28 research outputs found
The senescence-dependent spatial reorganization of H3K9me3-marked chromatin differs at the perinucleolar space and the nuclear periphery.
<p>(<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.s011" target="_blank">S11 Fig</a>). (<b>B</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). (<b>C</b>) 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. (<b>D</b>) 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<sub>Y</sub> = 88, n<sub>S</sub> = 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. (<b>E</b>) 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 (<b>D</b>). 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<sub>Y</sub> = 88, n<sub>S</sub> = 88. (<b>F</b>) 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 (<b>D</b>). Median: Y.lamina = 0.157, S.lamina = 0.137; Y.No = 0.056, S.No = 0.013. n<sub>Y</sub> = 88, n<sub>S</sub> = 88.</p
Comparative epigenomics of NADs and inter-NAD regions (iNADs) reveals specific heterochromatic features of NADs.
<p>(<b>A</b>) Distribution of different chromatin states in NADs and iNADs. Bar graphs show total and relative amounts of ChromHMM states in NADs and iNADs on the left and right, respectively. The chromatin states and their colour code correspond to the Primary Core Marks segmentation 15-state ChromHMM model of the Roadmap Epigenomics Project [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref041" target="_blank">41</a>]. Chromatin states specifically enriched in NADs are indicated. Red: Active Transcriptional Start Site (TSS), OrangeRed: Flanking Active TSS, LimeGreen: Transcription at gene ends, Green: Strong transcription, DarkGreen: Weak transcription, GreenYellow: Genic enhancers (Enh), Yellow: Enh, MediumAquamarine: ZNF genes & repeats, PaleTurquoise: Heterochromatin, IndianRed: Bivalent/Poised TSS, DarkSalmon: Flanking Bivalent TSS/Enh, DarkKhaki: Bivalent Enh, Silver: Repressed PolyComb, Gainsboro: Weak Repressed PolyComb, White: Quiescent/Low. (<b>B</b>) Boxplot of DNaseI accessibility in NADs (n = 1646) and iNADs (n = 1669). The average accessibility per base and segment were calculated from GSM468792. (<b>C</b>) Boxplot of RNA-seq read densities. For each NAD (n = 1646) and iNAD (n = 1669) the average number of reads per base in GSM438363 were calculated. (<b>D</b>) Replication timing profiles around NAD borders. Average percentage-normalized signals of different replication domains within a distance of 500 kb from aligned 5’ and 3’ NAD borders are shown. Repli-Seq signals from five different time points of the cell cycle (S1-S4 and G2) were taken from Pope <i>et al</i>.,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref042" target="_blank">42</a>] and averaged over 1 kb windows. Only NADs with a width >500 kb were considered (n = 652). The arrows drawn at the bottom show away from the aligned NAD/iNAD borders.</p
Comparative genomics of NADs, iNADs, lamina-associated domains (LADs) and inter-LADs (iLADs) uncovers NAD-specific genomic features.
<p>(<b>A</b>) Venn diagrams and Jaccard coefficients show the extent of overlap between NADs and LADs. LAD1: LADs of Tig3 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref044" target="_blank">44</a>], LAD2 and LAD3: LADs of IMR90 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref025" target="_blank">25</a>]. (<b>B</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. (<b>C</b>) RefSeq gene (ZNF, OR and DEF indicate zinc finger, olfactory receptor and defensin gene families, respectively) frequencies in NADs, iNADs, LADs, and iLADs. (<b>D</b>) Non-coding RNA gene (‘RNA genes’) and (<b>E</b>) 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.</p
Replicative senescence causes fusion of nucleoli, but only local, transcription-dependent changes in NADs.
<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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). (<b>E</b>) 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.</p
Weak nucleolus association is characteristic for escaper regions on the X chromosome.
<p>(<b>A</b>) Microarray signals of nucleolus-association (log2-fold difference of the nucleolar signal over the background, combined from two biological replicate experiments) and X escaper gene positions are shown on the top of the X chromosome ideogram. (<b>B</b>) Boxplot of NAD scores in ‘escaper’, ‘heterogeneous’ and ‘inactive’ genes (classification based on Carrel and Willard [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.ref045" target="_blank">45</a>]).</p
Map and genome features of nucleolus-associated chromosomal domains (NADs) in IMR90 primary human embryonic fibroblast cells.
<p>(<b>A</b>) 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>B</b>) Histogram of NAD sizes. Median = 361kb, a total of 1,646 NADs were identified. (<b>C</b>) 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). (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178821#pone.0178821.s015" target="_blank">S2 Table</a> for further details). The positions of the BAC clones used in 3D immuno-FISH experiments to monitor NADs and iNADs are shown also in (<b>A</b>) by red and grey circles, respectively.</p
Nucleolus—satellite repeat cluster interactions are impaired in senescent cells, whereas rDNA and telomeres do not display remarkable changes in nucleolus association frequency.
<p>Maximal intensity projections of Z-sections of representative confocal microscopy images are shown to aid the visualization of all repeat clusters within a single nucleus. Nucleolar staining is in red, DAPI counterstain in blue and FISH signals are in green on all images (scale bars: 1.6 μm). From top to bottom: The distal junction regions of NORs and rDNA repeat clusters remain associated with nucleoli in senescent cells. The signal intensity of telomeres is reduced in senescence, but their overall association with nucleoli does not show specific changes. HSATII and alpha-satellite repeat clusters display senescence-associated distension and reduced nucleolus association.</p
Chromatin-specific termination at the homotypic cluster of TTF-I.
<p>(A) Overview of the murine rRNA gene and the location of the TTF-I binding sites. A homotypic cluster of TTF-I sites is located in the terminator region. The distances between TTF-I binding sites, their orientation and the gene promoter are indicated. A comparison of the TTF-I binding sites T<sub>0</sub> and the termination sites T<sub>1</sub> to T<sub>10</sub> is depicted. (B) Increasing amounts of TTF-IΔN348 were incubated with 50 nM of either a fluorescently labelled 30-mer oligonucleotide containing a Sal-box motif (T<sub>2</sub>) or a control oligonucleotide of the same length. Protein-DNA interactions are quantified by microscale thermophoresis. Curve fitting with a Hill coefficient of 1 resulted in a K<sub>D</sub> of 500 nM+/−120 nM for the T<sub>2</sub> sequence. (C) Transcription reaction using the circular rDNA minigene plasmid pMr-SB containing a single termination site, a partially purified nuclear extract lacking most of the nuclear TTF-I (DEAE280), performed in the presence or absence of recombinant TTF-I. The positions of the long read-through and the terminated transcripts are indicated. (D) Transcription on free DNA and chromatin, using the pMrWT-T DNA containing the promoter with the TTF-I binding site T<sub>0</sub> and the full terminator with the 10 termination sites. DNA (lanes 1–8) and chromatin (lanes 9–16) were incubated with increasing concentrations of TTF-I as indicated and the DEAE280 extract. The position of the long, non-terminated read-through transcript (RT) and the terminated transcripts are indicated.</p
Clustered termination sites enhance transcription and are required for chromatin looping.
<p>(A) Overview to the stably integrated rDNA minigenes and the locations of the PCR amplicons. (B) Chromatin-immunoprecipitation (ChIP) assays on stably integrated rDNA reporter genes using the indicated antibodies. Occupancies were measured by qPCR, calculated as percentage of input chromatin and background signals as determined from control IPs with unspecific antibodies (α-HA or α-IgG) were subtracted. At least three independent biological replicates were performed. Error bars indicate the standard error of the mean. For statistical analysis, a two-sided, homoscedatic student's t-test was performed, stars denote significances. * p<0.05, ** p<0.01, *** p< = 0.001. (C) ChIP experiment using an rDNA reporter in which the Pol I spacer promoter, core promoter and enhancer regions of a pT<sub>10</sub> reporter construct were replaced by a Pol II promoter containing a canonical TBP binding site. The experiment was performed as described in (B).</p
Multiple termination sites enable cooperative binding of TTF-I to chromatin.
<p>(A) Electrophoretic mobility shift assays (EMSA) were performed with a single TTF-I binding site (T<sub>1</sub>, lanes 1–4), two binding sites (T<sub>1–2</sub>, lanes 5–9) and an array of five binding sites (T<sub>1–5</sub>, lanes 10–14) and increasing concentrations of TTF-I as indicated. Nucleoprotein complexes are resolved on native polyacrylamide gels and detected by autoradiography. The positions of the free DNA molecules and the TTF-I-DNA complexes (triangles) are indicated. (B) Monitoring TTF-I binding to the chromatinized terminator by DNase I footprinting. The pMr-T plasmid containing the full terminator was reconstituted into chromatin with <i>Drosophila</i> embryo extract. Chromatin was incubated with increasing concentrations of TTF-I as indicated and partially digested with DNase I. Footprints were analysed by a primer extension reaction using a radioactively labelled oligonucleotide and resolving the DNA on 6% sequencing gels. The marker was generated by partial digestion of the plasmid with the restriction enzyme <i>Sal</i>I and analysed by the same primer extension reaction. The <i>Sal</i>I sites (T<sub>1</sub> to T<sub>10</sub>) represent the TTF-I binding sites and the triangles indicate sites of DNase I protection. (C) Comparative footprinting of TTF-I binding to the promoter and terminator of free DNA and chromatin. Identical amounts of pMrWT-T were used as free DNA (lanes 1 to 4 and 8 to 11) or chromatin (lanes 5 to 7 and 12 to 14) and incubated with increasing amounts of TTF-I as indicated. DNA was partially digested with DNase I and the purified DNA was analysed by primer extension reactions, either using a radiolabelled oligonucleotide binding close to the promoter (lanes 1 to 7) or binding close to T<sub>1</sub> in the terminator region (lanes 8 to 14). DNA was separated on 8% sequencing gels, dried and analysed after autoradiography. The TTF-I binding sites T<sub>1</sub>, T<sub>2</sub> and T<sub>0</sub> and the protected DNase I cutting sites (triangles) are indicated.</p