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. Author manuscript; available in PMC: 2016 Mar 16.
Published in final edited form as: Cell Rep. 2015 Nov 25;13(9):1772–1780. doi: 10.1016/j.celrep.2015.10.070

H4K44 acetylation facilitates chromatin accessibility during meiosis

Jialei Hu 1,2, Greg Donahue 1,2, Jean Dorsey 1,2, Jérôme Govin 1,4, Zuofei Yuan 2,3, Benjamin A Garcia 2,3, Parisha P Shah 1,2,5, Shelley L Berger 1,2,5
PMCID: PMC4793274  NIHMSID: NIHMS734630  PMID: 26628362

Abstract

Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double strand breaks during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal double strand break formation and meiotic recombination.

Keywords: chromatin, histone, acetylation, sporulation, meiotic recombination

Graphical Abstract

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Introduction

Modulating accessibility of the nucleosomal DNA is critical for transcription, replication, recombination and DNA damage repair (Bell et al., 2011). In these processes, histones modifications are key players, functioning either as docking sites for recruiting trans-acting proteins, or to directly influence the chromatin structure, thus affecting DNA accessibility. In their trans-acting role, histone modifications commonly recruit effectors to influence the chromatin function, via association with specialized protein domains, such as PHD domains (Wysocka et al., 2006) or bromodomains (Musselman et al., 2012). In contrast, modifications on the histone globular cores directly modulate chromatin structure. For example, lysine acetylation on the nucleosome lateral surface, including H3K56, H3K64, H3K115, H3K122, H4K77 and H4K79, can regulate DNA accessibility by facilitating nucleosome mobility or histone eviction (Tropberger and Schneider, 2013). These findings support a model whereby lateral surface modifications alter nucleosome mobility and stability (Cosgrove et al., 2004).

An important biological function regulated by DNA accessibility is meiotic homologous recombination. In most sexual species, meiotic recombination ensures accurate chromosome segregation and generates genetic diversity in gametes. Meiotic recombination is triggered by the formation of programmed DNA double-strand breaks (DSBs), catalyzed by the conserved topoisomerase-related Spo11 (Keeney, 2001). In S. cerevisiae, at least nine additional factors are required for DSB formation (Keeney, 2008). Following DSB formation, Rad51 and Dmc1 recombinases are involved in the repair of meiotic DSBs (Hunter, 2007).

Multiple chromatin features are associated with meiotic recombination in S. cerevisiae. Meiotic recombination occurs preferentially at specific sites (hotspots), which often reside in open regions at most gene promoters (Pan et al., 2011); the open configuration is believed to contribute to initiation of recombination (Wu and Lichten, 1994). Some hotspots exhibit increased nuclease sensitivity shortly before DSB formation (Ohta et al., 1994), indicative of active chromatin remodeling to increase DNA accessibility. DSB hotspots are also enriched for specific histone modifications (Zhang et al., 2011), including H3K4me3, which may be a major determinant of DSB location (Borde et al., 2009; Sollier et al., 2004). The mechanistic link between histone methylation and DSB formation is achieved by the COMPASS subunit Spp1 (Acquaviva et al., 2013; Sommermeyer et al., 2013); however, it remains unclear how chromatin structure is regulated to favor DSB formation.

In a previous study, we carried out a mutational screen of modifiable residues on histones H3 and H4 to uncover substitutions that affect sporulation efficiency in S. cerevisiae (Govin et al., 2010a). Interestingly, a number of modifications were located on the nucleosome lateral surface, indicating an important function for chromatin structure regulation. Here we describe an acetylation site on Lys44 on histone H4 (H4K44ac) on the nucleosome lateral surface. We show that H4K44ac is associated with meiotic recombination, and our results suggest an important role for H4K44ac in promoting an accessible chromatin environment for efficient programmed DNA recombination.

Results

H4K44ac is important for yeast sporulation

We previously identified several modifiable residues on histone H3 and H4 required for yeast sporulation, including residues reside on the nucleosome globular core (Govin et al., 2010a). To determine which among these residues are modified in meiosis, we purified histones from S. cerevisiae meiotic cells and subjected them to chemical derivization via propionylation (pr) and nanoLC-MS/MS analyses. Tandem mass spectrometry revealed a small peptide from histone H4 core that was acetylated at K44 (prGGVKacR) (Fig. 1A). Accurate mass (307.688 m/z) matched the calculated mass of this peptide as acetylated (307.685 m/z), as opposed to tri-methylated (307.703 m/z), and retention time also indicated an acetylated rather than a tri-methylated peptide (Fig. S1A).

Figure 1. Histone H4K44 is acetylated in sporulation, and is important for normal sporulation efficiency.

Figure 1

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A. Mass spec analysis of histones during sporulation. Fragmentation of the parent ion (histone H4 41-GGVKacR-45) with m/z=307.688. The arrows bracket the peaks that de ne the presence of acetyl-lysine (Kac).

B. Representative western blot (left) of flag-tagged histone H4 immunoprecipitated with flag antibody from WT and H4K44 mutants probed with antibodies against H4K44ac (top) or total H4 (bottom). H4K44ac is detectable in WT after cells are transferred into acetate (pre-sporulation) medium; H4K44ac is not detectable in H4K44A or K44R mutants. Quantification (right) was performed using two biological replicates.

C. Presence of pre-sporulation H4K44ac, followed by peak of H4K44ac during meiosis (4h), and then loss of H4K44ac. Representative western blot (left) for each histone modification throughout sporulation; quantification of each modification (right; n=3).

D. H4K44 mutants have reduced sporulation efficiency, as measured by percentage of tetrads from an initial population of cells induced to sporulate (mean ± SEM of three independent experiments). The difference is statistically significant: **P-value<0.01.

E. H4K44R spores are mostly inviable. 4-spore tetrads from three different yeast isolates were dissected for WT and K44R strains; distribution of spore viabilities was plotted per tetrad for each strain. n=100.

See also Figure S1.

To characterize the H4K44ac modification, we raised an antibody against a synthetic peptide containing acetylated H4K44. Antibody specificity was measured by western blot and dot blot analyses (Fig. 1B and S1B). Using this antibody, we observed enrichment of H4K44ac during growth in the pre-sporulation medium and at prophase I in meiosis (Fig. 1C and S1C). This pattern is unique compared to other meiosis-associated histone modifications, such as H4S1ph that increases following meiosis, or H3K4me3 that is constant through sporulation (Fig. 1C) (Govin et al., 2010a; Krishnamoorthy et al., 2006).

To characterize the function of H4K44ac during sporulation, we engineered H4K44 mutant strains harboring non-modifiable H4K44R. WT and H4K44R strains were sporulated and cells were collected throughout sporulation to determine the overall sporulation frequency. H4K44R sporulation was significantly lower than WT (63% of WT; Fig. 1D). Importantly, most of the resulting tetrad spores in the H4K44R mutant were inviable (Fig. 1E). H4K44R spore inviability suggests a defect in chromosome segregation (Keeney, 2001), implicating that meiotic recombination may be compromised in H4K44R.

H4K44ac is important for meiotic recombination

H4K44ac enrichment during meiosis (Fig. 1C) and the extremely low spore viability in H4K44R (Fig. 1E) led us to focus on the role of H4K44ac during meiotic recombination. First, we examined the effect of H4K44R in a random spore analysis assay measuring recombination frequency between heteroalleles of HIS4 locus during meiosis. H4K44R displayed a significant decrease in meiotic recombination events at HIS4, to less than 50% of WT H4 recombination (Fig. 2A, bottom panels).

Figure 2. H4K44ac facilitates meiotic recombination.

Figure 2

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A. Top: Schematic diagram of intragenic recombination at his4G and his4R heteroalleles. Bottom left: Random spore analysis from WT and H4K44R strains. Spores were plated on control YPD plates (left) and – HIS selection plates (right); ten times as many spores were plated on –HIS. H4K44R shows reduced recombination compared to WT. Bottom right: Intragenic recombination was calculated as the percent of HIS+ colonies relative to total viable colonies on YPD plate (mean ± SEM of three independent experiments). The difference is statistically significant: **P-value<0.01.

B–C. H4K44R strains exhibit fewer DSBs than WT (sae2Δ background). Top: Schematic map of the FRS2-GAT1 (left) and BUD23-ARE1 (right) loci. The positions of the HindIII restriction sites and the sizes of the DSB fragments are indicated; probe locations marked by the black bars. Bottom: Representative Southern blot for DSB formation across a meiotic time course in WT and K44R. n=3. Arrows indicate the HindIII fragment without DSBs and fragments corresponding to meiotic DSBs. Quantification of DSBs levels at the bottom of each blot.

D. Pie chart representation of Rad51 peaks loss in H4K44R (78% loss, 330 out of 423).

E. Box plot representation of Rad51 enrichment (relative to input) at Rad51 peaks. Rad51 reduction in H4K44R is significant at the 330 lost peaks (Wilcoxon test: ** p < 2.2 × 10−16), while there is no significant change in the set of 93 retained peaks (p = 0.1702). Area-under-curve (AUC) over the Rad51 peaks used to estimate p-value.

F. UCSC track shows decreased Rad51 at DSB hotspots in H4K44R relative to WT. Black bars represent DSB hotspots (Pan et al., 2011).

See also Figure S2 and Table S1.

We next analyzed whether DSB formation itself was affected in H4K44R. We directly analyzed meiotic DSB formation at two well-studied DSB hotspots, FRS2-GAT1 and BUD23-ARE1 (Fig. 2B and 2C) (Acquaviva et al., 2013; Yamashita et al., 2004). Meiotic DSBs at each hotspot were reduced in H4K44R compared to WT, determined by Southern blot using hotspot probes (Fig. 2B and 2C). We also performed pulsed-field gel electrophoresis (PFGE) to detect genome-wide meiotic DSBs (EtBr stain; Fig. S2A) and DSBs on Chr. III (Southern blot using probe to CHA1; Fig. S2B and S2C). These analyses confirmed that fewer DSBs form in H4K44R compared to WT.

To advance this observation and to compare genome-wide DSB formation and repair, we performed Rad51 ChIP-seq (Smagulova et al., 2011). Recombinases Rad51 and Dmc1 form nucleoprotein filaments on ssDNA at processed DSBs and are required for repair (Neale and Keeney, 2006); thus, we utilized Rad51 enrichment as an independent marker for DSB formation and repair. Rad51 ChIP-seq data were highly reproducible between biological replicates (Fig, S2D) and showed reproducible reduction of Rad51 enrichment in H4K44R compared to WT (Fig. 2F, S2E and S2F).

To more specifically determine differences in Rad51 peaks between WT and H4K44R, we identified 423 Rad51 peaks in WT (Supplemental Table S1) using MACS (see methods). Of these, 330 (78%) were undetected in H4K44R, with 93 Rad51 peaks maintained in H4K44R (Fig 2D and 2E), indicating significant Rad51 reduction in the mutant. Consistent with Rad51 enrichment at DSBs (Neale and Keeney, 2006), Rad51 peaks were highly correlated with Spo11 DSB hotspots (WT p-value = 0.018; K44R p-value < 0.001; Fig. S2F). We note that 153 Rad51 peaks were identified in H4K44R only; however, Rad51 enrichment at these peaks is lower than at Rad51 peaks in only WT or shared between WT and H4K44R (Fig, S2G and S2H). Thus, H4K44R-only Rad51 peaks may be an artifact of MACS, due to the low Rad51 signal in H4K44R (Fig. S2E) or may mark regions of transient Rad51 binding in H4K44R. The overall observation of significant Rad51 reduction in H4K44R, taken together with the Southern blot and PGFE analysis above, indicate that DSB formation and meiotic recombination are reduced in H4K44R and suggest that H4K44ac is required for normal levels of DSB formation.

H4K44ac marks meiotic DSB hotspots

Based on our observations, we postulated that H4K44ac may be specifically enriched at DSB hotspots. To test this, we performed H4K44ac ChIP-seq in WT meiotic cells at peak DSB formation (4h) to determine the genomic distribution of the modification. Strikingly, H4K44ac mapped preferentially to intergenic regions containing promoters (Fig. 3A), indicating a possible association with DSB hotspots, which generally are formed in intergenic promoters (Pan et al., 2011). To address this directly, we examined the H4K44ac enrichment at DSBs, using a recent high-resolution genome-wide DSB map (Pan et al., 2011). H4K44ac showed significantly strong enrichment at hotspot centers (Fig. 3B, p < 0.01). We note that H4K44ac enrichment at DSB hotspots is due in part to co-occurrence of DSBs at promoters. To determine whether H4K44ac is more specific to the promoters with DSB hotspots, we divided promoters into two classes: those overlapping (4,350) or not (2,367) with DSB hotspots (defined as the region 400 bp upstream from transcription start sites, TSS) (Tischfield and Keeney, 2012). On average, promoters with hotspots showed obvious and significantly higher H4K44ac enrichment than non-hotspot promoters (Fig. 3C and 3D). This observation was validated by ChIP-qPCR, randomly examining two promoters from each class (Fig. S3A).

Figure 3. H4K44ac is enriched at recombination hotspots.

Figure 3

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A. Metaplot representation of the distribution of H4K44ac around all TSS in S. cerevisiae.

B. Metaplot representation of the distribution of H4K44ac around meiotic recombination hotspots.

C. Metaplot representation of the distribution of H4K44ac around TSS with DSB hotspot (blue, n=4,350) vs those without (green, n=2,367). The maximum value for the H4K44ac profile for each gene between 500bp upstream and TSS was calculated for p-value.

D. Boxplot representation of the distribution of H4K44ac enrichment at 500bp window upstream of TSS from each group in C. H4K44ac enrichment is significantly different at TSS with DSB hotspot vs without (Wilcoxon test: ** P < 2.2 × 10−16). P-value was estimated as in (C).

E. H4K44ac enrichment for promoter hotspots divided into quintiles by Spo11 strength. Box plots (right) show distribution of Spo11 within each quintile.

F. Boxplot representation of H4K44ac enrichment at each quintile from E. H4K44ac enrichment is significant different at lowest and highest Spo11 quintiles (Wilcoxon test: ** P = 1.5 × 10−9). P-value estimated from AUCs of 400 bp around the Spo11 peak centers.

G. UCSC track shows distribution of H4K44ac enrichment at selected DSB hotspots (left) and control regions (right). Black bars represent DSB hotspots (Pan et al., 2011).

H. ChIP-qPCR validation of H4K44ac enrichment at DSB hotpots. PCR primers are designed within the regions marked in Fig. 3C. Mean ± SEM of three independent experiments. The difference is statistically significant: **P-value<0.01.

I. Scatter plot of H4K44ac enrichment intensities over 3,600 Spo11 DSBs between two independent H4K44ac ChIP-seq experiments shows a high degree of correlation (R2 = 0.89).

J. Boxplot representation of H4K44ac over 3,341 fragile nucleosomes and 58,673 stable nucleosomes (Xi et al., 2011) shows enrichment at fragile nucleosome sites. H4K44ac enrichment is significantly different between fragile and stable nucleosomes (Wilcoxon test: ** P < 1 × 10−16).

See also Figure S3.

To further determine a quantitative relationship between H4K44ac levels and DSBs, we subdivided promoter hotspots into five quintiles based on Spo11 enrichment (Fig. 3E, right panel). Average H4K44ac enrichment profiles (Fig. 3E, left panel) show that H4K44ac correlates to DSB “hotness”; we observe higher H4K44ac enrichment in the hottest DSB quintile (most Spo11) relative to the coldest (least Spo11) (Fig. 3F). We note that the difference in H4K44ac enrichment across quintiles is modest relative to the extreme difference in Spo11 enrichment per quintile (~20-fold difference in medians; Fig. 3E, right panel); therefore, correlation between H4K44ac levels and DSB strength is small (R2=0.003) when measuring H4K44ac enrichment at individual promoter hotspots (Fig. S3B). Thus, our results indicate that H4K44ac is a histone modification that occurs specifically at DSB hotspots. However, enrichment of the modification alone is unlikely predictive of levels of DSB formation, as is the case for H3K4me3 (Tischfield and Keeney, 2012).

We further determined whether non-modifiable H4K44R has an impact on other important meiotic histone modifications, thus acting indirectly through other modifications. We analyzed H3K4me3, important for meiotic recombination and enriched at DSB hotspots (Acquaviva et al., 2013; Borde et al., 2009; Sollier et al., 2004; Sommermeyer et al., 2013) and H3K56ac associated with meiosis (Fig. 1C) (Govin et al., 2010a; Recht et al., 2006). We performed western blots to examine levels of H3K4me3 and H3K56ac during sporulation in WT and H4K44R, and found no obvious change in timing and relative abundance of each modification (Fig. S3C). We also investigated genome-wide distributions of H3K4me3 and H3K56ac using ChIP-seq following the same time profiling in the H4K44ac studies. We observed a modest decrease (less than 20%) of either H3K4me3 or H3K56ac enrichment at gene promoters or DSB hotspots in H4K44R compared to WT (Fig. S3, D–H), which may be due to a secondary effect of abnormal DSB formation in H4K44R cells. The H3K4me3 and H3K56ac analyses indicate that H4K44ac appears to function in meiotic recombination independent of a known chromatin pathway.

We note that H4K44ac enrichment perplexingly appears almost directly over the DSB hotspots, which are previously-defined promoter nucleosome-depleted regions (Pan et al., 2011). To address this possible paradox, we first validated the ChIP-seq data and con rmed the specificity of the H4K44ac antibody for ChIP. We randomly examined three DSB hotspots - BIO2, ELO1 and PYC1 (track views shown in Fig. 3G), which are within gene promoters - by ChIP-qPCR. As expected based on ChIP-seq results, H4K44ac is enriched at these DSB promoters relative to control loci within gene bodies. Importantly, H4K44ac enrichment was greatly reduced in H4K44R mutants at all measured loci (Fig. 3H). To further validate H4K44ac ChIP-seq enrichment, we performed an independent biological H4K44ac ChIP-seq replicate, which yielded consistent results (Fig. 3I).

We then considered how H4K44ac enrichment occurs in regions expected to be nucleosome-depleted. Previous reports have described highly sensitive nucleosomes located within nucleosome-depleted regions, suggesting that these regions are not completely devoid of nucleosomes but are associated with “fragile nucleosomes” (Weiner et al., 2010; Xi et al., 2011). We measured H4K44ac enrichment in regions of fragile and stable nucleosomes and observed significantly higher H4K44ac at fragile versus stable nucleosomes in both biological replicate ChIP-seq datasets (Fig. 3J). This observation suggests that H4K44ac may be associated with nucleosome fragility at certain sites and may contribute to nucleosome instability or weak histone/DNA interaction, as investigated below.

H4K44ac influences chromatin structure

Lateral surface histone modifications generally influence nucleosome stability (Tropberger and Schneider, 2013). H4K44 exists at the L1 loop linking α-helix1 and α-helix2 of histone H4, located close to the DNA entry-exit region of the nucleosome (Fig. 4A) and thus could contribute to chromatin organization. Meiotic micrococcal nuclease (MNase) digestion analysis has shown that some recombination hotspots exhibit an increase in MNase accessibility prior to the appearance of meiotic DSBs (Ohta et al., 1994). Therefore, we tested whether H4K44ac regulates chromatin accessibility during meiosis using MNase-seq to determine nucleosome positioning in WT and H4K44R during peak meiotic DSB formation (4h) compared to vegetative growth (YPD media) and pre-sporulation (0h; YPA media). Overall nucleosome occupancy was elevated in HK44R compared to WT in the regions surrounding all TSS (Fig. 4B) and DSB hotspots at 4h (Fig. 4C). There were no detectable nucleosome occupancy changes observed between H4K44R and WT in YPD or 0h (Fig. S4, A–D), suggesting that H4K44ac promotes chromatin accessibility when meiotic recombination is initiated. The increased nucleosome occupancy observed at 4h in H4K44R is more clearly visualized in overlaid WT and H4K44R tracks at representative DSB hotspots (Fig. 4F).

Figure 4. H4K44ac contributes to the chromatin openness.

Figure 4

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A. Nucleosome image of the full histone octamer; side chain containing H4K44 shown in both unmodified and acetylated states (PyMOL, PDB code 1ID3).

B–C. Nucleosome occupancy pro les of WT (red) and H4K44R (blue) at peri-TSS regions (B) and DSB hotspots (C) indicate significantly increased occupancy in H4K44R. P-values estimated from maximum value 500bp downstream of TSS and 500bp around Spo11 hotspots for (B) and (C) respectively.

D. Nucleosome occupancy pro les of WT (red) and H4K44R (blue) at TSS with DSB hotspot (top) vs without (bottom). Nucleosome occupancy is significantly increased in H4K44R at promoters with DSBs only. P-values estimated as in (B).

E. Nucleosome occupancy pro les of WT (red) and H4K44R (blue) at Spo11 quintiles. Box plots (right) show distribution of Spo11 enrichment within quintiles. P-values estimated as in (C).

F. Example overlaid MNase-seq track of WT (orange) and H4K4R (blue) shows increased nucleosome occupancy of H4K44R (blue over black curve) around DSB hotspots. Black bars represent DSB hotspots; track was smoothed with 15 pixels.

G. MNase-accessibility assay indicates increased chromatin accessibility in H4K44Q (right) compared to WT (left) and K44R (middle). A representative of three biological replicates is shown. The quantitative densitometric analysis of the indicated lanes is shown on the right.

H. Spot dilutions of WT, K44R, and K44Q strains carrying URA3 at the silent mating locus HMR indicate a growth defect in K44Q on 5-FOA (right) compared to YPD control (left). A representative result of two biological replicates is shown. H4K91A mutant (Ye et al., 2005) is a positive control.

I. Model for H4K44ac in chromatin regulation and meiotic recombination. H4K44ac may promote chromatin ‘openness’ in parallel with H3K4me3 at recombination hotspots to create accessible chromatin to facilitate meiotic recombination.

See also Figure S4.

We further investigated the correlation between nucleosomal occupancy changes and DSB hotspots between H4K44R and WT. As with the H4K44ac analyses(Fig. 3C and 3E), we divided all promoters into those with and without hotspots and then divided all hotspot promoters into quintiles of DSB strength. We observed that nucleosome occupancy in H4K44R at 4h is specifically increased at promoters with DSB hotspots (Fig. 4D) and that increased nucleosome occupancy correlates with DSB strength (Fig. 4E). Taken together, these observations support a function for H4K44ac in facilitating or maintaining chromatin accessibility at DSB hotspots in meiosis.

In contrast to H4K44R, we speculated that the H4K44Q constitutive acetylation mimic mutant should exhibit decreased nucleosome density. H4K44Q cells displayed severe growth limitation under pre-sporulation acetate conditions and failed to initiate premeiotic DNA replication or enter meiosis (Fig. S4, E–G; compare to H4K44R). Thus, we could not address the consequence of H4K44Q during meiosis and sporulation, noting that the failure of meiotic induction may result from a deleterious effect of mimicking constitutive acetylation. As an alternative, we characterized H4K44Q in logarithmically growing cells. First, we examined nucleosome accessibility by MNase digestion in H4K44Q, H4K44R, and WT strains. An equal number of nuclei from cycling cells were treated with an increasing concentration of MNase and the resulting digestion profiles were compared between strains. H4K44Q cells showed extensive digestion compared to WT and H4K44R cells (Fig. 4G, lane 5), indicating a more accessible chromatin structure in the constitutive acetylation-like state at H4K44.

Finally, we tested functional effects of H4K44 substitutions using a classic heterochromatin silencing assay (van Leeuwen et al., 2002), reasoning that increased accessibility would de-silence a reporter placed in normally closed chromatin. We examined effects of H4K44R and H4K44Q substitutions on expression of URA3 integrated (1) near the left telomere of chromosome VII (Tel VII-L), (2) within the silent MAT loci and (3) within the rDNA repeats. Expression of URA3 causes conversion of 5-FOA in the growth medium into toxic 5-uorouracil. Based on our other observations, we predicted that H4K44Q would display defective silencing, and indeed de-silencing occurred in H4K44Q at Tel VII-L, HMR (the effect was subtle at HML) and rDNA loci (Fig. 4H). Importantly, silencing was not affected in H4K44R mutation (Fig. 4H), as expected since acetylation is not likely occurring at silenced heterochromatin (Richards and Elgin, 2002) and since H4K44ac is not enriched in mitotic cells (Fig. 1B and S1C). For comparison, we tested H4K91A, which has a strong silencing phenotype and is important for chromatin structure (Ye et al. 2005), and we found similar silencing defects between H4K44Q and H4K91A (Fig. 4H). Thus, constitutive acetylation mimic at H4K44 results in increased MNase accessibility, compromised heterochromatin maintenance, and failure to induce sporulation. Together with the data above, these results underscore an important role for acetylation at H4K44 in regulating chromatin accessibility during meiosis.

Discussion

In this study, we identified and characterized the function of H4K44ac, a previously undiscovered histone modification enriched in pre-meiotic and meiotic yeast, and located on the nucleosomal lateral surface adjacent to the DNA backbone. Substitution of this residue to non-modifiable H4K44R confers reduction in meiotic recombination rate (Fig. 2A), DSB formation (Fig. 2B, 2C, and S2, A–C), and manifests in severe spore inviability (Fig.1E). In addition, genome-wide H4K44ac enrichment shows high correlation with DSB hotspots (Fig. 3, A–F), and with locations of fragile nucleosomes (Fig. 3J). H4K44R mutants show specific increased nucleosome occupancy around DSB hotspots during meiosis (Fig. 4, B-E and S4, A–D), and in contrast, H4K44Q mutants show increased MNase accessibility during mitosis (Fig.4G). Together, these data support a model in which acetylation at H4K44 on the histone globular core results in increased chromatin accessibility during meiosis.

Interestingly, this role is distinct from our previous study of post-meiotic histone H4 tail acetylation in chromatin compaction (Govin et al., 2010a). H4K44ac peaks during meiotic DSB formation and repair, which may be favorable for chromatin accessibility of key DSB-associated complexes to initiate and complete meiotic recombination. We propose that H4K44ac may play an important role in increasing chromatin accessibility via nucleosome-DNA destabilization, as has been shown for H3K64ac and H3K122ac on the histone H3 globular domain (Di Cerbo et al., 2014; Tropberger et al., 2013), whereas H4K16ac on the histone H4 tail inhibits inter-fiber interaction (Shogren-Knaak et al., 2006).

We observed preferential enrichment of H4K44ac at gene promoters with DSB hotspots than those without hotspots, and an increase of nucleosome occupancy around hotspot promoters in H4K44R at 4h rather than YPD or 0h, in support of a direct role of H4K44ac in meiosis. However, we note that H4K44ac alone is an insufficient indicator of the quantitative levels of DSB activity (Fig. S3B). Likewise, the overall nucleosome occupancy increase in H4K44R, while significant, is not as high as may be expected given the significant reduction in DSB formation compared to WT. This observation is likely due to the average effect of analyzing nucleosome occupancy at hotspots over the cell population, obscuring larger changes that may be occurring in individual H4K44R mutant cells. There are thousands of Spo11 binding sites across the yeast genome, but only an estimated ~160 actual DSBs occur per meiotic cell (Pan et al., 2011). We speculate that general increased chromatin accessibility at 4h in meiosis, mediated in part by acetylation at H4K44, is likely a prerequisite for DSB formation, and local chromatin structural changes at individual hotspots may vary within a population due to cell-to-cell variability.

While our observations highlight a role for H4K44ac during meiosis, we note that H4K44ac is not meiosis-specific, but is also observed in pre-meiosis (Fig.1B and 1C). The reduction between 0h and 4h during meiosis is possibly caused by passive loss via DNA replication during meiotic S phase. Interestingly, H4K44ac is observed only after switching the yeast into pre-sporulation acetate medium, indicative of a potential response from the absence of normal glucose growth conditions. We note that H3K4me3, which is important for meiotic DSB formation, is present in exponentially growing cells and is maintained at all sporulation stages (Fig. 1C) (Borde et al., 2009), and the switch between the vegetative and meiotic functions may be due to its binding factor Spp1 (Acquaviva et al., 2013; Sommermeyer et al., 2013). Thus, we speculate that H4K44ac may be associated with pre-meiotic transcriptional changes upon the switch into acetate medium. Considering that we observe no difference in nucleosome occupancy in H4K44R at 0h when H4K44ac is enriched (Fig. S4, C and D), another possibility is that the enzyme for H4K44ac is activated upon pre-meiotic induction (see below), but an additional Spp1-like factor may regulate the function of this modification.

Although K-to-R/Q substitution is a widely used strategy to characterize the function of lysine acetylation, these mutants may still potentially cause unrelated phenotypes from differences in the amino acid structures. To overcome this limitation, it is important to determine the enzyme that acetylates H4K44 in meiosis to further characterize the modification. Based on previous yeast meiotic transcriptome data (Primig et al., 2000), we surveyed expression of known histone acetyltransferases (Gcn5, Hat1, Hat2, Hpa1, Hpa2, Hpa3, Sas2, Sas3, Sas4, Sas5, Rtt109, Spt10, Esa1). We identified only Rtt109 having a similar expression pattern to H4K44ac during meiosis; however, Rtt109 does not show enzymatic activity on histone H4 (Driscoll et al., 2007; Han et al., 2007), and thus further investigation is required to identify the specific enzyme for H4K44ac.

Our observations provide additional insight into the direct role of histone modifications on meiotic recombination and support previous studies of chromatin openness at DSB hotspots (Pan et al., 2011). We further note that H4K44ac occurs near the DNA entry-exit point of the nucleosome and may play a role in nucleosome stability, supported by H4K44ac enrichment at fragile nucleosomes (Fig. 3J). Interestingly, the other known acetylated lysine residues located on the lateral surface, H3K64 and H3K122, also impact nucleosome dynamics and regulate gene transcription (Di Cerbo et al., 2014; Tropberger et al., 2013). These functions appear to be distinct from H4K44ac, which contributes to normal programmed recombination in sporulation. Hence, acetylation at different sites on the nucleosome lateral surface may exhibit specificity in molecular function based on location. It is of great interest to further characterize the mechanisms of these modifications in regulating distinct biological processes.

Materials and Methods

Yeast strains

The genotypes of all yeast strains and applications are listed in Table S2.

ChIP and ChIP-seq analysis

Chromatin immunoprecipitation (ChIP) assays were performed as described (Govin et al., 2010b). Sequences of primers used for ChIP-qPCR can be found in Table S3. ChIP-seq libraries were prepared using NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® and were single-end sequenced using Illumina® HiSeq 2000 or NextSeq 500 platforms.

MNase digestion and preparation of mononucleosomal DNA for sequencing

MNase digestion assays were performed as described previously (Rando, 2010). Mononucleosomal DNA was isolated and end-repaired for library preparation; libraries were constructed with NEBNext® Ultra™ DNA Library Prep Kit for Illumina® and single-end sequenced using Illumina® HiSeq 2000 or NextSeq 500 platforms.

Genomics analyses

Raw sequenced data were produced via Illumina® sequencing (Hi-Seq for WT and H4K44R MNase-seq, histone PTM, and H4 data; NextSeq 2000 for all others). ChIP-seq and MNase-seq data were aligned to the yeast sacCer2 assembly using bowtie v1.0 (parameters -m 1 and --best). All sequencing data are supplied on GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=qjcxwkoeflghjez&acc=GSE59005

Supplementary Material

1
2
3

Acknowledgments

We thank the IDOM Functional Genomics Core Sequencing Facility for ChIP-seq and MNase-seq. This work was supported by NIH grants GM055360 and U54-HD068157 awarded to S.L.B.

Footnotes

Detailed experimental methods and data analyses are provided in Supplemental Experimental Procedures.

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References

  1. Acquaviva L, Szekvolgyi L, Dichtl B, Dichtl BS, de La Roche Saint Andre C, Nicolas A, Geli V. The COMPASS subunit Spp1 links histone methylation to initiation of meiotic recombination. Science (New York, NY. 2013;339:215–218. doi: 10.1126/science.1225739. [DOI] [PubMed] [Google Scholar]
  2. Bell O, Tiwari VK, Thoma NH, Schubeler D. Determinants and dynamics of genome accessibility. Nat Rev Genet. 2011;12:554–564. doi: 10.1038/nrg3017. [DOI] [PubMed] [Google Scholar]
  3. Borde V, Robine N, Lin W, Bonfils S, Geli V, Nicolas A. Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites. The EMBO journal. 2009;28:99–111. doi: 10.1038/emboj.2008.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cosgrove MS, Boeke JD, Wolberger C. Regulated nucleosome mobility and the histone code. Nature structural & molecular biology. 2004;11:1037–1043. doi: 10.1038/nsmb851. [DOI] [PubMed] [Google Scholar]
  5. Di Cerbo V, Mohn F, Ryan DP, Montellier E, Kacem S, Tropberger P, Kallis E, Holzner M, Hoerner L, Feldmann A, et al. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription. Elife. 2014;3 doi: 10.7554/eLife.01632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Driscoll R, Hudson A, Jackson SP. Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science (New York, NY. 2007;315:649–652. doi: 10.1126/science.1135862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Govin J, Dorsey J, Gaucher J, Rousseaux S, Khochbin S, Berger SL. Systematic screen reveals new functional dynamics of histones H3 and H4 during gametogenesis. Genes & development. 2010a;24:1772–1786. doi: 10.1101/gad.1954910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Govin J, Schug J, Krishnamoorthy T, Dorsey J, Khochbin S, Berger SL. Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae. Nucleic acids research. 2010b;38:4599–4606. doi: 10.1093/nar/gkq218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Han J, Zhou H, Horazdovsky B, Zhang K, Xu RM, Zhang Z. Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication. Science (New York, NY. 2007;315:653–655. doi: 10.1126/science.1133234. [DOI] [PubMed] [Google Scholar]
  10. Hunter N. Meiotic recombination. In: Aguilera A, Rothstein R, editors. Molecular Genetics of Recombination. Springer; Berlin Heidelberg: 2007. pp. 381–442. [Google Scholar]
  11. Keeney S. Mechanism and control of meiotic recombination initiation. Curr Top Dev Biol. 2001;52:1–53. doi: 10.1016/s0070-2153(01)52008-6. [DOI] [PubMed] [Google Scholar]
  12. Keeney S. Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis. Genome dynamics and stability. 2008;2:81–123. doi: 10.1007/7050_2007_026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krishnamoorthy T, Chen X, Govin J, Cheung WL, Dorsey J, Schindler K, Winter E, Allis CD, Guacci V, Khochbin S, et al. Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis. Genes & development. 2006;20:2580–2592. doi: 10.1101/gad.1457006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Musselman CA, Lalonde ME, Cote J, Kutateladze TG. Perceiving the epigenetic landscape through histone readers. Nature structural & molecular biology. 2012;19:1218–1227. doi: 10.1038/nsmb.2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neale MJ, Keeney S. Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature. 2006;442:153–158. doi: 10.1038/nature04885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ohta K, Shibata T, Nicolas A. Changes In Chromatin Structure at Recombination Initiation Sites during Yeast Meiosis. Embo Journal. 1994;13:5754–5763. doi: 10.1002/j.1460-2075.1994.tb06913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pan J, Sasaki M, Kniewel R, Murakami H, Blitzblau HG, Tischfield SE, Zhu X, Neale MJ, Jasin M, Socci ND, et al. A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation. Cell. 2011;144:719–731. doi: 10.1016/j.cell.2011.02.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Primig M, Williams RM, Winzeler EA, Tevzadze GG, Conway AR, Hwang SY, Davis RW, Esposito RE. The core meiotic transcriptome in budding yeasts. Nature genetics. 2000;26:415–423. doi: 10.1038/82539. [DOI] [PubMed] [Google Scholar]
  19. Rando OJ. Genome-wide mapping of nucleosomes in yeast. Methods in enzymology. 2010;470:105–118. doi: 10.1016/S0076-6879(10)70005-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Recht J, Tsubota T, Tanny JC, Diaz RL, Berger JM, Zhang X, Garcia BA, Shabanowitz J, Burlingame AL, Hunt DF, et al. Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:6988–6993. doi: 10.1073/pnas.0601676103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell. 2002;108:489–500. doi: 10.1016/s0092-8674(02)00644-x. [DOI] [PubMed] [Google Scholar]
  22. Shogren-Knaak M, Ishii H, Sun JM, Pazin MJ, Davie JR, Peterson CL. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science (New York, NY. 2006;311:844–847. doi: 10.1126/science.1124000. [DOI] [PubMed] [Google Scholar]
  23. Smagulova F, Gregoretti IV, Brick K, Khil P, Camerini-Otero RD, Petukhova GV. Genome-wide analysis reveals novel molecular features of mouse recombination hotspots. Nature. 2011;472:375–378. doi: 10.1038/nature09869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sollier J, Lin W, Soustelle C, Suhre K, Nicolas A, Geli V, de La Roche Saint-Andre C. Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression. The EMBO journal. 2004;23:1957–1967. doi: 10.1038/sj.emboj.7600204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sommermeyer V, Beneut C, Chaplais E, Serrentino ME, Borde V. Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes. Molecular cell. 2013;49:43–54. doi: 10.1016/j.molcel.2012.11.008. [DOI] [PubMed] [Google Scholar]
  26. Tischfield SE, Keeney S. Scale matters The spatial correlation of yeast meiotic DNA breaks with histone H3 trimethylation is driven largely by independent colocalization at promoters. Cell cycle (Georgetown, Tex. 2012;11:1496–1503. doi: 10.4161/cc.19733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tropberger P, Pott S, Keller C, Kamieniarz-Gdula K, Caron M, Richter F, Li G, Mittler G, Liu ET, Buhler M, et al. Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer. Cell. 2013;152:859–872. doi: 10.1016/j.cell.2013.01.032. [DOI] [PubMed] [Google Scholar]
  28. Tropberger P, Schneider R. Scratching the (lateral) surface of chromatin regulation by histone modifications. Nature structural & molecular biology. 2013;20:657–661. doi: 10.1038/nsmb.2581. [DOI] [PubMed] [Google Scholar]
  29. van Leeuwen F, Gafken PR, Gottschling DE. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell. 2002;109:745–756. doi: 10.1016/s0092-8674(02)00759-6. [DOI] [PubMed] [Google Scholar]
  30. Weiner A, Hughes A, Yassour M, Rando OJ, Friedman N. High-resolution nucleosome mapping reveals transcription-dependent promoter packaging. Genome research. 2010;20:90–100. doi: 10.1101/gr.098509.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wu TC, Lichten M. Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science (New York, NY. 1994;263:515–518. doi: 10.1126/science.8290959. [DOI] [PubMed] [Google Scholar]
  32. Wysocka J, Swigut T, Xiao H, Milne TA, Kwon SY, Landry J, Kauer M, Tackett AJ, Chait BT, Badenhorst P, et al. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature. 2006;442:86–90. doi: 10.1038/nature04815. [DOI] [PubMed] [Google Scholar]
  33. Xi Y, Yao J, Chen R, Li W, He X. Nucleosome fragility reveals novel functional states of chromatin and poises genes for activation. Genome research. 2011;21:718–724. doi: 10.1101/gr.117101.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yamashita K, Shinohara M, Shinohara A. Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:11380–11385. doi: 10.1073/pnas.0400078101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ye J, Ai X, Eugeni EE, Zhang L, Carpenter LR, Jelinek MA, Freitas MA, Parthun MR. Histone H4 lysine 91 acetylation a core domain modification associated with chromatin assembly. Molecular cell. 2005;18:123–130. doi: 10.1016/j.molcel.2005.02.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang L, Ma H, Pugh BF. Stable and dynamic nucleosome states during a meiotic developmental process. Genome research. 2011;21:875–884. doi: 10.1101/gr.117465.110. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

1
NIHMS734630-supplement-1.docx (13.9KB, docx)
2
NIHMS734630-supplement-2.xlsx (18.7KB, xlsx)
3
NIHMS734630-supplement-3.pdf (7MB, pdf)

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