Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe

Failure to grow at 36°C and elongated cell shape in an <i>S. pombe asf1</i> mutant.

<p>(A) L972 (<i>asf1⁺</i>) and SKP605-33 (<i>h⁻ leu1-32 ura4-D18 asf1-33-13myc-kan^r</i>) were grown on YES plates containing phloxine B at 26°C or 36°C for 24 h. Cell morphology was observed by a microscope. (B) Cdc2 (Y15) was highly phosphorylated in SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) at the restrictive temperature. L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated at 26°C or 36°C for 6 h. Cells were collected by centrifugation and washed once with STOP buffer. Protein extraction was performed by the glass-beads method. Samples were suspended into SDS-sample buffer. Ten micrograms of total proteins was used for western blotting. The relative intensity of each band (Cdc2Y15P) relative to the control (Cdc2) was measured using ImageJ (<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>). (C) Schematic representation of the strategy to identify protein kinases responsible for activation of the cell cycle checkpoint in the <i>asf1-33</i> mutant.</p

Interaction between Asf1 and histone H3 was lost and Asf1-33 proteins were mislocalized at 36°C in the <i>asf1-33</i> mutant.

<p>(A) Immunoprecipitation assay to investigate the interaction between Asf1 and histone H3. L972 (<i>asf1⁺</i>), SKP561-15 (<i>asf1⁻-13myc-kan^r</i>) and SKP605-33 (<i>asf1-33-13myc-kan^r</i>) strains were incubated at 26°C and 36°C for 6 h. The cells were collected by centrifugation and washed once with STOP buffer. Two milligrams of total proteins were used. After incubation with Protein G sepharose at 4°C for 1 h, the samples were washed five times with HB buffer. The samples were suspended in SDS-sample buffer and boiled at 100°C. Proteins were detected by western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. (B) Extraction of histone proteins from <i>asf1</i> mutants. L972 (<i>asf1⁺</i>) and SKP593-33P (<i>asf1-33-13myc-kan</i>^r) strains were incubated at 26°C for 24 h. The temperature was increased to 36°C and cells were incubated for a further 6 h. Extraction of histone proteins was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. Extracted histone proteins were separated by SDS-PAGE and stained with Coomassie Blue. (C) Immunofluorescence images showing the localization of Asf1-13myc in the <i>asf1⁺</i> strain and <i>asf1-33</i> mutant. SKP561-15 (<i>asf1⁺-13myc-kan^r</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated at 26°C or 36°C for 6 h and immunofluorescence analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>.</p

Histone loading in the <i>asf1-33</i> mutant and heterochromatic silencing at the outer centromeric repeat.

<p>(A) Cultures of SKP551-6 (<i>otr1</i>::<i>ura4⁻</i>) and SKP593-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r otr1</i>::<i>ura4⁺</i>) were subjected to serial dilution with sterilized water and spotted on YES plates containing 5-FOA. Each strain was incubated at 26, 34, and 36°C for several days. (B) RT-qPCR analysis of the <i>asf1-33</i> mutant. L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were cultured in YES medium at 26°C or 36°C for 6 h. After incubation, cellular RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. RT-qPCR was performed by using primer sets amplifying <i>ura4</i> and <i>act1</i>. (C) ChIP analysis of the <i>asf1-33</i> mutant. L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated in YES medium at 36°C for 6 h and the cells were collected by centrifugation. Immunoprecipitation was performed using an anti-C terminal H3 antibody. After immunoprecipitation, DNA was extracted and amplified by PCR with specific primers for quantitative analysis.</p

DNA double-stranded breaks occurred in the <i>asf1-33</i> mutant.

<p>(A) Pulse-field gel electrophoresis analysis of the <i>asf1-33</i> mutant. L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated in YES medium at 26°C and 36°C for 6 h, and 5.0×10⁸ cells were collected by centrifugation. Pulse field gel electrophoresis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. The intensity of DSB bands was measured using ImageJ. (B) Rad22-GFP foci in SKP558-7 (<i>asf1⁻</i>) and KT166 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were observed after incubation at 26°C or 36°C for 6 h. Fluorescence of Rad22-GFP foci was observed with a Leica TCS-SP5 confocal laser scanning microscope (Leica Microsystems, Japan). Arrows indicate the Rad22-GFP foci. (C) The number of Rad22-GFP foci was counted.</p

<i>asf1</i> mutations caused defects in chromatin structure at 36°C.

<p>L972 (<i>asf1⁺</i>) and SKP593-33P (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were treated with Zymolyase to make spheroplasts. MNase was added to the spheroplasts and incubated at 37°C for 5 min. Digested chromatin DNA was resolved by agarose gel electrophoresis and detected with ethidium bromide staining.</p

<i>S. pombe</i> strains used in this study.

<p><i>S. pombe</i> strains used in this study.</p

DNA damage checkpoint was activated in the <i>asf1-33</i> mutant at 36°C.

<p>(A&B) Chk1, but not Cds1, was phosphorylated in the <i>asf1-33</i> mutant at 36°C. Phosphorylation of Chk1 and Cds1 proteins was observed by the mobility shift of phosphorylated proteins during electrophoresis. TH1 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r cds1⁻-3HA</i>) and TH9 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r chk1⁻-13myc</i>) were incubated in YES medium at 26°C and 36°C for 6 h. 30 µg and 15 µg of total proteins were used for detecting Chk1 and Cds1, respectively, by western blotting. HU is used as a DNA replication inhibitor which arrests cell cycle progression at G1/S phase. MMS is used as a DNA damaging agent. Addition of HU and MMS induced mobility shift of phosphorylated Cds1 and Chk1 proteins, respectively. (C) L972 (<i>asf1⁺</i>), SKP593-33P (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>), TH19 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r Δchk1</i> mutant), TH20 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i> Δ<i>cds1</i> mutant), and TH34 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r chk1S345A</i> mutant) were grown on YES plates containing phloxine B at 26°C and 36°C for 24 h. Cell morphology was observed by a microscope. (D) Cultures of L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were serially diluted with sterilized water. The cells were spotted on YES plates containing 10 mM HU (DNA replication inhibitor) and 0.0075% MMS (DNA damaging agent) and cultured at respective temperature for 3 days.</p