Journal: Genome Research

Article Title: Reorganization of the host epigenome by a viral oncogene

doi: 10.1101/gr.132308.111

Figure Lengend Snippet: Small e1a causes global deacetylation and redistribution of H3K18ac. ( A ) Venn diagram showing the overlap between significant peaks of H3K18ac in mock- (dark blue) and dl 1500-infected cells (light blue). ( B ) Venn diagram showing the overlap between significant peaks of H3K9ac in mock- (dark orange) and dl 1500-infected cells (yellow). ( C ) Patterns of H3K18ac and H3K9ac in the intergenic region between COPS8 and COL6A3 genes in mock-, dl 1500-infected, and asynchronous IMR90 cells. ( D ) Quantitative PCR (% of input) for EP300 and CREBBP in mock- and dl 1500-infected cells for the COL6A3 intergenic region, and CCNE2 and POLD3 promoters are shown as bar plots. Patterns of H3K18ac in mock- and dl 1500-infected cells at CCNE2 and POLD3 loci are also shown. For each histone modification, the y -axis indicates the number of input-normalized ChIP-seq reads across the locus ( x -axis). (Light blue arrows) New peaks of acetylation in e1a-expressing cells. (Dark arrows) Direction of transcription. ( E , F ) Overview of distribution of H3K18ac and H3K9ac peaks in mock- and dl 1500-infected cells in relation to gene structure are shown as pie charts. ( G ) Distribution of significant peaks of H3K18ac with respect to TSS in mock- (dark blue) and dl 1500-infected (light blue) cells. ( H ) Distribution of significant peaks of H3K9ac with respect to TSS in mock- (dark orange) and dl 1500-infected (yellow) cells.

Article Snippet: IMR90 human primary lung embryo fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Infection, Real-time Polymerase Chain Reaction, Modification, ChIP-sequencing, Expressing

Journal: Genome Research

Article Title: Reorganization of the host epigenome by a viral oncogene

doi: 10.1101/gr.132308.111

Figure Lengend Snippet: Analyses of RB-family protein genome-wide binding in contact-inhibited IMR90 fibroblasts. ( A ) Overview of RB1, RBL2, and RBL1 peak distributions in mock-infected cells in relation to gene structure are shown as pie charts. ( B ) Average binding profiles (significant counts) of the indicated RB-family members across the TSS regions of their respective target genes. ( C ) A 600-bp region around the peaks of RB1, RBL2, and RBL1 in mock-infected cells were analyzed for TF binding motifs using sitepro (Galaxy). Top three significant motifs for each family member are shown. All P -values are less than 1 × 10 −10 . ( D ) The distributions of the RB-family proteins across ±5 kb of TSS for genes with at least one RB-family member bound are shown as heat maps. The seven clusters are based on combinatorial binding patterns of the three proteins. ( E ) Relative gene expression changes of each of the seven clusters after e1a expression at 24 h p.i. are shown as box plots.

Article Snippet: IMR90 human primary lung embryo fibroblasts (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 .

Techniques: Genome Wide, Binding Assay, Infection, Gene Expression, Expressing

Journal: ACS chemical biology

Article Title: Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-inducing Virus-like Particles

doi: 10.1021/acschembio.8b00490

Figure Lengend Snippet: (a) Schematic of Qβ(ZZ)42@CD13 viral coat protein expression and particle assembly, followed by VLP labeling with SSEA-5 antibodies. (b) Electrophoretic, (c) FPLC, and (d) dynamic light scattering analyses of Qβ(ZZ)42@CD13 VLPs. (%Đ = % dispersity; Rh = hydrodynamic radius). (e) EC50 curves showing dose-dependent decreases in human induced pluripotent stem cell (hiPSC) and human embryonic stem cell (hESC) survival after 24 h treatment with both 8 nM Qβ(ZZ)42@CD13+SSEA-521 VLPs in the presence of the prodrug 5-fluorocytosine (5-FC) or the cytotoxin 5-fluorouracil (5-FU). (f) EC50 curves show dose-dependent decreases in human dermal fibroblast (hDF) survival percentages after 24 h treatment with 5-FU, but negligible cell death after treatment with 8 nM Qβ(ZZ)42@CD13+SSEA-521 VLPs in the presence of 5-FC. (g) Representative microscopy images show co-cultures of hPSC colonies and MEFs that were treated for 15 h with 8 nM of unlabeled, IgG1-labeled (isotype control), and SSEA-5-labeled Qβ(ZZ)42@CD13 VLPs in the presence of 100 μM 5-FC as test conditions, and 100 μM 5-FU as a positive control. Red fluorescence indicates dead cells and green fluorescence indicates expression of the pluripotent stem cell-specific TRA-1–60 marker.

Article Snippet: Human induced pluripotent stem cells [iPS(IMR90)-1, WiCell] 29 and human embryonic stem cells (H7, WiCell) 30 were cultured either on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs), where they were maintained in hPSC medium (KnockOut DMEM, 20% KnockOut Serum Replacement, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 U/mL Penicillin-Streptomycin) supplemented with 8 ng/mL human basic fibroblast growth factor (bFGF), or in feeder-free hPSC conditions as previously described.

Techniques: Expressing, Labeling, Microscopy, Positive Control, Fluorescence, Marker

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Distribution of NADs along human autosomes. NADs are indicated by red rectangles over the ideograms of the chromosomes. Note that the p-arms of the five acrocentric chromosomes (13, 14, 15, 21 and 22), centromeres and some pericentromeric regions were not analysed because they are not present in the current human genome assembly. ( B ) Histogram of NAD sizes. Median = 361kb, a total of 1,646 NADs were identified. ( C ) 3D immuno-FISH analysis of NAD and inter-NAD regions (iNADs) in IMR90 cells. Nucleolus association of a chromosomal domain is illustrated by showing the Z-projection of an IMR90 nucleus on the left and the corresponding single light optical sections with the associated and non-associated allele on the right. BAC hybridization signals are shown in green, nucleolar staining in red and DAPI counterstain in blue (scale bar: 1.6 μm). ( D ) Hybridization signals (percentage of nucleolus-associated alleles) are plotted against the according microarray signals (average log2-fold difference of the nucleolar signal over the background). Red and grey circles indicate genomic regions that reside in NADs and iNADs, respectively (see for further details). The positions of the BAC clones used in 3D immuno-FISH experiments to monitor NADs and iNADs are shown also in ( A ) by red and grey circles, respectively.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Hybridization, Staining, Microarray, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Venn diagrams and Jaccard coefficients show the extent of overlap between NADs and LADs. LAD1: LADs of Tig3 cells , LAD2 and LAD3: LADs of IMR90 cells [ , ]. ( B ) Bar graphs show Gencode v19 and UniProt gene frequencies in NADs (red), iNADs (grey), LADs (black), and iLADs (white) based on UCSC Table Browser data. ( C ) RefSeq gene (ZNF, OR and DEF indicate zinc finger, olfactory receptor and defensin gene families, respectively) frequencies in NADs, iNADs, LADs, and iLADs. ( D ) Non-coding RNA gene (‘RNA genes’) and ( E ) repeat frequencies in NADs, iNADs, LADs, and iLADs. The SINE repeat bars are divided with a horizontal line into MIR (bottom) and Alu (top) sub-groups.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques:

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Bar graph of nucleolus number in young, proliferating (‘Y’) and senescent (‘S’) IMR90 cells. Proliferating cells have 3.0±1.2 and senescent cells 1.7±1.1 nucleoli per nucleus. Z projections of mid-sections of representative confocal microscopy images are shown on the top. Nucleolar staining is shown in red and DAPI counterstain in blue (scale bars: 1.6 μm). ( B ) Maps of NADs on chromosome 5 from young and senescent cells. Genomic regions associated with nucleoli only in young (¬S) or senescent (¬Y) cells are shown also as individual tracks. ( C ) Y-only and S-only NADs are enriched in protein-coding genes compared to all NADs and the genome. RefSeq gene data were obtained from the UCSC Table Browser. ( D ) Boxplots show positive correlation of senescence-related loss of nucleolus association and gene activation. Global gene expression changes (log2 fold change in senescent versus young cells) in constitutive (Y∧S), Y-only and S-only NAD genes are shown. The notches are defined as +/-1.58*IQR/sqrt(n) and represent the 95% confidence interval for each median. Group means are significantly different for all comparisons (p-value < 0.05, Tukey HSD). ( E ) Stacked columns show that the association frequency of five selected genomic regions is similar in young and senescent cells. Nucleolus-association data were collected from 50 cells for each category. BAC clones 1 to 5: RP11-44B13, RP11-173M10, RP11-828F4, RP11-125O21, RP11-81M8.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Confocal Microscopy, Staining, Activation Assay, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Semi-quantitative immunoblots show severely decreased H3K9me3 and Lamin B1 levels, less strongly decreased H3 levels and no detectable alterations in Lamin A/C, tubulin and GAPDH levels in senescence. The same amounts of whole cell extracts of young and senescent cells were loaded as serial two-fold dilutions on SDS-PA gels and analysed on immunoblots (see the entire dataset from three independent experiments in ). ( B ) H3K9me3 is accumulated at spatially compact satellite repeat clusters. 3D immuno-FISH shows strong co-localization of H3K9me3 and HSATII staining. Mid-section of a representative confocal microscopy image is shown. HSATII FISH signals are in red, DAPI counterstain in blue, and H3K9me3 immunofluorescence signals are in green (scale bar: 1.6 μm). ( C ) Quantitative immunofluorescence analysis of H3K9me3 distribution. The areas of interest are illustrated on a light optical section of a representative confocal microscopy image. The lamina- and nucleolus-associated areas label 240 nm distances from the edges of the DAPI and nucleolus staining, respectively. ( D ) Bee swarm plots of relative fluorescence intensities show senescence-dependent small decrease in H3K9me3 levels at the nuclear periphery and strong reduction at the perinucleolar space. Proliferating and senescent IMR90 cells were stained for H3K9me3 and the relative immunofluorescence intensities were measured at the nuclear periphery (lamina) and at the perinucleolar space (No). Values measured in proliferating cells (‘Y’) are shown in red, values measured in senescent cells (‘S’) are shown in blue. Results from individual cells are illustrated as single data points (n Y = 88, n S = 88). A solid line indicates the median, and thin lines the upper and lower quartile. Median: Y.lamina = 0.095, S.lamina = 0.084; Y.No = 0.052, S.No = 0.022. ( E ) Bee swarm plots indicate more heterogeneous H3K9me3 staining in the nucleus and at the nuclear periphery of senescent cells, but no change in the perinucleolar space. The heterogeneity of staining was calculated as coefficient of variation (C.V. = standard deviation/mean of fluorescence intensity) for the total nucleus (Nu), the nuclear periphery (lamina) and the perinucleolar space (No). Plot labels are as in ( D ). Median: Y.Nu = 0.573, S.Nu = 0.677; Y.lamina = 0.632, S.lamina = 0.709; Y.No = 0.576, S.No = 0.555, n Y = 88, n S = 88. ( F ) Bee swarm plots illustrate robust rearrangement of the most heterochromatic regions in the perinucleolar space. The distribution of the 10% brightest pixels was quantified at the nuclear periphery (lamina) and the perinucleolar space (No). Ratios were calculated compared to the whole nucleus. Plot labels are as in ( D ). Median: Y.lamina = 0.157, S.lamina = 0.137; Y.No = 0.056, S.No = 0.013. n Y = 88, n S = 88.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Western Blot, Staining, Confocal Microscopy, Immunofluorescence, Fluorescence, Standard Deviation