Changes in chromatin organization occur during aging. Overexpression of histones partially alleviates these changes and promotes longevity. We report that deletion of the histone H3-H4 minor locus HHT1-HHF1 extended the replicative life span of Saccharomyces cerevisiae. This longevity effect was mediated through TOR signaling inhibition. We present evidence for evolutionarily conserved transcriptional and phenotypic responses to defects in chromatin structure, collectively termed the chromatin architectural defect (CAD) response. Promoters of the CAD response genes were sensitive to histone dosage, with HHT1-HHF1 deletion, nucleosome occupancy was reduced at these promoters allowing transcriptional activation induced by stress response transcription factors Msn2 and Gis1, both of which were required for the life-span extension of hht1-hhf1Δ. Therefore, we conclude that the CAD response induced by moderate chromatin defects promotes longevity.

Cellular responses to stress, such as starvation, DNA damage, oxidation, and protein aggregation, antagonize aging. The attenuation of these pathways during aging causes accumulation of damaged macromolecules that impair cellular function, while boosting these stress response pathways often promotes longevity. Target of rapamycin (TOR) signaling is a highly conserved regulatory node of cellular stress response, which integrates nutrient and cellular stress signals with cellular metabolism, cell cycle progression, and stress response activation. Starvation or high levels of cellular stress inhibit TOR signaling, leading to the inhibition of protein synthesis and the initiation of the general control protein Gcn4-mediated transcriptional response ( 5 ). At the chromatin level, there are two distinct changes associated with strong stress signals: Overall transcriptional activity is decreased due to reduced histone acetylation levels and increased chromatin compaction associated with slowed or arrested cell cycle progression, whereas stress response genes are activated by specific transcription factors due to chromatin remodeling at their promoters. However, the response of cells to chromatin-associated changes, especially histone loss, remains poorly understood. Here, we explored the relationship between histone gene expression dosage and longevity in the budding yeast Saccharomyces cerevisiae and unexpectedly discovered that moderate reductions in H3-H4 histone gene expression can extend the yeast RLS by activating a distinct stress response and inhibiting the TOR signaling pathway.

The basic structural unit of a eukaryotic genome, the nucleosome, comprises 146–base pair (bp) DNA segments wrapped around core histones. Proper dynamics of nucleosome formation and higher-level chromatin organization regulate the accessibility of DNA and ensure normal genomic functions ( 1 ). Thus, histones also function as an indispensable regulatory platform for cellular and biological processes, including aging. Altered histone modifications, which occur during aging, contribute to the aging process ( 2 ). A decrease in histone protein levels is a hallmark of aging cells ( 3 ). Overexpression of genes encoding histones H3 and H4 from a galactose-inducible promoter extends the replicative life span (RLS) of the budding yeast, suggesting that histone gene expression capacity may be a limiting factor in aged cells ( 4 ). However, considering that histone gene expression is cell cycle regulated, the relationship between longevity and histone gene expression dosage cannot be delineated without a careful dosage titration using the native promoters.

RESULTS

Deleting the minor H3-H4–coding gene pair HHT1-HHF1 extends the RLS The budding yeast S. cerevisiae contains two copies of core histone gene pairs under the control of bidirectional promoters. Despite encoding identical amino acid sequences, the two copies differ in their nucleotide sequences and expression levels. Consistent with a previous report (6), HHT1-HHF1, the minor H3-H4–coding gene pair, provided approximately 15% of the total H3-H4–encoding mRNA (fig. S1A), whereas the major copy HHT2-HHF2 accounted for the rest of the H3-H4 transcripts. Although removing both H3-H4 copies is lethal, knockout strains for either gene pair show no observable growth defects (7). Deletion of single histone genes disrupted the stoichiometry of nucleosome assembly and reduced the RLS (fig. S1B). Therefore, we genetically manipulated only the complete H3-H4 gene pairs rather than single histone genes. Removal of the minor copy HHT1-HHF1 (htf1Δ), representing 85% of wild-type (WT) H3-H4 dosage, significantly extended the RLS, whereas deletion of HHT2-HHF2 (htf2Δ), cutting down the dosage to 15%, reduced the RLS (Fig. 1A). Consistent with the lack of longevity effects upon H2A-H2B overexpression (4), deletion of the nonessential H2A-H2B–encoding gene pair had no effect on the life span (Fig. 1B). Reintegrating the deleted copies of HHT1-HHF1 or HHT2-HHF2 at the URA3 locus restored the life span of htf1Δ or htf2Δ, respectively, to the WT level, suggesting that the life span effects in both deletion strains were the direct result of altered histone gene dosage (Fig. 1, C and D). We ruled out the possibility of Ty transposon–mediated duplication (8) in the htf1Δ and htf2Δ strains by verifying the histone gene copy number via quantitative polymerase chain reaction (qPCR) using genomic DNA (fig. S1C). To further investigate the life-span effects when reducing a histone dosage to between 15 and 85%, we constructed another strain with two copies of HHT1-HHF1 (htf2Δ HTF1OE), which provides 30% of WT H3-H4 gene dosage. This strain showed an RLS similar to that of the WT and between those of the htf1Δ and htf2Δ (fig. S1D), suggesting a dosage-dependent change in life span between 15 and 85% of H3-H4 dosage. Fig. 1 RLS of histone copy variant strains. (A) RLS of the WT (n = 81), htf1Δ (n = 51), and htf2Δ (n = 61) strains. Values in parentheses on the graph are mean RLS. (B) RLS of WT (n = 101) and hta2-htb2Δ (n = 97). (C) RLS of WT (n = 55) and htf1Δ rescue strain HTF1 (n = 51). (D) RLS of WT (n = 59) and htf2Δ rescue strain HTF2 (n = 50).

Reduction of the H3-H4 gene copy number increases chromatin accessibility We next assessed the effect of deleting the histone gene pairs on histone protein levels. In nonsynchronized cells, deletion of either the minor or the major copy of the H3-H4 gene pair had no impact on overall H2B, H3, and H4 protein levels (Fig. 2A), despite the changes in the mRNA levels (Fig. 2B). These observations were similar to a previous report (9). Consistent with the findings of reduced histone synthesis capacity in htf1Δ and htf2Δ, when synchronized, these mutant cells showed significantly lower rates of histone synthesis following the release from α-factor–induced cell cycle arrest (Fig. 2C). Subsequently, we tested whether histone gene deletion affects age-dependent histone depletion. The htf2Δ, bearing deletion of the major histone gene copy, showed a greater degree of histone depletion relative to similarly aged htf1Δ or WT cells (Fig. 2D and fig. S1E), suggesting that histone synthesis capacity becomes a critical issue in aged cells. Fig. 2 Reduction in H3-H4 gene copies increases chromatin accessibility. (A) Steady-state level of H3 protein, with glyceraldehyde phosphate dehydrogenase (GAPDH) as a loading control (left). No differences in H2B, H3, and H4 levels were found (right). Error bars here and below represent SD, unless noted otherwise. (B) qPCR analysis of H3 transcript abundance normalized against H2B transcript level. ***P < 0.001 compared to WT. (C) H3 protein levels in synchronized cells at indicated times following α-factor release. *P < 0.05 and #P < 0.001 compared to WT. (D) H3 protein levels in old and young cells. *P < 0.5; ns, not significant compared to WT. (E) MNase digestion assay with designated concentration of MNase. (F) Fold change (log 2 FC) in expression of telomeric and subtelomeric genes in htf1Δ, htf2Δ, and HTF2OE strains. Error bars represent the 25 and 75% percentiles. (G) qPCR analysis of HMLα1 transcript abundance. *P < 0.05 and ***P < 0.001 compared to WT. (H) α-Factor (αF) sensitivity test. Error bars represent the 25 and 75% percentiles. Serving as the building blocks of the nucleosomes, histones greatly affect chromatin architecture. Thus, we assessed histone-dependent effects on chromatin accessibility using micrococcal nuclease (MNase). Young cells of htf1Δ and htf2Δ strains contained more MNase-accessible chromatin than the WT (Fig. 2E). The htf2Δ strain demonstrated a greater increase in chromatin accessibility than htf1Δ, commensurate with the respective changes in histone dosages. To further assess local chromatin changes and their effects on transcription of normally silenced regions, we performed RNA sequencing (RNA-seq) to compare the transcriptomes of htf1Δ-, htf2Δ-, and HHT2-HHF2–overexpressing (HTF2OE) strains to that of the WT. The subtelomeric genes located within 30 kb of the chromosome ends, which are known to be sensitive to chromatin changes (10), were up-regulated in both deletion strains, but not in HTF2OE (Fig. 2F), indicating derepression of subtelomeric chromatin. Another heterochromatic region in the budding yeast includes the hidden mating-type loci HML and HMR, containing silenced copies of mating-type genes MATα and MATa, respectively. Both of the histone gene deletion strains exhibited derepression of the HML loci, as evidenced by increased expression of HMLα1 and decreased sensitivity to the mating pheromone α-factor (Fig. 2, G and H). These changes, however, were not observed in HTF2OE. These data indicated a more open chromatin structure in strains with histone gene deletions. It is particularly interesting that, with the deletion of different histone copies, htf1Δ and htf2Δ show markedly different life span. Since we observed that knockout of major histone copy leads to substantial chromatin opening (Fig. 2, E and G) compared to htf1Δ, we propose that this may lead to a more disturbed transcriptome. The RNA-seq analysis indeed detected more changes in the transcriptome of htf2Δ compared to htf1Δ, indicating that the extensive loss of histones in this strain leads to higher transcriptional noise (fig. S2A) and may eventually result in the short-lived phenotype. Note that the transcriptome profiles of htf1Δ and htf2Δ are highly similar, with just a few genes differentially expressed in one mutant but not the other (fig S2B). This fact suggests that deletion of minor or major H3-H4–coding gene leads to similar transcriptional response, while the level of this disturbance is the determinant of life span. Given that overexpression of either H3-H4–coding gene pair extends the RLS (fig. S2C) (4), we asked whether shared underlying mechanisms exert the anti-aging effects in htf1Δ and the histone gene overexpression strains. The Pearson correlation between the transcriptomic patterns of htf1Δ and HTF2OE was substantially lower than that between the transcriptomes of htf1Δ and htf2Δ (fig. S2, B and D). In contrast to htf1Δ, the overexpression strain also showed no loss of subtelomeric gene or HML locus silencing (Fig. 2, F and G). Furthermore, we found that the deletion of the histone transcription regulator 1 (HIR1) gene, which by itself increases H3-H4 levels and mimics the H3-H4 overexpression phenotype (4), reduced the RLS when combined with deletion of the minor H3-H4 gene pair (htf1Δ hirΔ; fig. S2E). These data suggested that the life-span extension mechanism of histone overexpression is likely distinct from what we see in htf1Δ cells.

Life-span extension in htf1Δ is mediated through the TOR pathway To determine whether the RLS extension of htf1Δ is mediated through known aging regulatory pathways, we performed epistasis analyses. Considering that the aging regulator Sir2 is a chromatin modifier (11), we tested whether it is required for the longevity of the htf1Δ cells. The sir2Δ single mutant has extremely short life span due to the dominant ribosomal DNA instability (12), we tested Sir2 epistasis in a sir2Δ fob1Δ strain that shows a life span close to the WT. As shown in Fig. 3 (A and B), htf1Δ extended life span in the sir2Δ fob1Δ background, as well as the SIR2 overexpression background, suggesting that the life span–extending effect of HHT1-HHF1 deletion is completely independent of the Sir2-Fob1 pathway. Fig. 3 Life-span extension in htf1Δ is mediated through the TOR pathway. (A) RLS of WT (n = 51), htf1Δ (n = 50), sir2Δ fob1Δ (n = 55), and htf1Δ sir2Δ fob1Δ (n = 68). (B) RLS of WT (n = 50), htf1Δ (n = 52), SIR2OE (n = 50), and htf1Δ SIR2OE (n = 50). (C) RLS of WT (n = 62), htf1Δ (n = 50), tor1Δ (n = 63), and htf1Δ tor1Δ (n = 50). (D) RLS of WT (n = 59), htf1Δ (n = 50), rpl20bΔ (n = 50), and htf1Δ rpl20bΔ (n = 50). (E) RLS of WT (n = 50) and htf1Δ (n = 50) cells under caloric restriction (CR; 0.05% glucose), P = 0.455. (F) RLS of WT (n = 101), htf1Δ (n = 68), TOR1L2134M (n = 87), and htf1Δ TOR1L2134M (n = 100). (G) Average cell cycle duration of WT, htf1Δ, htf2Δ, and tor1Δ. ***P < 0.001 compared to WT. Error bars represent the 25 and 75% percentiles. Another well-recognized and evolutionarily conserved aging regulatory pathway is the TOR. The key component of the TOR pathway is the kinase Tor1, which integrates nutrient signaling and cellular stress to elicit global downstream metabolic changes and stress response pathways (13) . We found negative epistasis between tor1Δ and htf1Δ in RLS effects: Despite the extended life spans of the single mutants, the double mutant had a life span similar to that of the single mutants, with no further RLS increase (Fig. 3C). To verify this epistatic relationship, we performed similar life-span analysis using either the rpl20bΔ strain, which mimics the effects of TOR inhibition (14), or calorie restriction conditions. In both cases, HHT1-HHF1 deletion failed to further extend the life span (Fig. 3, D and E). Furthermore, when introduced into a hyperactive Tor1 mutant, TOR1L2134M (15), the HHT1-HHF1 deletion failed to extend the life span (Fig. 3F), underscoring that inhibition of TOR signaling is required for longevity induced by altered histone dosage. Moreover, the histone mutants and tor1Δ have similar growth phenotypes, with slower cell cycles compared to the WT (Fig. 3G). We reasoned that if TOR signaling is inactivated in htf1Δ, then deletion of HHT1-HHF1 and inhibition or ablation of TOR signaling should induce similar transcriptional responses. Therefore, we used RNA-seq to compare the transcriptomes of htf1Δ and htf2Δ mutants, calorie-restricted WT cells, and TOR1-deleted cells. Gene ontology (GO) analysis revealed categories of genes, including glycolysis and translation, that were similarly down-regulated in histone dosage mutants and calorie-restricted cells (Fig. 4A and fig. S3, A and B) (16). Further RNA-seq analysis revealed a substantial number of genes that were similarly down-regulated in htf1Δ and tor1Δ cells (Fig. 4B). Together, these data supported the hypothesis that TOR signaling inhibition mediates the longevity effect in htf1Δ cells. Fig. 4 Transcriptome analysis of histone deletion strains and characterization of stress-related features. (A) GO analysis of down-regulated genes in htf1Δ. GO categories with Benjamini P < 0.05 were included here and in GO analyses below. Underlined categories are enriched in transcriptomic changes of htf1Δ and cells under CR. Numbers in all GO figures indicate fold enrichment of the corresponding category. (B) Venn diagram showing genes down-regulated in tor1Δ cells (total, 1496), in htf1Δ cells (total, 269), and in both (total, 131). (C) GO analysis of genes up-regulated in htf1Δ. (D) Overlap between stress-related category genes and genes up-regulated in htf1Δ. (E) Flow cytometry–based cell cycle analysis of indicated strains. Cells were synchronized with α-factor. (F) qPCR analysis of MRE11 and RAD51 transcript abundance. *P < 0.05 and ***P < 0.001 compared to WT. (G) Flow cytometry–based cell cycle analysis of indicated strains. Cells were unsynchronized. Percent values represent fraction of cells within the shaded region containing 1N genome.

Stress experienced by the histone deletion strains is distinct from replication or mitotic stress The htf1Δ cells demonstrated up-regulation of genes belonging to GO categories that have been previously linked to stress responses (Fig. 4C), including transposable (17) element, proteolysis (18), the tricarboxylic acid (TCA) cycle (19), and cell wall components (20); htf2Δ also shows up-regulation of similar categories (fig. S3C). Moreover, we observed a significant overlap between genes up-regulated in htf1Δ and those induced by stress (Fig. 4D). Defects in chromatin assembly due to mutations in the histone chaperone chromatin assembly factor 1 (CAF-1) complex cause replication stress, manifested by S phase arrest and DNA damage (21). We thus asked whether reduced histone supplies pose similar challenges to DNA replication. However, we did not observe slowed cell cycle progression or stalled S phase in either of the histone deletion strains (Fig. 4E). The histone deletion strains also did not exhibit sensitivity to DNA damaging agents such as hydroxyurea, methyl methanesulfonate (MMS), or phleomycin that cause replication stress (fig. S3D). Furthermore, in contrast to the MMS treatment, inactivation of either of the H3-H4 gene pairs did not induce expression of the genes encoding the replication stress sensor Mre11 (22) and the DNA damage responder Rad51 (Fig. 4F) (23). These data suggested that the histone dosage mutants do not experience DNA damage and replication stress. Given that functional chromatin is also essential for separation of sister chromatids during cell division, we next assessed the histone deletion strains for potential mitotic stress. Deletion of the histone chaperone ASF1 gene causes cells to arrest in the G 2 /M phase (24), corresponding to a profound decrease in the haploid cell fraction. However, this phenotype was not evident in either the htf1Δ or the htf2Δ strain (Fig. 4G). The lack of mitotic stress in the histone deletion strains was also underscored by their insensitivity to benomyl, a drug that disrupts microtubule formation and causes mitotic stress (fig. S3D). A recent study of Caenorhabditis elegans reported that a decrease in histone levels induces the mitochondrial unfolded protein response (UPRmt) to promote life-span extension (25). Hence, we assessed UPRmt induction in the yeast histone deletion mutants. Induction of UPRmt corresponds to the activation of mitochondrial heat shock proteins (HSPs) (25), such as Hsp60 and Hsp10 (26). Neither of these mitochondrial HSPs was up-regulated in the htf1Δ and htf2Δ strains according to RNA-seq analysis (fig. S3E). Therefore, the form of stress experienced by the histone deletion mutants was distinct from the above well-characterized stress states.

Disruptions in chromatin architecture trigger a distinct type of stress response Responses to various stresses can trigger nucleosome position–dependent alterations in chromatin structure and consequent changes in gene transcription (27). However, these chromatin changes, especially those occurring during aging, have never been shown to trigger stress responses. Our present data suggest that reduced histone dosage induces a distinct type of stress response. We next asked whether such a stress response can also be observed in other mutants with chromatin alterations. Disruptions of histone- and chromatin-related factors have negative impacts on yeast and higher eukaryotes, inducing chromosomal rearrangement, promoting genome instability, and potentially leading to diseases such as cancer (28, 29). Mutations affecting chromatin regulators, such as the Snf2 family chromatin remodeling enzymes and histone chaperones, result in common phenotypic manifestations: increase in adenosine triphosphate (ATP) biosynthesis and mitochondrial biogenesis accompanied by induction of genes involved in the TCA cycle (30). Hence, we tested whether the histone dosage mutants also exhibit these phenotypes. Elevated cellular ATP levels (Fig. 5A) and higher mitochondrial DNA copy numbers (Fig. 5B) were detected in both histone gene pair deletion strains, although to a lesser extent compared with histone chaperone deficient strain msi1Δ, rtt106Δ, and asf1Δ. Moreover, as indicated by the transcriptomic analysis, TCA cycle–related genes were significantly up-regulated in htf1Δ cells (Fig. 4C), indicating potentially up-regulated respiration functions. Fig. 5 Similarities between transcriptomic responses and metabolic phenotypes of htf1Δ, htf2Δ, and other chromatin regulator mutants. (A) Relative ATP level in indicated strains. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to WT. (B) COX1 copy number analysis by qPCR in indicated strains. *P < 0.05 and **P < 0.01 compared to WT. (C) Overlap in genes significantly down-regulated in H3 depletion and htf1Δ strains. (D) Deletion strains with transcriptomes demonstrating the highest-ranking correlations with the htf1Δ transcriptome. Names are provided for strains with defects in chromatin-related factors. Genes targeted by the deletion mutations are color-coded according to their molecular function. (E) GO analysis of genes down-regulated in a histone knockout fly strain. Categories expected to be affected by TOR inhibition are underlined. (F) GO analysis of genes down-regulated in CAF-1 p150 knockdown MES cells. Categories expected to be affected by TOR inhibition are underlined. (G) Down-regulated genes overlapping between CAF-1 p150 KD and INK128-treated cells. (H) GO analysis of genes down-regulated in an H2A knockdown worm. Categories expected to be affected by TOR inhibition are underlined. tRNA, transfer RNA. (I to J) Volcano plots showing up-regulation of proteolysis-related and oxidation-reduction (OR)–related genes in htf1Δ yeast cells and CAF-1 p150 KD MES cells. Red dots represent genes with significantly (false discovery rate < 0.01) up-regulated expression level. KD, knockdown. To further investigate the similarities in the metabolic phenotypes of the histone and chromatin factor mutant strains, we compared the changes in their transcriptomes. There was a significant overlap in the genes differentially regulated in htf1Δ and in a strain depleted of H3 (Fig. 5C) (31). For a more comprehensive examination, we performed a correlation analysis using the transcriptome of the hft1Δ mutant and a recently published dataset containing transcriptomic data for 697 transcriptional responsive single gene deletion yeast strains (32). The Pearson correlation between the transcriptomes of htf1Δ and every deletion strain in the dataset was calculated and ranked (see Fig. 5D for the top correlated strains and table S3 for complete list). Among those with the highest similarity to htf1Δ (R ≥ 0.20), strains with chromatin factor deletions were significantly enriched (P < 10−6, χ2 test). The highest-ranked mutants included strains inactivated in all three subunits of the CAF-1 complex (Rlf1, Cac2, and Msi1) and in Rtt106, all of which are essential players in DNA replication–dependent nucleosome assembly. We also observed a significant overlap in the genes up-regulated in hft1Δ and these mutants (fig. S3, F and G). Together, these data suggested that defects in either histone expression or chromatin regulating factors elicit a similar cellular response, manifested by transcriptomic and metabolic changes, which we collectively refer to as the chromatin architectural defect (CAD).

CAD stress induces a conserved response in different eukaryotic models Nucleosome and chromatin structures are highly evolutionarily conserved. We thus asked whether the CAD response was conserved in higher eukaryotes as well. We compared the transcriptome of a Drosophila melanogaster strain with reduced histone gene copies (33) to age- and tissue-matched WT samples (34). Consistent with the changes observed here for htf1Δ and previously reported for TOR inactivation (35), genes associated with translation were significantly down-regulated in the fly histone mutant strain (Fig. 5E). For mammalian cell comparisons, we analyzed an RNA-seq dataset for mouse embryonic stem (MES) cells with a knockdown of p150, which represents the largest catalytic subunit in the CAF-1 histone chaperone complex (36). In a manner similar to the histone gene inactivation in htf1Δ, defects in CAF-1 in the MES cells resulted in the down-regulation of genes associated with ribosome biogenesis, protein translation, and amino acid biosynthesis (Fig. 5F). Using a published RNA-seq dataset for MES cells treated with the mammalian TOR inhibitor INK128 (37), we also detected an overlap in the 1000 most down-regulated genes resulting from TOR pathway inhibition and CAF-1 complex disruption (Fig. 5G). During the preparation of this manuscript, knockdown of H2A expression was found to extend the life span of C. elegans (25). Transcriptomic analysis of the H2A knockdown worm also revealed significant down-regulation of translation-related processes (Fig. 5H). Thus, inhibition of the TOR pathway in response to CAD stress is likely a conserved phenomenon among eukaryotes. Despite greater, likely organism-specific, differences between the up-regulated gene categories in the analyzed transcriptomes, we did detect comparable up-regulation of genes related to proteolysis and oxidation-reduction in the yeast and MES transcriptomes (Fig. 5 I and J). These observations suggest possible conservation of the CAD response in other eukaryotic organisms.