Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a , b EAF1 C-terminal region and MED26 N-terminal region are required for CB–HLB association. Wild-type cells and EAF1-mutant cells ( a ) or MED26-mutant cells ( b ) were fixed with paraformaldehyde, and immunofluorescence staining for Coilin and NPAT was performed. Scale bar, 5 μm. Enlarged images for representative particles are shown in the lower part of each image. Evaluation of CB–HLB association in each mutant cell line is shown in the right panels. The NPAT particles were extracted from images. The area of NPAT particles occupied by Coilin particles and the intensity of the particles of NPAT and Coilin were calculated. The density shows the degree to which dots overlap with others. Signal intensities were evaluated using more than 200 nuclei for each condition. Wild-type, n = 308; EAF1 mutant, n = 204 in a , and wild-type, n = 278; MED26 mutant, n = 318 in b . c , d Quantification of the number and intensity of Coilin particles. All detected Coilin particles in EAF1-mutant cells (EAF1-mutant) ( c ) or MED26-mutant cells (MED26-mutant) ( d ) are plotted. NPAT-associating Coilin particles are indicated by black dots and free Coilin particles are indicated by white dots. Quantification was performed using 300 ( c ) or 200 ( d ) Coilin particles. e Super-resolution images showing relative positions of HLBs, Mediator, and CBs. Wild-type cells (WT, upper) and EAF1-mutant cells (EAF1 mut, lower) were fixed with paraformaldehyde, and triple immunofluorescence staining for NPAT (green), MED26 (red), and Coilin (gray) was performed. Enlarged images for representative particles are shown in the lower part of each image. f Calculation of the distance between HLBs and CBs. Immunofluorescence staining for NPAT and Coilin was performed with wild-type and EAF1-mutant HEK293T cells. For Coilin-associated NPAT particles, the distance between the center of the NPAT particle and Coilin particle was measured, and the distance between each associating particle is displayed as boxplots. The center line of each boxplot represents the median. Upper and lower fences of each boxplot represent upper and lower quartiles, respectively. Wild-type, n = 465 particles from 301 nuclei; EAF1 mutant, n = 145 particles from 354 nuclei. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Mutagenesis, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a – f HeLa cells were fixed with paraformaldehyde and subjected to triple immunofluorescence staining. MED26 (red), MED1 (red), ICE1 (red), ELL (red), LSM11 (red), and FLASH (red) were co-stained with NPAT (green) and Coilin (gray) using specific antibodies. Scale bar, 5 μm. Averaged signals of immunofluorescence centered at NPAT signal and respective line plots for each immunofluorescence experiment are shown on the right. Scale bar, 1 μm. g , h HCT116 cells were stained with anti-MED26 (green), anti-NPAT (green), and anti-Coilin (magenta) antibodies, and the nuclear positions of MED26 (Mediator), NPAT (HLBs), and Coilin (CBs) were observed by stimulated emission depletion (STED) microscopy. Scale bar, 1 μm. Line intensity plots across the particles of NPAT (green), Coilin (magenta), and MED26 (magenta) are shown in the lower panels. i Ratios of ICE1, ELL, LSM11, MED1, MED26, or FLASH colocalization with CBs, HLBs, or both CBs and HLBs. The ratio was calculated using more than 100 nuclei. ICE1, n = 71 particles from 196 nuclei; ELL, n = 122 particles from 240 nuclei; LSM11, n = 161 particles from 242 nuclei; MED1, n = 84 particles from 237 nuclei; MED26, n = 83 particles from 204 nuclei; FLASH, n = 187 particles from 291 nuclei. Source data are provided as a Source Data file. j Model of the relative position of each protein at CBs and HLBs. Mediator was located between HLBs and CBs.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Immunofluorescence, Staining, Microscopy

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a Schematic illustration of the strategy for in situ biotinylation of nuclear bodies. Cells were fixed and permeabilized, and HRP-conjugated antibodies were deposited onto target proteins (Coilin or LSM11). Then, the proximal chromatin was biotinylated by adding biotin–phenol and H 2 O 2 . After cell lysis, biotinylated proteins and their associated DNA were avidin-purified, followed by qPCR or high-throughput DNA sequencing. b – d CBs were dissociated from RDH gene clusters in EAF1-mutant cells. b Heatmap showing the CB (Coilin)-association profile in wild-type HEK293T cells. The avidin-purified DNA was analyzed by high-throughput sequencing. Two RDH gene clusters located at chromosome 1 and chromosome 6 are indicated by arrowheads. c Genome browser tracks showing the distribution of in situ biotinylation-seq reads using anti-Coilin antibodies at RDH gene cluster 2 in wild-type (WT) and EAF1-mutant (mut) cells. d Total counts of Coilin in situ biotinylation-seq reads at RDH genes, snRNA genes and snoRNA genes, and CB enrichment levels were compared between wild-type (WT) and EAF1-mutant (EAF1 mut) cells. Each value is the mean of two independent experiments. Source data are provided as a Source Data file. e Genome browser tracks showing the distribution of in situ biotinylation-seq using anti-Coilin antibodies at snRNA gene RNU2 in wild-type (WT) and EAF1-mutant (mut) cells. f Heatmap showing the U7 snRNP (LSM11)-association profile in wild-type HEK293T cells. The avidin-purified DNA was analyzed by high-throughput sequencing. Two RDH gene clusters located at chromosome 1 and chromosome 6 are indicated by arrowheads. g Genome browser tracks showing the distribution of in situ biotinylation-seq reads using anti-LSM11 antibodies at RDH gene cluster 2 in wild-type (WT) and EAF1-mutant (mut) cells. h Heatmap showing CB enrichment and mRNA expression levels of RDH genes in wild-type (WT) HEK293T cells, and fold-change of CB enrichment and mRNA expression levels of RDH genes in EAF1-mutant (EAF1 mut) cells. i Immunofluorescence images showing CDK11 localization at CBs. HCT116 cells were fixed with methanol and subjected to immunofluorescence staining. CDK11 (green) and Coilin (red) were stained using specific antibodies. Scale bar, 10 μm. j Meta-gene analysis of PRO-seq reads around TESs. Coilin (light blue) and CDK11 (yellow) iCLIP data by Machyna et al. and Sathyan et al. are overlaid with PRO-seq data. Approximate positions of stem-loop (SL) and HDE are indicated in the lower panel.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: In Situ, Lysis, Avidin-Biotin Assay, Purification, High Throughput Screening Assay, DNA Sequencing, Mutagenesis, Next-Generation Sequencing, Expressing, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a Wild-type HEK293T (WT) and EAF1-mutant cells (mut) were fixed with paraformaldehyde containing 0.5% Triton X-100 and subjected to DNA-FISH. Four-color microscopy images are shown. RDH gene clusters 1 (cyan) and 2 (red) were stained using Cy5- or Cy3-labeled probe. NPAT (blue) and Coilin (green) were stained using specific antibodies. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 10 μm. The frequencies of each RDH gene cluster 2 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the left panel. The frequencies of each RDH gene cluster 1 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the right panel. b Representative four-color microscopy images showing the overlap of CB, HLB, and two RDH gene clusters in wild-type HEK293T cells. RDH gene clusters, NPAT, and Coilin were stained as described above. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 5 μm. The frequency of inter-chromosome interaction between two RDH gene clusters is shown in the right panel (WT, n = 1130 RDH gene cluster 2; mut, n = 771 RDH gene cluster 2). c – f 4C-seq analysis revealed that the higher-order inter-chromosome structure between two RDH gene clusters was abolished in EAF1-mutant cells. c , e Representative 4C-seq profile using RDH gene cluster 1 ( HIST1H2BK , chromosome 6) bait or RDH gene cluster 2 ( HIST2H2AB , chromosome 1) bait. The detailed contact profile at RDH gene cluster 2 (chromosome 1) ( c ) or at RDH gene cluster 1 (chromosome 6) ( e ) is shown in the lower panels. d , f Comparisons of 4C-seq counts detected in each RDH gene cluster region in wild-type (WT) or EAF1-mutant (EAF1 mut) cells under each 4C-seq condition. The read counts of 4C-seq using chromosome 6 HIST1H2BK bait ( d ) and chromosome 1 HIST2H2AB bait ( f ) are shown. These plots represent the mean of n = 2 experiments. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Mutagenesis, Microscopy, Staining, Labeling

Journal: PLoS Pathogens

Article Title: Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

doi: 10.1371/journal.ppat.1009231

Figure Lengend Snippet: A . BCBL1 cells untreated or treated for 10 min with 3.5% 1,6-Hexanediol were imaged by IF for Coilin (green), LANA (red) or DAPI (blue). Scale bar = 10 μm. B . BCBL1 cells treated as in panel A were quantified for >2 coilin or >4 LANA NBs. The bar graph represents means ± s.d, ** p value < 0.01, *** p value < 0.001 using two-tailed student t-test. C . iSLK RFP-LANA cells untreated or treated for 10 min with 3.5% 1, 6-Hexanediol or 2,5-Hexanediol were imaged by IF for Coilin (green), RFP-LANA (red) or DAPI (blue). Scale bar = 10 μm. D . iSLK RFP-LANA cells treated as in panel C were quantified for >2 coilin or >4 LANA-NBs. The bar graph represents means ± s.d, * p value < 0.05, ** p value < 0.005, *** p value < 0.001 using two-tailed student t-test.

Article Snippet: The following antibodies were used for immunofluorescence, Co-IP, and Western blotting studies: rat polyclonal anti-HHV8 or anti-LANA [LN53] (Abcam, ab4103), rabbit anti-Coilin (Cell Signaling technology,14168S), rabbit anti-DAXX (Sigma, D7810), rabbit anti-PML (Bethyl, A301167A), mouse anti-ORF50 (provided by Erle Robertson, UPENN), mouse anti-ORF45 (provided by Yan Yuan, UPENN), mouse anti-EBV (BIO-RAD, 4260–0906), anti-RAD21 (Abcam ab992), rabbit polyclonal anti-PARP1 (Enzo ALX-210-302-R100), rabbit EZH2 (Cell Signaling, 4905), rabbit H3K27me3 (Active Motif, 39155), rabbit IgG (Santa Cruz Biotechnology), AlexaFluor594 or AlexaFluor488 (Invitrogen), and mouse anti-Actin-HRP (Sigma, A23852).

Techniques: Two Tailed Test

Journal: PLoS Pathogens

Article Title: Phase separation and DAXX redistribution contribute to LANA nuclear body and KSHV genome dynamics during latency and reactivation

doi: 10.1371/journal.ppat.1009231

Figure Lengend Snippet: A . Immunoblotting of LANA, RAD21, PARP1, Coilin, DAXX and actin in BCBL1 cells exposed to 3.5% 1,6-Hexanediol for the indicated times. B . RT-qPCR for lytic transcripts ORF50, PAN and latent transcript LANA relative to cellular actin in BCBL1 cells treated as in panel A. The data are expressed as fold change of the treated versus untreated cells. C . BCBL1 and iSLK RFP-LANA cells treated for 1 hr with 3.5% 1,6-Hexanediol were assayed by ChIP for LANA or IgG. ChIP-qPCR primer positions indicated on x-axis are relative to KSHV genome. GAPDH primers were used as internal control. * p value < 0.05, two-tailed t-test.

Article Snippet: The following antibodies were used for immunofluorescence, Co-IP, and Western blotting studies: rat polyclonal anti-HHV8 or anti-LANA [LN53] (Abcam, ab4103), rabbit anti-Coilin (Cell Signaling technology,14168S), rabbit anti-DAXX (Sigma, D7810), rabbit anti-PML (Bethyl, A301167A), mouse anti-ORF50 (provided by Erle Robertson, UPENN), mouse anti-ORF45 (provided by Yan Yuan, UPENN), mouse anti-EBV (BIO-RAD, 4260–0906), anti-RAD21 (Abcam ab992), rabbit polyclonal anti-PARP1 (Enzo ALX-210-302-R100), rabbit EZH2 (Cell Signaling, 4905), rabbit H3K27me3 (Active Motif, 39155), rabbit IgG (Santa Cruz Biotechnology), AlexaFluor594 or AlexaFluor488 (Invitrogen), and mouse anti-Actin-HRP (Sigma, A23852).

Techniques: Western Blot, Quantitative RT-PCR, Two Tailed Test