Journal: Nucleic Acids Research

Article Title: Human snRNA genes use polyadenylation factors to promote efficient transcription termination

doi: 10.1093/nar/gkt892

Figure Lengend Snippet: Human snRNA genes do not use an Nrd1-like pathway to terminate transcription in vivo . ( A ) Western blot analysis of HeLa whole-cell extracts from control cells or cells transfected with an siRNA specific for SCAF8, senataxin and Xrn2. Antibodies used for detection were α-SCAF8 (left), α-Senataxin (middle) and α-Xrn2 (right). α-TATA-binding protein was used as a control for protein levels. ( B ) Schematic of the β-actin gene with the location of the primers used in ChIP assays indicated. The 5′ pause is described in . The arrow represents the TSS, exons are denoted by boxes and the position of the poly A site (pA) is indicated by a vertical line. Graphs represent results of pol II ChIP analysis before and after siRNA-mediated knockdown of Xrn2 (left graph), Senataxin (middle graph) and SCAF8 (right graph). ( C ) Schematics of the U1 and U2 snRNA genes are labelled as in . Graphs represent results of pol II ChIP analysis of the U1 (left panel) and U2 (right panel) snRNA genes before and after siRNA-mediated knockdown of Xrn2 (top), Senataxin (middle) and SCAF8 (bottom). Levels of pol II quantitated in (A) and (B), in control and knockdown cells, are normalized to levels quantitated on a non-transcribed region of the genome (120 bp region ∼2 kb upstream of the U2 snRNA gene). Error bars represent standard deviation of at least three independent experiments.

Article Snippet: Antibodies used in western blot studies were purchased from the following manufacturers: anti-SCAF8 (Bethyl, A301-036 A), anti-Senataxin (Bethyl, A301-105 A), anti-Int5 (Bethyl, A301–268 A), anti-Int9 (Bethyl, A300-412 A) and anti-Int11 (abcam, ab75276); anti-Xrn2 was a gift from Natalie Gromak ( ).

Techniques: In Vivo, Western Blot, Transfection, Binding Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Human snRNA genes use polyadenylation factors to promote efficient transcription termination

doi: 10.1093/nar/gkt892

Figure Lengend Snippet: Transcription termination pathways used by pol II transcribing human mRNA and snRNA genes. Termination factors that are common between both genes types are labelled in the same colour. Pol II CTD phosphorylation state is indicated as Phospho-Ser2 (S2P) and Phospho-Ser7 (S7P) ( A ). For protein-coding genes, the CPSF complex and the CstF complex recognize the poly A signal (AAUAAA) and U/GU-rich elements, respectively, as they emerge from the elongation complex. The CPSF-73 subunit, aided by CPSF-100, catalyzes cleavage at the poly A site (indicated by a lightning bolt) creating an entry site for the 5′–3′ exoribonuclease, Xrn2. Xrn2 degrades the downstream RNA product and displaces pol II. The CTD interacting domain of Pcf11 associates with the S2P form of the pol II CTD and bridges the polymerase with the nascent transcript, leading to pol II pausing and dismantling of the elongation complex. ( B ) The gene-specific PTF complex is recruited to the snRNA promoter and clears the transcription unit of nucleosomes to facilitate passage of pol II. The NELF complex is recruited at the end of the nucleosome-depleted region to terminate pol II where nucleosomes are encountered. CPSF-73 is not recruited to human snRNA genes but instead the Integrator complex directs cleavage upstream of the 3′ box processing element (indicated by a lightning bolt). The Int11 subunit is likely to catalyze the cleavage reaction with the help of Int9, as these subunits are homologous to CPSF-73 and CPSF-100, respectively. Thus, Integrator substitutes for the CPSF complex on snRNA genes. Ssu72 and Pcf11 are recruited to snRNA genes but do not have a major function in snRNA 3′-end processing. Instead, Ssu72 and Pcf11 act as transcription terminators for this gene class. CstF64 is also recruited, but its role in snRNA processing and transcription termination is not clear. Xrn2 is not implicated in snRNA transcription termination, but as cleavage upstream of the 3′ box would create an entry site for a 5′–3′ exoribonuclease, we cannot rule out that an alternative enzyme is recruited to promote termination.

Article Snippet: Antibodies used in western blot studies were purchased from the following manufacturers: anti-SCAF8 (Bethyl, A301-036 A), anti-Senataxin (Bethyl, A301-105 A), anti-Int5 (Bethyl, A301–268 A), anti-Int9 (Bethyl, A300-412 A) and anti-Int11 (abcam, ab75276); anti-Xrn2 was a gift from Natalie Gromak ( ).

Techniques:

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Immunoblots showing levels of FLAG-tagged MYCN (amino acids [aa] 2–137) and the six subunits of the TFIIIC complex after a pull-down assay using anti-FLAG affinity columns. Multiple columns labelled ‘Wash’ represent the sequential washings (n=2). ( B ) Size exclusion chromatography graph of MYCN (aa 1–137)/TauA (τA) (black trace) or MYCN alone (red trace). The blue box marks the fractions used for panels C and D (n=2). ( C ) Coomassie staining of fractions of the MYCN (aa 1–137)/τA complex (fractions marked with blue box in panel B). ( D ) Immunoblot of fractions of the MYCN (aa 1–137)/τA complex (fractions marked with blue box in panel B). ( E ) Growth curve (measured as % confluence) of SH-EP-MYCN-ER cells expressing doxycycline (Dox)-inducible shRNA targeting TFIIIC2 , TFIIIC3, or TFIIIC5 under the indicated conditions. Data show mean ± standard deviation (SD) (n=3). Figure 1—source data 1. Raw unedited gels for . Figure 1—source data 2. Uncropped and labelled gels for . Figure 1—source data 3. Raw unedited Coomassie images for . Figure 1—source data 4. Uncropped and labelled Coomassie images for . Figure 1—source data 5. Raw data for graphs shown in .

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Western Blot, Pull Down Assay, Size-exclusion Chromatography, Staining, Expressing, shRNA, Standard Deviation

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) SDS-PAGE gel of recombinant purified MYCN (amino acids [aa] 1–137) expressed in Escherichia coli cells. ( B ) SDS-PAGE gel of recombinant purified TauA (τA) subcomplex which was expressed in Spodoptera frugiperda (Sf9) cells. ( C ) Deconvoluted spectra of native mass spectrometry for the τA/3x FLAG-MYCN complex. Masses are shown in red, identities of complexes are marked in blue. ( D ) Boxplot of the 30 possibleτA/3x FLAG-MYCN complex molecular weights based on the masses observed using intact mass spectrometry. The red line indicates the mass observed for the complex by native mass spectrometry (204,763 Da). ( E ) Immunoblot showing levels of TFIIIC2, TFIIIC3, or TFIIIC5 and MYC in SH-EP-MYCN-ER cells expressing doxycycline (Dox)-inducible shRNAs targeting the different TFIIIC subunits (n=3). Where indicated cells were treated with Dox (1 µg/ml, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (200 nM, 4 hr), respectively. EtOH was used as control. In the following panels addition of 4-OHT is indicated by ‘+MYCN’ and Dox treatment by ‘– TFIIIC’. ( F ) Immunoblot of MYC and MYCN in SH-EP-MYCN-ER cells after induction of MYCN with 4-OHT (200 nM, 4 hr). VCL was used as a loading control. Figure 1—figure supplement 1—source data 1. Raw unedited Coomassie images for . Figure 1—figure supplement 1—source data 2. Uncropped and labelled Coomassie images for . Figure 1—figure supplement 1—source data 3. Raw data for graphs shown in . Figure 1—figure supplement 1—source data 4. Raw unedited gels for . Figure 1—figure supplement 1—source data 5. Uncropped and labelled gels for .

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: SDS Page, Recombinant, Purification, Mass Spectrometry, Western Blot, Expressing, Control

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Browser tracks for non-phosphorylated RNAPII (top) and RNAPII pSer2 (bottom) ChIP-Rx at the indicated gene loci. SH-EP-MYCN-ER cells were treated with doxycycline (Dox) (1 µg/ml, 48 hr) and/or 4-hydroxytamoxifen (4-OHT), respectively. EtOH was used as control. ( B ) Average density plot of ChIP-Rx signal for non-phosphorylated RNAPII. Data show mean (line) ± standard error of the mean (SEM indicated by the shade) of different gene sets based on an RNA-sequencing (RNA-seq) of SH-EP-MYCN-ER cells ± 4-OHT. The y-axis shows the number of spike-in normalised reads and it is centred to the TSS ± 2 kb. N=number of genes in the gene set defined in the Methods (n=2). ( C ) Density plot of ChIP-Rx signal for RNAPII pSer2 as described for panel B. The signal is centred to the transcription end site (TES) ± 2 kb (n=2). ( D ) Average bin dot plot showing fold change for RNAPII pSer2 ChIP-Rx reads over TES ± 2 kb and RNA-seq of SH-EP-MYCN-ER for the same genes ± MYCN + TFIIIC5 (blue) or + MYCN ± TFIIIC5 (red). The plot shows 20 bins representing a total of 13,239 and 12,330 genes for ± MYCN + TFIIIC5 and + MYCN ± TFIIIC5 datasets, respectively (n=3 for RNA-seq, n=2 pSer2 RNAPII ChIP-Rx). ( E ) Average bin dot plot for RNA-seq of SH-EP-MYCN-ER showing log2 mRNA expression normalised by control per bin. Cells were treated with 1 µg/ml Dox (‘– TFIIIC5’, 48 hr) and/or 4-OHT (‘+MYCN’, 4 hr) or EtOH as control. Expression was normalised by its control. Each bin represents 150 genes of a total of 14,085 genes. Dotted line marks the relative expression at 0 (n=3). ( F ) Density plot of ChIP-Rx signal for TFIIIC5. Data show mean (line) ± SEM (shade) for 14,722 genes. The signal is centred to the TSS ± 2 kb (n=2).

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Control, RNA Sequencing, Expressing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Metagene plot of ChIP-Rx signal for non-phosphorylated RNAPII. Data show mean (line) ± standard error of the mean (SEM indicated by the shade) of different gene sets based on an RNA-sequencing (RNA-seq) of SH-EP-MYCN-ER cells ± 4-hydroxytamoxifen (4-OHT). ( B ) Metagene plot of ChIP-Rx signal for RNAPII pSer2. Data are presented as described in (A). ( C ) Average density plot of ChIP-Rx signal for non-phosphorylated RNAPII in SH-EP cells treated with 4-OHT. Data show mean (line) ± SEM (indicated by the shade) of different gene sets based on an RNA-seq of SH-EP-MYCN-ER cells ±4 OHT. The signal is centred to the TSS ± 2 kb (n=2). ( D ) Average bin dot plot for RNA-seq of SH-EP-MYCN-ER showing mRNA expression normalised by control per bin. Cells were co-treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC3’, 48 hr) and/or 4-OHT (‘+ MYCN’, 4 hr) as EtOH as control. Expression was normalised by its control. Each bin represents 100 genes of a total of 12,091 genes. Dotted line marks the relative expression at 1 (n = 3). ( E ) Browser tracks for TFIIIC5 ChIP-Rx at the indicated gene loci. SH-EP-MYCN-ER cells were treated with 5,6-dichlorobenzimidazole-1-β-D-ribofuranoside (DRB) and/or 4-OHT, respectively. EtOH was used as control.

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: RNA Sequencing, Expressing, Control

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Diagram showing the workflow of p hosphorylated l inker HiChIP (pLHiChIP). The workflow corresponds to the original HiChIP protocol. For a detailed description, see Methods. ( B ) MYCN and MYC ChIP in SH-EP-MYCN-ER cells before and after activation of MYCN with 4-hydroxytamoxifen (4-OHT) (200 nM, 24 hr). Shown is the mean and points of technical triplicates normalised to an intergenic region (n=2). ( C ) (Top) Diagram illustrating the difference between valid and invalid pairs after mapping of s pike-in p hosphorylated l inker HiChIP (spLHiChIP) data. Valid pairs harbour non-contiguous elements of chromosomal DNA, whereas invalid pairs harbour contiguous stretches of chromosomal DNA. (Bottom) Bar graph showing percentage of valid and invalid reads in our dataset (right) compared to a published HiC protocol (left). ( D ) Table showing quality controls for MYCN and TFIIIC5 spLHiChIP compared to the original Oct4 HiChIP. ( E ) Representative example of pLHiChIP track for MYCN (red) and a phosphorylated linker Hi-C (pLHi-C) track (blue) showing the strongly increased PETs number of the MYCN interactions after MYCN immunoprecipitation relative to input (pLHi-C). Data are superimposed with a browser view of an MYCN ChIP-sequencing (ChIP-seq). ( F ) Table showing interactions involving tRNA genes in MYCN pLHiChIP. ( G ) Table showing the top three terms, their p-values, and the false discovery rate (FDR) from enrichment analysis for MSigDB C5 collection of all MYCN network. Figure 3—figure supplement 1—source data 1. Raw data for data shown in .

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: HiChIP, Activation Assay, Hi-C, Immunoprecipitation, ChIP-sequencing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Top: Representative browser track of MYCN three-dimensional chromatin interactions. Height shows the number of p aired e nd t ags (PETs) indicating the interaction intensity and the width of the line shows the start and end positions of each anchor. Middle and bottom: Browser tracks showing the number of reads of MYCN and total RNAPII ChIP-Rx, respectively. Unless stated, all experiments were performed in SH-EP-MYCN-ER cells treated with 4-hydroxytamoxifen (4-OHT) (200 nM, 4 hr). The ruler at the bottom shows the genomic coordinates (n=3 independent biological replicates for MYCN p hosphorylated l inker HiChIP [pLHiChIP]; n=2 for RNAPII ChIP-Rx). ( B ) Bar chart listing functional annotations of all binary MYCN interactions (N=4591; N indicates total number). ( C ) Boxplots showing relative binding of the indicated proteins (RNAPII, MYCN, TFIIIC5) to promoter regions or expression levels of the corresponding genes (mRNA by RNA-sequencing [RNA-seq]; 4sU by 4sU-seq). Red boxes: Genes bound by MYCN and part of MYCN-hubs. Blue boxes: Genes bound by MYCN that are not part of MYCN-hubs. Each pair was normalised to the median of the corresponding ‘blue’ gene set. p-Values were obtained by pairwise comparisons using Student’s t - test (n=2 for TFIIIC5 and RNAPII ChIP-Rx). ( D ) Boxplot showing the number of promoters in each cluster, with each red dot representing one cluster. ( E ) Network reconstruction of the three biggest clusters based on MYCN pLHiChIP interactions. Each anchor is represented by a node (‘triangle’) and the lines show interactions between the anchors. The colours are indicating the different functional annotation.

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: HiChIP, Functional Assay, Binding Assay, Expressing, RNA Sequencing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Representative example of p hosphorylated l inker HiChIP (pLHiChIP) track for MYCN (red) and TFIIIC5 (blue) interactions (conventions as in ) (n=2). ( B ) Bar chart listing the total number of functional annotations for all TFIIIC5 binary interactions (N=3499). ( C ) Venn diagram showing the number of interactions shared between MYCN and TFIIIC5. The diagram at the left shows the types of overlaps between connections. ( D ) Bar chart listing the interaction functional annotations for MYCN anchors not overlapping with TFIIIC5 anchors (‘MYCN only’) as well as TFIIIC5 anchors without overlapping MYCN anchors (‘TFIIIC5 only’) and their joint anchors. ( E ) Representative example of MYCN s pike-in p hosphorylated l inker HiChIP (spLHiChIP) track for MYCN interactions in the presence (blue) or absence (red) of TFIIIC5 (n=2). ( F ) Bar graph showing the fold change of all MYCN spLHiChIP interactions comparing ‘+TFIIIC5’ and ‘– TFIIIC5’ in SH-EP-MYCN-ER cells expressing a doxycycline (Dox)-inducible shRNA targeting TFIIIC5 . n1,2 indicates two independent biological replicates. ( G ) Representative example of TFIIIC5 spLHiChIP track without (blue) or with (red) induction of MYCN for SH-EP-MYCN-ER cells (conventions as in ). ( H ) Bar graph showing the number of TFIIIC5 interactions normalised by the relative binding of TFIIIC5 ChIP-Rx signals for the same coordinates. Coordinates defined as TSS ± 2 kb of 14,722 genes.

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: HiChIP, Functional Assay, Expressing, shRNA, Binding Assay

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Heatmap showing analysis of sequence motifs characteristic for interacting boxes on both anchors of the interactions. Colour reflects the number of interactions. ( B ) Table summarising numbers of MYCN interactions in the presence and absence of TFIIIC5 in SH-EP-MYCN-ER cells and the TFIIIC5-dependent change. Data merged of two independent biological replicates. ( C ) Table displaying total numbers of TFIIIC5 interactions in SH-EP-MYCN-ER±MYCN + 5,6-dichlorobenzimidazole-1-β-D-ribofuranoside (DRB).

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Sequencing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Volcano plot depicting changes in intron retention upon TFIIIC3 knock-down in the absence (left) and presence (right) of MYCN. Triangle reflects the effect of the splicing error after TFIIIC knock-down. ( B ) Volcano plot depicting changes in exon skipping upon TFIIIC3 knock-down in the absence (left) and presence (right) of MYCN. Triangle reflects the effect of the splicing error after TFIIIC knock-down. ( C ) Controls for proximity ligation assays (PLAs) shown in . Immunofluorescence show specificity of the antibody. Single antibodies PLAs were performed in parallel to each PLA. Mean of signal in the nucleus and cytoplasm was calculated and compared to mean of the signal per nucleus of the PLA. ( D ) Boxplots showing the number of PLA signals between RNAPII and TFIIIC5. SH-EP-MYCN-ER cells were treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC5’, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (‘+MYCN’). EtOH was used as control. For clarity purposes, 500 cells pooled from different replicates were plotted. p-Values were calculated comparing the PLA signal of all cells using unpaired Wilcoxon rank sum test. The grey dotted line indicates the median in the control condition (n=3). ( E ) BRCA1 ChIP in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT. Data show fold change of BRCA1 binding after induction of MYCN in the presence (blue) or absence (red) of TFIIIC5 (n=2). Figure 5—figure supplement 1—source data 1. Raw data for plots shown in .

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Knockdown, Ligation, Immunofluorescence, Control, Expressing, shRNA, Binding Assay

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Boxplots showing the number of proximity ligation assay (PLA) signals between RNA polymerase II (RNAPII) and NELFE, PP2A, PNUTS, or XRN2. SH-EP-MYCN-ER cells were treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC5’, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (‘+MYCN’). EtOH was used as control. For clarity purposes, 500 cells pooled from different replicates were plotted. p-Values were calculated comparing the PLA signal of all cells using unpaired Wilcoxon rank sum test. The grey dotted line indicates the median in the control condition (n=3). ( B ) Density plot of CUT&RUN for EXOSC5 binding (N=14,704 genes) in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT. Data show mean ± SEM (shade). ( C ) BRCA1 ChIP in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT (4 hr). Shown is the mean of technical triplicates of one representative experiment with identical results (n=2). Figure 5—source data 1. Raw data for plots shown in .

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Proximity Ligation Assay, Control, Binding Assay, Expressing, shRNA

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet:

Article Snippet: SH-EP-MYCN-ER cells were plated in 384-well plates (PerkinElmer), treated with Dox and/or 4-OHT and fixed with methanol for 20 min. After blocking for 30 min with 5% BSA in PBS, cells were incubated overnight at 4°C with primary antibodies: Total RNAPII (F12): sc-55492; TFIIIC5: A301-242A, Bethyl Laboratories; NELFE: ABE48, Merck; PP2A: 2038, Cell Signaling; PNUTS: A300-439-1, Bethyl Laboratories; XRN2: A301-103A, Bethyl Laboratories.

Techniques: Recombinant, Expressing, Plasmid Preparation, Variant Assay, Sequencing, shRNA, ChIP-qPCR, In Situ, Affinity Purification, Fluorescence, Isolation, Multiplex Assay, Transfection, Magnetic Beads, Software, Imaging