H2B ubiquitylation and the histone chaperone Asf1 cooperatively mediate the formation and maintenance of heterochromatin silencing

Abstract

Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin factors results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication and repair by anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications that modify nucleosome dynamics, such as H2B ubiquitylation (H2Bub). We report here that H2Bub and Asf1 cooperatively promote transcriptional silencing at yeast telomeres and mating loci. Through real time monitoring of HML (Hidden MAT Left) locus silencing, we found that transcriptional repression was slowly initiated and never fully established in mutants lacking both Asf1 and H2Bub. These findings are consistent with impaired HML silencer-binding and spreading of repressor proteins, Sir2 and Sir3. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing at mating loci. Thus, the interplay between H2Hbub and Asf1 may fine-tune nucleosome dynamics and SIR protein recruitment, and represent an ongoing requirement for proper formation and maintenance of heterochromatin.

Figure 1.

Cells lacking both anti-silencing function 1 (Asf1) and H2Bub exhibit defects in stress-resistance and gene silencing. (A) Specific genetic interactions between H2Bub and the Asf1 histone chaperone. Cell growth was observed for 10-fold serial dilutions of yeast cells spotted onto non-selective YPD plates, or plates containing one of the following chemicals at 30°C for 3–5 days: methyl methanesulfonate (MMS; causes DNA damage), hydroxyurea (HU; source of replication stress), UV irradiation (leads to DNA damage) or 6-Azauracil (6-AU; affects transcriptional elongation). The rad52Δ strain served as a control for MMS, HU and UV treatment, while rtf1Δ served as a control for 6-AU treatment. (B) Positional cluster analysis shows gene expression changes in asf1Δ, htb-K123R and asf1Δ htb-K123R mutant cells. Only genes whose expression was increased or decreased by at least 1.75-fold in at least one of these strains are shown (775 upregulated genes and 826 downregulated genes). Genes located within 20 kb of their respective telomeres are marked on the left as the telomere region. (C) H2Bub and Asf1 are required for gene silencing within the proximity of telomeres. Transcripts from each strain were isolated and analyzed by Phalanx Yeast OneArray^®. The numbers of genes affected in the mutants compared to wild-type (WT) were plotted against their position from telomeres in kilobase pairs (kb). The numbers of upregulated and downregulated genes are shown as red and green bars, respectively.

Figure 2.

H2Bub and Asf1 are important for cellular response to sex pheromone. (A) Loss of mating type silencing in asf1Δ htb-K123R mutant cells (Halo assay). The indicated strains (mating type a) were treated with various doses of α-factor (1, 5 and 25 mg/ml) and the inhibition of cell growth indicates that mating type silencing was well maintained. (B) Cells lacking both Asf1 and H2Bub cannot respond to alpha factor. Isogenic WT (HTB1), htb-K123R, asf1Δ and asf1Δ htb-K123R cells were arrested at G1 phase with α-factor at 30°C for 2–5 h; exp: cells at exponential stage; h: the time in hours under α-factor treatment. Cells at each time point were stained with SYBR Green and the DNA content was determined by flow cytometry. (C) Transcriptional profiles of htb-K123R, asf1Δ and asf1Δ htb-K123R mutants under α-factor treatment. Cells were treated with α-factor for 3 h and transcripts were isolated and analyzed by Phalanx Yeast OneArray^®. Red and green represent upregulated and downregulated genes, respectively. Genes that were not significantly affected appear black in the heat map. Genes that exhibited a 1.75-fold change or greater between the WT and at least one of the mutants were selected and used in gene clustering analyzes. Table A: downregulated genes were clustered into two predominant GO terms, sexual reproduction and response to pheromone. Table B: upregulated genes were clustered into several GO terms.

Figure 3.

Asf1 and H2Bub are required for the silencing of HML loci. (A) Deletion of the HML cassette restores the response to pheromone in asf1 htb-K123R double mutant cells. The halo assay was performed using WT, htb-K123R, asf1Δ and asf1Δ htb-K123R cells, all with HML deletion. (B) Deletion of the HML cassette restores gene expression of MAT a in response to pheromone in asf1 htb-K123R double mutant cells. The expression levels of several sexual reproduction genes (PRM2, PRM3, PRM6, FIG1, FUS2) were measured in strains with a WT (HML) or hmlΔ background by reverse transcriptase-polymerase chain reaction and normalized to ACT1. The value for the WT was set as 1 for each group and all values are shown as the mean ± SEM (n = 3). Means with different letters are significantly different (P < 0.05).

Figure 4.

Asf1 and H2Bub cooperate to regulate the establishment of HML repression. (A) Schematic of the HML replacement by GFP encoding gene. A gene encoding a nuclear-localized, destabilized version of GFP expressed from the URA3 promoter was integrated into the HML locus, replacing the α1 and α2 genes. (B) Fluorescence intensity of cells containing the hml::GFP reporter. Fluorescence intensity was measured by flow cytometry; profiles are shown for both nicotinamide (NAM, a Sir2 inhibitor)-treated (red column, NAM+) and untreated (blue column, NAM−) cells. Data are shown as the mean ± SEM (n = 3). Means with different letters are significantly different (P < 0.05). (C) The kinetics of silencing establishment are disrupted in the asf1 and htb-K123R double mutant. Isogenic cultures of the indicated strains were grown in 5 mM NAM to de-repress hml::GFP::PEST::NLS. NAM was removed by washing prior to measuring the establishment of silencing. The mean fluorescence intensity in relative fluorescence units was plotted against time for the four cultures; intensity is shown as the mean ± SEM (n = 3).

Figure 5.

Asf1 and H2Bub fine-tunes nucleosome occupancy and Sir protein recruitments at silenced mating loci. (A) Agarose gel image of chromatin DNA following micrococcal nuclease (MNase) digestion. Chromatin extracted from each strain was treated with different doses of MNase (0.01, 0.25, 0.5 and 0.1 U MNase/OD unit of cells) for 15 min at 37°C. MNase-undigested (0.5 OD unit) and -digested chromatin DNA (2.5 OD unit) were loaded onto a 2% agarose gel. (B) Nucleosome occupancy at the HML locus in different strains. Schematic of the primer pairs against the indicated positions of the HML locus. Nucleosomal DNA enrichment (normalized to signals derived from undigested genomic DNA) is shown at the indicated positions along the HML locus in each strains after treated with MNase (0.5 U MNase/OD unit of cells) for 15 min at 37°C. Data are shown as the mean ± SEM (n = 3). (C) The recruitment of silent information regulatory (SIR) proteins (Sir2 and Sir3) is decreased in asf1Δ and htb-K123R mutant cells. Top: schematic of the relative locations of the primer pairs used. Primers against SPS22 were used for normalization. Bottom: chromatin immunoprecipitation (ChIP) was performed using anti-Sir2 antibody (Ab) or anti-myc Ab (myc-tagged Sir3) to detect the relative occupancy of Sir2 or Sir3, respectively, on chromatin in isogenic WT (HTB1), asf1Δ, htb-K123R and asf1Δ htb-K123R cells. Data are shown as the mean ± SEM (n = 3). Means with different letters are significantly different (P < 0.05).

Figure 6.

H2Bub and Asf1 are critical for higher-order heterochromatin organization at silenced mating loci. (A) Chromosome conformation capture (3C) assay reveals a more flexible chromatin structure in asf1Δ and htb-K123R double mutants as compared to WT. Schematic of the primers used in 3C assay. Log phase cells were fixed with 3% formaldehyde at room temperature for 20 min and then digested with the Sau3A restriction enzyme. The linear range for the quantitative PCR reactions was determined by titrating the cross-linked and control templates after intra-molecular ligation and cross-link reversal. The products were separated on agarose gels; primers at site P were used as a positive control. (B) Agarose gel image of WT (HTB1) and asf1Δ htb-K123R chromatin DNA prior to chromatin immunoprecipitation. Chromatin was cross-linked for 15 min and treated with 0.5 or 0.25 U MNase/OD (WT and asf1Δ htb-K123R cells, respectively) for 10 min at 37°C. Sheared chromatin DNA fragments were then visualized on a 2% agarose gel. (C) Nucleosome accessibility at the HML locus is increased in double mutants. ChIP was performed using an antibody against the H3 C-terminus to detect the relative occupancy of histone H3 across the HML locus in WT (HTB1) and asf1Δ htb-K123R cells. Primers against telomere (VI-R) were used for normalization. Data are shown as the mean ± SEM (n = 3). The differences between nucleosome DNA of WT and asf1Δ htb-K123R cells at each position are shown as Δ relative IP.

Figure 7.

A proposed model. Anti-silencing function 1 (Asf1) and histone H2B ubiquitylation (H2Bub) collaboratively maintain transcriptional silencing at the HML locus by fine-tuning nucleosome assembly and Sir protein recruitment, both of which are critical for heterochromatin maintenance. The recruitment of general repressors Sir2 and Sir3 to HML E/I silencers and subsequent spread into α1/α2 genes is significantly reduced in double mutants. In addition, the loss of nucleosomes at HML, which in turn affects higher-order heterochromatin organization, is pronounced in the absence of H2Bub and Asf1.