Depletion of p150CAF-1 Leads to Loss of Clustering, Altered Localization, and Decondensation of Pericentric Heterochromatin Domains

<div><p>Distribution of pericentric (red) and centric (green) domains was analyzed in the interphase nuclei of mouse ES cells by DNA FISH, using major satellite (pSAT) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>] and minor satellite (pMR150) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b048" target="_blank">48</a>] DNA probes, respectively. (A) In ES cells expressing control (cont) siRNA, pericentric regions from several chromosomes associate in clusters (red). These chromocenters form foci as revealed by DAPI staining (left-hand image), while centric regions (green) remain independent entities at the periphery of these domains. The right-hand image shows the merge between the pericentric and centric FISH signals.</p><p>(B) The organization of pericentric domains was altered in cells expressing p150CAF-1 siRNA. Instead of forming well-defined chromocenters, pericentric domains were found either isolated or associated in heterogeneous aggregates of various sizes, often at the nuclear periphery. Scale bar = 10 μm.</p><p>(C) Control ES cells. Fluorescence was quantified along a line randomly drawn across the nucleus in the merged image and data were plotted. One can distinguish clear peaks corresponding to chromocenters (red) and the condensed minor satellites (green).</p><p>(D) ES cells expressing p150CAF-1 siRNA. p150CAF-1 depletion led to a lower fluorescence intensity and a broader distribution of signals corresponding to DAPI (blue) and major satellite hybridization (red, plot) while the organization of the minor satellites remained unaffected. Insets in the right-hand images show a typical chromocenter in control cells (C) and a disrupted chromocenter in p150CAF-1-depleted cells (D).</p></div

Nucleosomal Organization Is Not Altered in p150CAF-1-Depleted ES Cells

<div><p>Nuclei were prepared from ES cells transfected with control or p150CAF-1 siRNA vector. Nuclei were digested with increasing amounts of DNase I or MNase. (A) After digestion with the indicated nucleases, total DNA was prepared and run onto an agarose gel which was stained with ethidium bromide to reveal bulk genomic DNA.</p><p>(B) The DNA was blotted onto a nylon membrane, which was then hybridized with the α-³²P-labeled pSAT major satellite repeat probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>].</p></div

Alteration of Epigenetic Marking at Pericentric Heterochromatin in p150CAF-1-Depleted Cells

<div><p>(A) p150CAF-1 depletion leads to reduced H4K20me3 and H3K9me3 at pericentric heterochromatin. Enrichment of histone marks at major satellite repeats was determined by ChIP from control (cont) and p150CAF-1 (p150) siRNA-expressing ES cells. DNA prepared from the input and the antibody-bound fraction were run onto an agarose gel and analyzed by Southern blot with the pSAT major satellite repeat probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>].</p><p>(B) Hybridization signals were quantified using an Instant Imager. After autoradiography, the membrane was stripped and rehybridized with a minor satellite probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b049" target="_blank">49</a>]. After quantification, the membrane was stripped and rehybridized with an IAP LTR probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b050" target="_blank">50</a>]. Results are presented as the amount of DNA immunoprecipitated from p150CAF-1-depleted ES cells divided by the DNA obtained from control cells. The figure shows the mean value and standard deviation of three independent ChIP experiments.</p><p>(C and D) H3K9me3 and H4K20me3 fluorescence patterns are severely altered in p150CAF-1-depleted ES cells. Immunodetection of H3K9me3 (C, green), H4K20me3 (D, green), and HP1α (red) in control and p150CAF-1 siRNA-expressing ES cells. Merging of HP1α with H3K9me3 (C) and H4K20me3 (D) is shown in yellow. Scale bars represent 10 μm.</p></div

DNA CpG Methylation at Pericentric Heterochromatin Is Not Altered in p150CAF-1-Depleted ES Cells

<p>Total DNA was isolated from cells transfected with a control or the p150CAF-1 siRNA vector and digested with <i>Mae</i>II, whose recognition sequence is present in major satellite repeats. Digested DNA was run onto an agarose gel and analyzed by Southern blotting using the pSAT major satellite probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>].</p

Depletion of p150CAF-1 in ES Cells Results in a Severe Alteration of Heterochromatin Organization

<div><p>(A) Strategy used to deplete p150CAF-1 by RNAi in ES cells. The siRNA expression vector includes a puromycin selection cassette (Puro), the mouse H1 promoter, and the siRNA encoding sequence. ES cells were kept under puromycin selection during 48 h following transfection.</p><p>(B) Abnormal heterochromatin organization in p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 (green) and HP1α (red) in ES cells transfected with control (cont) and p150CAF-1 siRNA. siRNA expression results in efficient p150CAF-1 depletion. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue. Scale bar = 10 μm.</p><p>(C) Heterochromatin organization is not altered in p150CAF-1-depleted MEFs. Immunodetection of p150CAF-1 (green) and HP1α (red) in MEFs transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue.</p><p>(D) PML bodies are not altered in p150CAF-1-depleted ES cells. Immunodetection of PML (red) in ES cells transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between PML fluorescence and DAPI-stained DNA in blue.</p></div

p150CAF-1 Depletion and Loss of Heterochromatin Organization Are Compatible with Active DNA Replication in ES Cells

<div><p>(A) ES cells were transfected with p150CAF-1 or control siRNA vectors. After 3 d under puromycin selection, cells were pulse-labeled for 10 min with BrdU and immediately analyzed by IF. No significant difference in BrdU incorporation could be detected between control and p150CAF-1-depleted cells. The right-hand image shows the merge between the p150CAF-1 and BrdU fluorescence. Scale bar = 10 μm.</p><p>(B) Immunodetection of PCNA (red) revealed no significant difference in the formation of replication foci between control and p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 is shown in green and the merging of p150CAF-1 and PCNA appears in yellow.</p><p>(C) Flow cytometry analysis showed a similar cell-cycle profile for control and p150CAF-1-depleted ES cells. Results are presented as the percentage of cells in each phase of the cell cycle (G1, S, G2/M), as defined by BrdU incorporation and DNA content. Data presented are the mean of three independent experiments; error bars indicate the standard deviation. All experiments were performed 3 d after transfection of the siRNA vectors.</p></div

Loss of p150CAF-1 Function Leads to Early Developmental Arrest and Alteration of Heterochromatin Organization

<div><p>(A) Generation of <i>Chaf1a</i>^+/<i>−</i> mice by homologous recombination in ES cells. (Top) Scheme of the p150CAF1 protein indicating the interacting domains (ID) for HP1 and p60CAF-1, and the acidic domain (AD). (Middle) Structure of the murine <i>Chaf1a</i> gene. Blocks and lines represent exons and introns, respectively. A star indicates the translation initiation site. Below is shown the <i>Chaf1a</i> targeting vector, which includes genomic DNA homology regions (5 HR and 3 HR), the diphtheria toxin (DT), and neomycin selection genes. Recombined (Rec) ES cells and mice were identified by Southern blot using <i>Sca</i>I (S) and the indicated probe.</p><p>(B) Breeding strategy used for the production of E4 <i>Chaf1a</i>^{−/<i>−</i>} embryos. Genotyping was performed by PCR using the three oligonucleotide primers indicated in (A) (1, 2, and 3). An example of the result of a genotyping experiment is shown.</p><p>(C) Immunodetection of p150CAF-1 (green) and HP1α (red) in E4 embryos derived from <i>Chaf1a</i>^+/<i>−</i> intercrosses. Because <i>Chaf1a</i>^+/+ and <i>Chaf1a</i>^+/<i>−</i> embryos both stain positively for the presence of p150CAF-1, they are both designated as wild-type. p150CAF-1 expression, which indicates ongoing S phase, can be detected in most cells within the wild-type blastocyst (upper panel). The lower panel shows a 12-cell embryo labeling negatively for p150CAF-1. Only nonspecific background labeling can be observed. In each panel, the right-hand image shows the merge between the HP1α fluorescence and DAPI-stained DNA in blue. Pink color indicates the association of HP1 with DAPI-dense material. The bottom of each panel shows the magnification of a nucleus selected from the above embryo (white square). The arrowhead indicates the typical heterochromatin foci revealed by DAPI and HP1α staining in wild-type embryos. These foci are not visible in <i>Chaf1a</i>^−/− embryos. DAPI and HP1α staining are diffuse within the nucleus of p150CAF-1-depleted embryos, with enrichment at the nuclear periphery, revealing abnormal heterochromatin organization. Scale bars represent 10 μm.</p><p>(E) Wild-type embryos isolated at embryonic day 2 (E2, two cells), E2.5 (four cells), E3 (eight cells), E3.5 (16 cells), and E4 (32 cells) are shown. Heterochromatin was monitored by DAPI staining (upper panel) and HP1 immunolabeling (in red, lower panel).</p><p>(F) Magnification of a nucleus (DAPI-stained) representative of each stage. The right-hand panel shows the nucleus of an ES cell. Scale bar represents 10 μm.</p></div