.

Article Snippet: Antibodies used were as follows: anti-Paxillin and anti-GM130 antibodies (BD Transduction Laboratories, New Jersey, USA), anti-Akap8 and anti-Nup153 (Abcam, Cambridge, UK), anti-FAK, anti-PDI, anti-p-Atf2 T71, anti-Atf2, anti-Brg1, anti-Rpb1, anti-Cdk8, anti-Cdk9, anti-Histone H4, anti-H3K4me2, anti-H3K4me3, anti-H3K27Ac, anti-Histone H3, anti-Lamin A/C and anti-GAPDH (Cell Signaling Technologies, Danvers, MA, USA), as well as anti-Ambra1 antibody (Millipore, Billerica, MA, USA).

Techniques: Transfection, Western Blot, Marker

Journal: bioRxiv

Article Title: A novel transcriptional signalling pathway mediated by the trafficking protein Ambra1 via scaffolding Atf2 complexes

doi: 10.1101/2020.01.08.899328

Figure Lengend Snippet: (A) SCC FAK-WT cells were transfected with siControl and siAkap8 (siGENOME pool). After 48 h whole cell lysates and chromatin extracts were analysed by Western blot using anti-Ambra1, anti-FAK, anti-p-Atf2 T71, anti-Atf2 and anti-Akap8. Anti-Histone H4 served as a marker for chromatin as well as a loading control. (B) The graph shows relative chromatin protein levels normalised to Histone H4. (C) SCC FAK-WT cells were transfected with siControl, siCdk8 and siCdk9 (siGENOME pool). After 48 h whole cell lysates, nuclear and chromatin extracts were analysed by Western blot using anti-pAtf2 T71, anti-Atf2, anti-Cdk8 and anti-Cdk9. Anti-Lamin A/C, anti-GAPDH and anti-Histone H4 served as a control for the purity of nuclear and chromatin extracts as well as a loading control. (D) The graph shows relative nuclear or chromatin protein levels normalised to Lamin A/C or Histone H4 respectively. (E) Hypothesis model. In the nucleus of SCC cells Ambra1 and Akap8 form a complex and contribute to the recruitment of active Atf2 (p-Atf2 T71) to chromatin, most likely downstream via the Mediator complex component Cdk9. This strongly suggests that Ambra1 might be involved in chromatin remodeling and transcription. Further, together with Akap8 and Atf2 it might co-regulate the expression of a sub-set of genes.

Article Snippet: Antibodies used were as follows: anti-Paxillin and anti-GM130 antibodies (BD Transduction Laboratories, New Jersey, USA), anti-Akap8 and anti-Nup153 (Abcam, Cambridge, UK), anti-FAK, anti-PDI, anti-p-Atf2 T71, anti-Atf2, anti-Brg1, anti-Rpb1, anti-Cdk8, anti-Cdk9, anti-Histone H4, anti-H3K4me2, anti-H3K4me3, anti-H3K27Ac, anti-Histone H3, anti-Lamin A/C and anti-GAPDH (Cell Signaling Technologies, Danvers, MA, USA), as well as anti-Ambra1 antibody (Millipore, Billerica, MA, USA).

Techniques: Transfection, Western Blot, Marker, Expressing

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: STAT1 and STAT3 HyIL-6-Induced Ser727 Phosphorylation Is CDK8/9 Mediated (A and B) Spider plots showing pTyr701 STAT1 (A) or pTyr705 STAT3 (B) (blue line) and pSer727 STAT1 (A) or pSer727 STAT3 (B) (red line) MFI normalized to HyIL-6-treated cells in the presence of different inhibitors in human primary CD4 + Th-1 cells. (C) Effect of different mTOR inhibitors on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (D) Effect of ATM inhibitor (KU53933) and DNA-PK inhibitor (KU57788) on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (E) Effect of different CDK inhibitors on the STAT3 Tyr705 (top panel) and STAT3 Ser727 (bottom panel) phosphorylation induced by HyIL-6 in human primary CD4 + T cells. For all experiments, quantitative data were calculated from three individual biological replicates. Error bars show mean ± SEM.

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques:

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: PLA Analysis of the Interaction of STAT3 and CDK8/9 Induced upon HyIL-6 Stimulation in Human Primary CD4 + Th-1 Cells (A and B) Kinetics of the STAT3/CDK8 (A) or STAT3/CDK9 (B) interaction induced by 20 nM HyIL-6 in human primary CD4 + Th-1 cells. Scale bars, 20 μm. Statistical significance was calculated by one-way ANOVA. (C and D) STAT3/CDK8 (C) or STAT3/CDK9 (D) interactions were analyzed by PLA upon 20 nM HyIL-6 stimulation in the absence or presence of 2 μM MSC2530818 or 2 μM flavopiridol or upon treatment with the inhibitor only. Scale bars, 20 μm. Statistical significance was calculated by unpaired t test. White arrows in A to D indicate examples of cells where interaction signal was detected. Cumulative plots from n = 15 pictures alongside show the percentage of positive cells. Error bars show mean ± SEM. The p values were calculated based on non-parametric two-tailed Wilcoxon rank-sum test against the control group (first bar on the left). (E) STAT3/CDK9 interaction analyzed by PLA upon 20 nM HyIL-6 stimulation in STAT3 KnD Hut78 cells reconstituted with STAT3 WT-GFP (top panel) or STAT3 S727A-GFP (bottom panels). White arrows indicate examples of cells expressing the recombinant protein and where the STAT3/CDK9 interaction was detected by PLA. Scale bars, 20 μm. Graphs alongside show the nuclear GFP MFI normalized to unstimulated cells (top graph) or the nuclear STAT3/CDK9 PLA MFI in GFP-positive cells normalized to unstimulated cells (bottom graph). Quantitative data generated from n = 15 pictures. Error bars show mean ± SEM.

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques: Two Tailed Test, Expressing, Recombinant, Generated

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Regulation of the Phosphoproteomic Landscape of IL-6 in Human Primary CD4 + Th-1 Cells by CDK8 (A) Volcano plot of the CDK8-dependent HyIL-6-upregulated phosphosites in human primary Th-1 cells (top panel) and the 24 more affected phosphosites (bottom panel). (B) Volcano plot of the CDK8-dependent HyIL-6-downregulated phosphosites in human primary Th-1 cells (top panel) and the 24 more affected phosphosites (lower panel). Phosphopeptides identified in three biological replicates are shown as log-transformed SILAC ratios plotted against log-transformed p values (two-sided t test). Select phosphopeptides are labeled (see for full list). For (A) and (B), phosphosites regulated by HyIL-6- in a CDK8-dependent way and changed more than 1.5-fold with a p value of <0.05 are shown in red (decreased) or blue (increased), and highlighted in dark gray is the effect of MSC2530818 on those same phosphosites. (C) GO analysis showing the cellular location of the phosphosites regulated by HyIL-6 in a CDK8-dependent manner. (D) GO analysis showing the main pathways and cellular processes regulated by HyIL-6 in human primary Th-1 cells. (E) GO analysis showing the main pathways and cellular processes regulated by HyIL-6 in human primary Th-1 cells in a CDK8-dependent manner. (F) Pie charts showing the number of HyIL-6-regulated and CDK8-dependent phosphosites involved in the regulation of transcription (left graph) or RNA-Pol-II-mediated transcription (right graph). (G) The scheme shows the cellular location and molecular function of the proteins regulated by phosphorylation in response to HyIL-6 stimulation in human primary CD4 + Th-1 cells in a CDK8-dependent fashion as determined by DAVID analysis.

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques: Transformation Assay, Labeling

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Transcriptional Program Elicited by Interplay between HyIL-6 and CDK8 in Human Primary CD4 + Th-1 Cells (A) Number of differentially expressed genes (DEGs; fold chang,e >1.5; p < 0.05) between unstimulated versus HyIL-6-, mesenchymal stem cell (MSC)-, or HyIL-6+MSC-stimulated Th-1 cells in three biological replicates. (B) Scatterplot showing mean gene expression values (n = 3) before (x axis) and after indicated stimulation (y axis). Upregulated (red) and downregulated (blue) genes are highlighted. (C) Representative gene expression across different stimulation. Bars show mean ± SEM. (D) Gene set enrichment analysis (GSEA) ( Subramanian et al., 2005 ) plots for STAT3 upregulated genes (GEO: GSE21670) comparing stimulated versus unstimulated Th-1 transcriptomes. NES, normalized enrichment score; FDR, false discovery rate. (E) Violin plot showing the mean STAT3 binding intensity in n = 2,585 STAT3-bound regions across different stimulations. Peaks are identified by comparing HyIL-6+MSC stimulation and input. The p values were determined by two-tailed Wilcoxon rank-sum test ( ∗∗∗∗ p < 0.0001). (F) Representative loci showing STAT3 binding across different stimulations. The height of the tracks are indicated at bottom-right corner of the plots. (G) GSEA plots for 475 STAT3-bound genes comparing stimulated versus unstimulated Th-1 transcriptomes.

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques: Expressing, Binding Assay, Two Tailed Test

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Role of CDK8 Ser727 Phosphorylation of STAT3 in Th-17 Differentiation In Vitro (A) Experimental workflow for human Th-17 differentiation in vitro from isolated human resting CD4 + T cells. (B and C) Dot plot representations of IL-17- and IFNγ-positive cells in populations grown in the presence of HyIL-6 (B) or HyIL-6 + MSC2530818 (C). (D) IL-17-positive cells were identified by flow cytometry in untreated cells or cells treated with 2 μM MSC2530818. Data are percentage of positive cells ± SEM in four biological replicates; p values were calculated using a paired t test. (E) As in (D) but for IFNγ-positive cells. (F) Amount of IL-17 ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. (G) Amount of IFNγ ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. Statistical significance was calculated by unpaired t test.

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques: In Vitro, Isolation, Flow Cytometry

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: Rabbit anti-CDK8 , Invitrogen , Cat#PA1-21780; RRID: AB_2291488.

Techniques: Purification, Recombinant, Software

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A) A representative silver stained gel for FS2-FBXL19 purification and an empty vector control (EV) purification. Asterisk identifies the band corresponding to FS2-FBXL19 protein. (B) In order to visualise FBXL19 protein by western blot in and , the Fbxl19 gene was tagged by T7 knock-in in Fbxl19 fl/fl ES cells using the CRISPR Cas9 system. A schematic representation of the generation of the C-terminal T7 knock-in Fbxl19 is shown. HA1/2 indicate the homology arms of the targeting construct. (C) Western blot analysis of the expression of T7-FBXL19 from nuclear extract of the generated T7 knock-in ES cell line. (D) Western blot analysis of endogenous co-immunoprecipitation (IP) of FBXL19, CDK8 and MED12 from ES cell nuclear extracts. A control IP using a non-specific antibody (α-ΗΑ) was included. (E) A schematic illustration of the different FS2-FBXL19 truncation mutants and Western blot analysis of purification of FS2-FBXL19 mutants from HEK293T cells probed with the indicated antibodies. (F) Western blot analysis of FS2-FBXL19 and control purifications (EV) probed with the indicated antibodies. (G) Western blot analysis of histone extracts generated from Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells probed with two different antibodies recognizing ubiquitylated H2B K120 (H2Bub1). H4 was used as a loading control. (H) Western blot analysis of nuclear extracts from HEK293T cells transiently transfected with empty vector (EV) or Flag-FBXL19-expressing vector without (-) or following MG132 treatment (+). Blots were probed with the indicated antibodies. TBP was used as loading control.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Staining, Purification, Plasmid Preparation, Western Blot, Knock-In, CRISPR, Construct, Expressing, Generated, Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A) A Venn diagram showing the overlap between CDK8 peaks (n=24273) and NMIs (n=27698). (B) A scatter plot showing Spearman correlation between CDK8 signal and BioCAP at NMIs. (C) Heatmaps showing enrichment of CDK8, FS2-FBXL19 and BioCAP at FBXL19 peaks (n=11322) sorted by decreasing FBXL19 signal. (D) A schematic of the Fbxl19 fl/fl allele showing the location of the loxP sites. Treatment with tamoxifen (OHT) results in Cre-mediated deletion of the second exon, encoding for the ZF-CxxC domain but leaving the rest of the gene and protein intact. (E) RT-qPCR of FBXL19 expression in Fbxl19 fl/fl ES cells before (WT) and following tamoxifen treatment (OHT). Primers specific for the ZF-CxxC and LRR domains were used. Expression is relative to expression in WT ES cells. Error bars show SEM of three biological experiments. (F) A Western blot analysis of the expression of Mediator subunits in Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT) ES cells. HDAC1 and TBP were probed as loading controls. (G) Heatmaps showing enrichment of CDK8 in Fbxl19 fl/fl (WT) and Fbxl19 ΔCXXC (OHT), FS2-FBXL19 and BioCAP signal at ATAC peaks divided based on change in CDK8 binding in Fbxl19 ΔCXXC ES cells as in . The mean of the enrichment for each group is shown above the heatmaps. Left: zoomed heatmaps for ATAC peaks associated with a decrease (↓) or an increase (↑) in CDK8 binding and a differential heatmap representing log2 fold change of CDK8 signal between OHT and WT samples at the differential peaks. (H) Percent overlap between CDK8 peaks (as in ) and transcription start sites of genes (TSS, left) or FBXL19 peaks (right). (I) Metaplots showing enrichment of FS2-FBXL19, BioCAP and CDK8 ChIPseq signal at all CDK8 peaks (left), FBXL19-bound CDK8 peaks (FBXL19+, middle) and nonFBXL19 CDK8 peaks (FBXL19-, right). Peaks were divided based on change in CDK8 binding in Fbxl19 ΔCXXC ES cells as in .

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A) A schematic illustrating how addition of OHT (4-hydroxytamoxifen) leads to the generation of Fbxl19 ΔCXXC ES cells. (B) Western blot analysis of an OHT-treatment time course in the Fbxl19 fl/fl ES cell line. Hours of treatment are shown. FBXL19 protein was C-terminally tagged with a T7 epitope ( and ) to allow for Western blot detection with an anti-T7 antibody. TBP was probed as a loading control. (C) Metaplots showing CDK8 enrichment in Fbxl19 fl/fl ES cells (WT) and Fbxl19 ΔCXXC ES cells (OHT) at all CDK8 peaks (left), peaks showing decreased CDK8 occupancy (↑, middle) and peaks showing increased CDK8 occupancy (↓, right). The number of total peaks in each group is indicated. p-values denote statistical significance calculated by Wilcoxon rank sum test comparing ChIP-seq read counts between WT and OHT samples across a 1.5-kbp interval flanking the center of CDK8 peaks. (D) Screen shots showing ChIP-seq traces for CDK8 in wild type Fbxl19 fl/fl ES cells (WT) and Fbxl19 ΔCXXC ES cells (OHT). BioCAP and FS2-FBXL19 tracks are given for comparison. Left – CDK8 peaks showing reduced CDK8 binding in Fbxl19 ΔCXXC ES cells; Right – CDK8 peaks showing increased CDK8 binding in Fbxl19 ΔCXXC ES cells (indicated with rectangles). (E) Boxplots showing log2 fold change (log2FC) of CDK8 ChIP-seq signal (RPKM) at all CDK8 peaks (n=24273), and those with reduced CDK8 (n=783,↓) and increased CDK8 (n=379, ↑). p-values were calculated using a Wilcoxon rank sum test. (F) Boxplots showing size of CDK8 peaks as in (E). p-values were calculated using a Wilcoxon rank sum test. (G) Venn diagrams representing the overlap between CDK8 peaks and super enhancers (SE) at CDK8 peaks as in (E). Percent overlap of all SEs is indicated. (H) A metaplot (left) showing BioCAP enrichment at CDK8 peaks as in (E) and boxplot quantification (right) of BioCAP RPKM levels. p-values calculated using Wilcoxon rank sum test are indicated. (I) A metalplot (left) showing FS2-FBXL19 enrichment at CDK8 peaks as in (E) and boxplot quantification (right) of FS2-FBXL19 RPKM levels. p-values calculated using Wilcoxon rank sum test are indicated.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A) Gene ontology analysis of genes associated with a decrease in CDK8 binding. (B) A boxplot showing expression levels (log2FPKM) of CDK8-bound genes in wild type ES cells. Genes are divided based on CDK8 binding in Fbxl19 ΔCXXC ES cells as in . p-values were calculated using a Wilcoxon rank sum test. (C) A boxplot showing the change in gene expression (log2 fold change) observed by 4sU RNA-seq of CDK8-bound genes (as in ) following RA treatment. p-values were calculated using a Wilcoxon rank sum test. (D) A boxplot comparing gene expression levels (log2FPKM) of all (n=19310) and FBXL19-bound (FBXL19+, n=11368) genes separated by low (all genes n=7417; FBXl19+ genes n=2031) and high expression levels (all genes n=11893, FBXl19+ genes n=9337) in ES cells (based on ). (E) A boxplot showing CDK8 enrichment at all and FBXL19-bound genes separated by expression level as in (D). (F) A boxplot showing change in CDK8 binding at the TSSs of all and FBXL19-bound genes divided by expression level as in (D). p-values were calculated using a Wilcoxon rank sum test.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Binding Assay, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A)Metaplots showing enrichment of H3K27me3 (left) and H3K4me3 (right) ChIPseq signal at all (top), reduced(↓) and increased(↑) CDK8 peaks. (B) Heatmaps showing enrichment of CDK8, H3K27me3 and H3K4me3 ChIPseq signal at H3K27me3 peaks (n=5588) divided by overlap with CDK8 peaks (CDK8+ n= 3637, CDK8-n=1950) sorted by decreasing H3K27me3 signal. Distance from the peak center is shown. (C) Volcano plots showing alterations in expression (log2 fold change) comparing ES cells and RA-treated cells. Genes that rely on FBXL19 for normal CDK8 binding in the ES cells state are plotted. Top: reduced levels of CDK8 (red, n=673 of which 203 genes are significantly induced by RA and 55 genes are ES-specific). Bottom: increased levels of CDK8 (green, n=255 of which 28 genes are significantly induced by RA and 26 genes are ES-specific). (D) Genes for D-F were separated into two categories (low and high expression) based on FPKM value cut-off represented by the dashed line.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Expressing, Binding Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A)RT-qPCR gene expression analysis of developmental genes during an embryoid body differentiation time course. Expression is relative to an average of two house-keeping genes. Error bars represent SEM of three biological replicates. (B) A volcano plot showing differential expression (log2 fold change) comparing ES cells and RA-induced cells. Differentially expressed genes (log2FC < −0.5 or log2FC > 0.5, padj <0.05) are shown in red. The number of genes considered to be significantly different in expression are indicated. (C) Gene ontology analysis of the genes induced following RA treatment. (D) Gene ontology analysis of the up-regulated genes in Fbxl19 ΔCXXC cells following RA treatment. (E) Expression changes of CDK8-target genes (log2 fold change) in Fbxl19 ΔCXXC cells compared to WT cells in ES cells (ESC, left) and following RA treatment (right). Genes are divided based on the changes in CDK8 binding in Fbxl19 ΔCXXC ES cells-all CDK8 peaks, reduced CDK8 peaks (↓) and increased CDK8 peaks (↑). p-values were calculated using a Wilcoxon rank sum test.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A) A schematic illustrating the OHT treatment and differentiation approach. (B) RT-qPCR gene expression analysis of genes showing decreased CDK8 binding in Fbxl19 ΔCXXC ES cells before (ESC) and after RA induction. Expression is relative to the average of two house-keeping genes. Error bars show SEM of three biological replicates, asterisk represent statistical significance calculated by Student T-test: * p<0.05, ** p<0.01, *** p<0.001. (C) Volcano plots showing differential expression (log2 fold change) comparing WT and Fbxl19 ΔCXXC cells in the ES cell (top) and RA-induced state (bottom). Differentially expressed genes (log2FC < −0.5 or log2FC > 0.5, padj <0.1) are shown in red. The number of genes considered to be significantly altered in expression are indicated. (D) Gene ontology analysis of genes with decreased expression in Fbxl19 ΔCXXC ES cells following RA treatment. (E) A boxplot indicating the expression level (FPKM) based on 4sU RNA-seq in wild type ES cells of the gene groupings in (C): n.c. genes n=17869 (no change), up genes = 889, down genes = 552. p-values calculated using a Wilcoxon rank sum test are indicated. (F) A boxplot indicating the log2 fold change in gene expression of the gene groupings in (E) upon RA differentiation of ES cells. p-values calculated using a Wilcoxon rank sum test are indicated. (G) A boxplot indicating the change in CDK8 binding (log2FC) at the promoters of the gene groupings in (E) in ES cells. p-values calculated using a Wilcoxon rank sum test are indicated.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: FBXL19 recruits CDK-Mediator to the CpG islands of developmental genes to prime them for activation during lineage commitment

doi: 10.1101/299552

Figure Lengend Snippet: (A)A schematic illustrating the OHT treatment to generate MED13/13L KO ES cells and differentiation approach. (B)Western blot analysis showing the efficiency of MED13/13 knock-out upon 96h OHT treatment of Med13/13L fl/fl ES cells. Suz12 was blotted as a loading control. (C) ChIP-qPCR showing CDK8 enrichment in WT and MED13/13L KO ES cells. Enrichment is relative to gene desert control region. Error bars show standard deviation of two biological replicates. (D)RT-qPCR gene expression analysis in WT and MED13/13L KO ES cells before and after RA induction. Expression was normalised to the expression of the PolIII-transcribed gene tRNA-Lys and is represented as relative to WT ES cells (for pluripotency genes) or RA-treated WT cells (for differentiation markers). Error bars show SEM of three biological replicates, asterisks represent statistical significance calculated by Student T-test: * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: Antibodies used for Western blot analysis were mouse anti-Flag M2 (F1804, Sigma-Aldrich), mouse anti-SKP1 (sc-5281, Santa Cruz), goat anti-CDK8 (sc-1521, Santa Cruz), rabbit anti-MED12 (A300-774A, Bethyl laboratories), rabbit anti-MED13L (A302-420A, Bethyl laboratories), rabbit anti-MED13 (GTX129674, Genetex), rabbit anti-MED1, (A300-793A, Bethyl laboratories), rabbit anti-MED26 (A302-370A, Bethyl laboratories), rabbit anti-T7 (13246, Cell Signaling Technology), rabbit anti-TBP (ab818, Abcam), rabbit anti-RNF20 (11974, Cell Signaling Technology), rabbit anti-ubiquityl-Histone H2B (Lys120) (5546, Cell Signaling Technology), and mouse anti-ubiquityl-Histone H2B (Lys120) (05-1312, Millipore).

Techniques: Western Blot, Knock-Out, Standard Deviation, Quantitative RT-PCR, Expressing