Defective transfer of parental histone decreases frequency of homologous recombination by increasing free histone pools in budding yeast

Abstract Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3–H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells.


Supplemental Figure 1. A graphic outline of eSPAN experimental procedure.
The graphic outlines the hypothetical outcomes for the symmetric newly synthesized and parental histone H3-H4 eSPAN peaks, with new and parental (H3-H4)2 tetramers at two replicating strands.The sample collection procedure was as Fig 1A .The chromatin was digested into the majority of Mono/di-nucleosome with MNase.The digested chromatin was immunoprecipitated with H3K4me3 (parental) or H3K56ac(new) specific antibodies.ChIPed DNA was first denatured and then undergo BrdU-IP by BrdU specific antibody.The eSPAN samples were constructed into the library with a single strand specific method (1).The red and green lines represent the Watson and Crick strands, respectively.In this situation, both parental histone and new histone eSPAN peaks will have no strand bias.Bias calculation at individual nucleosomes (two in the cartoon) is shown at the bottom.Supplemental Figure 2. Combination of dpb3Δ and mcm2-3A mutations neutralizes the strand bias of new histone H3-H4 tetramers (H3K56ac) in single dpb3Δ or mcm2-3A mutants.
(A) Snapshot of H3K56ac ESPAN read enrichment at leading and lagging strands at early replication origin ARS1309 in wild-type (WT), dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.The sequence reads were mapped to both the Watson strand (red) and the Crick strand (green) of the reference genome.The eSPAN experimental procedure following Fig 1A . (B-E) Top: Heatmaps representing the bias ratio of newly synthesized histone H3 (H3K56ac) eSPAN peaks for WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains at each of the 10 nucleosomes surrounding each of the 134 early DNA replication origins.Individual nucleosomes are represented by the circles at the top of the heatmaps, and their positions are indicated relative to the origin (−10 to +10).Each row represents the average log2 Watson/Crick ratio of H3K56ac eSPAN sequence reads at one origin.Bottom: Average bias ratio of newly synthesized histone H3 (H3K56ac) eSPAN peaks for WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains at each of the 10 nucleosomes surrounding the 134 early replication origins.(A) Fluorescence images of colonies derived from WT, dpb3Δ, mcm2-3A, dpb3Δ/mcm2-3A, and cac1Δ strains containing the RFP-GFP cassette at the HMLα::cre locus.The bright green sector in GFP channel or dark sector in RFP channel represent the loss of silence.(B) WT, dpb3Δ, mcm2-3A, dpb3Δ/mcm2-3A, and cac1Δ strains showed different degrees of silencing at the HML locus when analyzed by CRASH assay.A one-way ANOVA analysis was used for comparing between two strains.Error bars depict standard error of the mean.Asterisks indicate statistical significance between two strains.*p < 0.05, **p < 0.01, ***p<0.001,****p<0.0001.

Supplemental Figure 5. Cell-cycle progression in the parental histone chaperone mutants.
Cell-cycle progression of the WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains were monitored by flow cytometry after cells were released from arrest in G1-phase.A mating pheromone was added 60 min after release from G1-phase so that completion of cell division could be monitored by the disappearance of 2C cells and the reappearance of 1C cells.Supplemental Figure 6.Chromatin accessibility assay on WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A mutants at late S phase.

Figure 3 .Supplemental Figure 4 .
New histone chaperone mutations (cac1Δ, asf1Δ and rtt106Δ) show little effect on parental histone transfer.(A)Snapshot of H3K4me3 eSPAN read enrichment at leading and lagging strands at early replication origin ARS1309 in WT, cac1Δ, asf1Δ and rtt106Δ strains.The sequence reads were mapped to both the Watson strand (red) and the Crick strand (green) of the reference genome.The H3K4me3 eSPAN experimental procedure following Fig 1A.(B-D) Top: heatmaps representing the bias ratio of parental histone H3 (H3K4me3) eSPAN peaks for cac1Δ, asf1Δ and rtt106Δ strains at each of the 10 nucleosomes surrounding each of the 134 early DNA replication origins.Individual nucleosomes are represented by the circles at the top of the heatmaps, and their positions are indicated relative to the origin (−10 to +10).Each row represents the average log2 Watson/Crick ratio of H3K4me3 eSPAN sequence reads at one origin.Bottom: average bias ratio of parental histone H3 (H3K4me3) eSPAN peaks for cac1Δ, asf1Δ and rtt106Δ strains at each of the 10 nucleosomes surrounding the 134 early replication origins.Effect of parental histone chaperone mutations on loss of silencing at the HML locus, as determined by CRASH assay.
with different MNase amount.Chromatin sensitivity assays were performed using digestion with various concentrations of MNase for 10 min followed by quenching with stop solution and DNA extraction.MNase amount used from lane 1 to lane 6 are: 40;10; 2.5; 0.6; 0.15;0 in Unit.High MNase mount led to smaller fragment and nucleosome bands (poly, tri, di, and mononuclesomes) mean strong nucleosome positioning.The lane 4 DNA was used for mononulclesome/undigested chromatin fragment calculation in (B).The lane 1 DNA was used for sequencing library preparation and sequencing data analysis in (C) and (D).(B) Calculated relative ratio of mononulclesome/undigested chromatin fragment fraction.The ImageJ software was used to quantify the band intensity.(C) MNase-seq profiles of mean nucleosome occupancy around transcription start sites (TSS) for the WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.(D) MNase-seq profiles of mean nucleosome occupancy around early replication origin sites for the WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.Supplemental Figure 7. Histone modifications and cell growth in the parental histone chaperone mutants.(A) Immunoblot analysis of histone modification levels in whole cell extracts (WCE) from WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.H3K4me3 (ab8580 Abcam); H3K56ac (2); H3K36me3 (ab9050 Abcam); PGK1(ab113687 Abcam); Sir2 (CS1102,(3)) antibodies were used in these analyses.(B) Calculated relative H3K36me3, H3K56ac, H3K4me3 and Sir2 level.The data shown in comes from three independent experiments.The signals were normalized to the signals obtained for soluble PGK1 on western blots.Error bars depict standard error of the mean.A one-way ANOVA analysis was used for comparing between two strains.(C) Serial dilution spot assay on YPD and YPD with 50 mM hydroxyurea medium for the WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.Supplemental Fig 8. Genetic interaction of parental histone chaperone mutants (dpb3Δ, mcm2-3A) and new histone chaperone mutants (cac1Δ, asf1Δ and rtt106Δ) on Rad52 foci formation.(A) Rad52 foci frequency in WT, the histone dosage regulation mutant (hht2-hhf2Δ), and new histone chaperone mutants (cac1Δ, asf1Δ and rtt106Δ).The Rad52 foci frequency of dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A in hht2-hhf2Δ (B), asf1Δ (C), cac1Δ (D), and rtt106Δ (E) background.A one-way ANOVA analysis was used for comparing between two strains.Error bars depict standard error of the mean.ns p>0.05, *p < 0.05, **p < 0.01, ***p<0.001,****p<0.0001.Supplemental Fig 9.Western blot test of rad53-phosphralation and H2AX levels.(A) Western blot test Rad53-phosphralation without/with 0.1% MMS treatment.Rad53 antibody (ab104232, Abcam) was used for Rad53 detection.(B) Immunoblot analysis of H2AX levels (anti-γ-H2A antibody (ab15083, Abcam)), a marker of the DNA damage response in yeast, before and after treatment with methylmethane sulphonate (MMS; a DNA damaging agent) in WT, dpb3Δ, mcm2-3A, and dpb3Δ mcm2-3A strains.Supplemental Figure 10.Parental histone chaperone mutations increase free histone levels.(A-B) Immunoblot of H3, H3K4me3(a marker for chromatin proteins) and PGK1 (a marker for soluble proteins) in the whole cell extract, soluble fraction, and chromatin fraction of WT, dpb3Δ, mcm2-3A, dpb3Δ mcm2-3A and rad53 Δ strains.The experiment procedure following Fig 5A except two time points (60minutes and 80minutes).H3K4me3 (ab8580 Abcam); PGK1(ab113687 Abcam) and H3-HA (12CA5 Sigma) were used for Western blot.(C-D) Quantitation of soluble H3 and H3K4me3.The signals obtained for soluble histones were normalized to the signals obtained for soluble PGK1 on western blots (C-D).(E-F) Soluble H3 and H3K4me3 levels are higher in dpb3Δ, mcm2-3A, dpb3Δ mcm2-3A and rad53 Δ.The data shown in comes from average of three independent time points (50 minutes, 60 minutes and 80 minutes) data.Error bars depict standard error of the mean.The signals obtained for soluble histones were normalized to the signals obtained for soluble PGK1 on western blots.Error bars depict standard error of the mean.A one-way ANOVA analysis was used for comparing between two strains.*p < 0.05, **p < 0.01, ***p < 0.001.Supplemental Figure 11 Overexpression and deletion of new histone chaperones (Caf1, Asf1, Rtt106) on the soluble histone H3 level and HR frequency of dpb3Δ mcm2-3A double mutant.(A) Immunoblot of H3 and PGK1 in the whole cell extract, soluble fraction, and chromatin fraction of dpb3Δ mcm2-3A strain with overexpressing Caf1, Rtt106 and Asf1.The experimental following Fig 5A.The empty-vector control pRS425 (High copy vector) and a plasmid containing all three genes of the Caf-1 complex (pCAF-1, alias pJR3418), ASF1 (pASF1, alias pJR3425), or RTT106 (pRTT106, alias pJR3419) (4) were transformed into dpb3Δ mcm2-3A mutant cells.(B) Quantitation of soluble H3.The data shown comes from two independent experiments.The signals obtained for soluble histones were normalized to the signals obtained for soluble PGK1 on western blots.Error bars depict standard error of the mean.A one-way ANOVA analysis was used for comparing between two strains.ns: p>0.05.(C) Overexpression of the new histone chaperone complex's effect on the HR efficiency of WT.The empty-vector control pRS425 (High copy vector) and a plasmid containing all three genes of the CAF-1 complex (pCAF-1, alias pJR3418), ASF1 (pASF1, alias pJR3425), or RTT106 (pRTT106, alias pJR3419) (42) were transformed into WT.(D) Overexpression of the new histone chaperone complexes cannot rescue the HR efficiency of dpb3Δ mcm2-3A.The same plasmids used in (C) were used in this panel.(E) Deletion of the new histone chaperones on the HR efficiency of WT and dpb3Δ mcm2-3A strains.A one-way ANOVA analysis was used for comparing between empty vector and each overexpressed histone chaperone (C and D).ns: p>0.05, **p < 0.01, ****p<0.0001.