Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly

SummaryChromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106

pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis

Preventing H4K16 acetylation rescues S phase progression defects without modulating telomere length or telomeric origin activity.

<p>(A-B) Reducing H4K16ac and H3K79me levels rescues S phase progression defects of <i>yku70Δ</i> mutants. Asynchronous cells were incubated in YPD for 8 hours at 30°C in the presence of 20 mM (A) or 5mM (B) NAM. Samples were taken at indicated time for flow cytometry-based DNA content analysis. (C-D) <i>SAS2</i> deletion does not prevent early activation of telomeric origins in <i>yku70Δ</i> cells. Cells were synchronized in G1 and released toward S phase in the presence of 200 mM hydroxyurea. 30 minutes before release, 400 ug/mL BrdU was added to cultures. Sonicated BrdU-labelled DNA was immunoprecipitated and recovered material from telomeric/subtelomeric origins was quantified by qPCR as described in materials and methods. Error bars: Standard error of the mean. (E) H4K16ac levels do not significantly influence telomere length. Telomere length was analysed by southern blotting using a probe that recognizes the telomeric TG_1-3 repeats.</p

An interplay between multiple sirtuins promotes completion of DNA replication in cells with short telomeres

<div><p>The evolutionarily-conserved sirtuin family of histone deacetylases regulates a multitude of DNA-associated processes. A recent genome-wide screen conducted in the yeast <i>Saccharomyces cerevisiae</i> identified Yku70/80, which regulate nonhomologous end-joining (NHEJ) and telomere structure, as being essential for cell proliferation in the presence of the pan-sirtuin inhibitor nicotinamide (NAM). Here, we show that sirtuin-dependent deacetylation of both histone H3 lysine 56 and H4 lysine 16 promotes growth of <i>yku70Δ</i> and <i>yku80Δ</i> cells, and that the NAM sensitivity of these mutants is not caused by defects in DNA double-strand break repair by NHEJ, but rather by their inability to maintain normal telomere length. Indeed, our results indicate that in the absence of sirtuin activity, cells with abnormally short telomeres, e.g., <i>yku70/80Δ</i> or <i>est1/2Δ</i> mutants, present striking defects in S phase progression. Our data further suggest that early firing of replication origins at short telomeres compromises the cellular response to NAM- and genotoxin-induced replicative stress. Finally, we show that reducing H4K16ac in <i>yku70Δ</i> cells limits activation of the DNA damage checkpoint kinase Rad53 in response to replicative stress, which promotes usage of translesion synthesis and S phase progression. Our results reveal a novel interplay between sirtuin-mediated regulation of chromatin structure and telomere-regulating factors in promoting timely completion of S phase upon replicative stress.</p></div

Preventing H4K16 acetylation rescues S phase progression defects without modulating telomere length or telomeric origin activity.

<p>(A-B) Reducing H4K16ac and H3K79me levels rescues S phase progression defects of <i>yku70Δ</i> mutants. Asynchronous cells were incubated in YPD for 8 hours at 30°C in the presence of 20 mM (A) or 5mM (B) NAM. Samples were taken at indicated time for flow cytometry-based DNA content analysis. (C-D) <i>SAS2</i> deletion does not prevent early activation of telomeric origins in <i>yku70Δ</i> cells. Cells were synchronized in G1 and released toward S phase in the presence of 200 mM hydroxyurea. 30 minutes before release, 400 ug/mL BrdU was added to cultures. Sonicated BrdU-labelled DNA was immunoprecipitated and recovered material from telomeric/subtelomeric origins was quantified by qPCR as described in materials and methods. Error bars: Standard error of the mean. (E) H4K16ac levels do not significantly influence telomere length. Telomere length was analysed by southern blotting using a probe that recognizes the telomeric TG_1-3 repeats.</p

Strains used in this study.

<p>Strains used in this study.</p

Elevated DDR signalling is deleterious for growth of <i>yku70Δ</i> cells upon NAM- and MMS-induced replicative stress.

<p>(A) Mutations that abolish H4K16ac cause a reduction in H3K79me3 levels. Protein samples from asynchronous cells were immunoblotted with indicated antibodies. Bar graph represents the ratio of H3K79me3 onto H4 signals as quantified by densitometry. Error bars: standard error of the mean (B) Deletion of <i>DOT1</i> rescues growth of <i>yku70Δ</i> mutants in NAM (C) <i>sas2Δ</i> and <i>dot1Δ</i> mutations reduce Rad53 activation in <i>yku70Δ</i> cells. Cells were exposed to 20 mM NAM for 8 hours at 30°C and samples were taken for Rad53 <i>in situ</i> autophosphorylation assays (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007356#sec009" target="_blank">materials and methods</a> for details). (D-E) <i>rad9Δ</i> (D) and <i>RAD53-HA</i> (E), which limit DDR signalling in response to replicative stress, rescue growth of <i>yku70Δ</i> mutants in NAM. Growth assay in 96-well plates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007356#sec009" target="_blank">materials and methods</a>). Error bars: standard deviation. (F) Deletion of <i>RAD9</i> rescues S phase progression defects of <i>yku70Δ</i> cells exposed to NAM. Asynchronous cells were incubated in YPD for 8 hours at 30°C in the presence of 20 mM NAM. Samples were taken at indicated time for flow cytometry-based DNA content analysis. (G-H) <i>yku70Δ</i> display synthetic sensitivity to MMS when combined with <i>slx4Δ</i> (G) and <i>pph3Δ</i> (H) mutants.</p

Multiple sirtuins permit growth of cells lacking Yku70/80.

<p>(A-B) <i>yku70Δ</i> causes synthetic growth defects when combined with <i>hst3Δ hst4Δ</i>. Five-fold serial dilution of cells were spotted on solid media and incubated at 25°C. (B) Doubling time for strains in A incubated in YPD at 30°C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007356#sec009" target="_blank">material and methods</a>). Error bars: standard deviation, p-value = 7.42x10^-7 (two-sided student’s T-test). (C-D) Preventing H4K16 acetylation rescues the growth of <i>yku70Δ</i> mutants in NAM. Yeast cells were incubated in a 96-well plate containing increasing concentrations of NAM. OD₆₃₀ readings were acquired after 48 h at 30°C and results were normalized to untreated controls. Error bars: standard deviation. (E) Deletion of genes encoding subunits of the Hst1-Sum1-Rfm1 complex causes synthetic lethality when combined with <i>hst3Δ hst4Δ yku70Δ</i>. (F) Lack of Sum1 causes synthetic sensitivity to MMS-induced replicative stress when combined <i>yku70Δ</i>. (G-H) H4K16ac is deleterious to the growth of <i>yku70Δ</i> mutants in the presence of MMS-induced replicative stress or at elevated temperatures.</p

Cells with short telomeres present Tel1-dependent defects in completing DNA replication upon NAM exposure.

<p>(A) <i>tel1Δ</i> does not cause NAM sensitivity and rescues the growth of <i>yku70Δ</i> cells in NAM (B) <i>tel1Δ</i> rescues the S phase progression defects of <i>yku70Δ</i> mutants in NAM. (C) Cells lacking telomerase subunits arrest in S-phase upon NAM-exposure. (D-E) <i>yku70Δ</i> cells do not present increased proportion of cells with Rad52-YFP (D) or Rfa1-YFP (E) foci compared to wild-type upon NAM exposure. (B-E) Asynchronous cells were incubated in YPD for 8 hours at 30°C in the presence of 20 mM NAM. Samples were taken at indicated time for flow cytometry DNA content analysis or fluorescence microscopy (Rad52-YFP or Rfa1-YFP foci). (F) <i>rrm3Δ</i> exacerbates the MMS-induced replicative stress sensitivity of <i>yku70Δ</i> cells. (G-H) Galactose-induced overexpression of <i>CDC45</i>, <i>SLD3</i> and <i>SLD7</i> (45/3/7) improves the growth of <i>yku70Δ</i> mutants in MMS (G) and NAM (H). A construct expressing a Myc-His Tag (MHT) was used for the control condition. (I) Overexpression of <i>CDC45</i>, <i>SLD3</i> and <i>SLD7</i> (45/3/7) rescues the synthetic growth defects of <i>yku70Δ rad52Δ</i> mutants exposed to MMS.</p