HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin

The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD⁺-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Δ hst4Δ mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Δ hst4Δ mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.     

Substitution as a mechanism for genetic robustness: the duplicated deacetylases Hst1p and Sir2p in Saccharomyces cerevisiae

How duplicate genes provide genetic robustness remains an unresolved question. We have examined the duplicated histone deacetylases Sir2p and Hst1p in Saccharomyces cerevisiae and find that these paralogs with non-overlapping functions can provide genetic robustness against null mutations through a substitution mechanism. Hst1p is an NAD⁺-dependent histone deacetylase that acts with Sum1p to repress a subset of midsporulation genes. However, hst1Δ mutants show much weaker derepression of target loci than sum1Δ mutants. We show that this modest derepression of target loci in hst1Δ strains occurs in part because Sir2p substitutes for Hst1p. Sir2p contributes to repression of the midsporulation genes only in the absence of Hst1p and is recruited to target promoters by a physical interaction with the Sum1 complex. Furthermore, when Sir2p associates with the Sum1 complex, the complex continues to repress in a promoter-specific manner and does not spread. Our results imply that after the duplication, SIR2 and HST1 subfunctionalized. The single SIR2/HST1 gene from Kluyveromyces lactis, a closely related species that diverged prior to the duplication, can suppress an hst1Δ mutation in S. cerevisiae as well as interact with Sir4p in S. cerevisiae. In addition, the existence of two distinct protein interaction domains for the Sir and Sum1 complexes was revealed through the analysis of a chimeric Sir2–Hst1 molecule. Therefore, the ability of Sir2p to substitute for Hst1p probably results from a retained but reduced affinity for the Sum1 complex that is a consequence of subfunctionalization via the duplication, degeneration, and complementation mechanism. These results suggest that the evolutionary path of duplicate gene preservation may be an important indicator for the ability of duplicated genes to contribute to genetic robustness.     

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.     

The role of MutY homolog (Myh1) in controlling the histone deacetylase Hst4 in the fission yeast Schizosaccharomyces pombe

The DNA glycosylase MutY homolog (Myh1) excises adenines misincorporated opposite guanines or 7,8-dihydro-8-oxo-guanines on DNA by base excision repair thereby preventing G:C to T:A mutations. Schizosaccharomyces pombe (Sp) Hst4 is an NAD⁺-dependent histone/protein deacetylase involved in gene silencing and maintaining genomic integrity. Hst4 regulates deacetylation of histone 3 Lys56 at the entry and exit points of the nucleosome core particle. Here, we demonstrate that the hst4 mutant is more sensitive to H₂O₂ than wild-type cells. H₂O₂ treatment results in an SpMyh1-dependent decrease in SpHst4 protein level and hyperacetylation of histone 3 Lys56. Furthermore, SpHst4 interacts with SpMyh1 and the cell cycle checkpoint Rad9-Rad1-Hus1 (9-1-1) complex. SpHst4, SpMyh1, and SpHus1 are physically bound to telomeres. Following oxidative stress, there is an increase in the telomeric association of SpMyh1. Conversely, the telomeric association of spHst4 is decreased. Deletion of SpMyh1 strongly abrogated telomeric association of SpHst4 and SpHus1. However, telomeric association of SpMyh1 is enhanced in hst4Δ cells in the presence of chronic DNA damage. These results suggest that SpMyh1 repair regulates the functions of SpHst4 and the 9-1-1 complex in maintaining genomic stability.     

Deacetylase Rpd3 Facilitates Checkpoint Adaptation by Preventing Rad53 Overactivation

The DNA damage checkpoint is tightly controlled. After its activation, the checkpoint machinery is inactivated once lesions are repaired or undergoes adaptation if the DNA damage is unable to be repaired. Protein acetylation has been shown to play an important role in DNA damage checkpoint activation. However, the role of acetylation in checkpoint inactivation is unclear. Here we show that histone deacetylase Rpd3-mediated deacetylation of Rad53 plays an important role in checkpoint adaptation. Deletion of Rpd3 or inhibition of its activity impairs adaptation. RPD3 deletion also leads to a higher acetylation level and enhanced kinase activity of Rad53. Replacement of two major acetylation sites of Rad53 with arginine reduces its activity and further suppresses the adaptation defect of rpd3Δ cells, indicating that Rpd3 facilitates adaptation by preventing Rad53 overactivation. Similar to its role in adaptation, deletion of RPD3 or inhibition of its activity also suppressed checkpoint recovery. Altogether, our findings reveal an important role of Rpd3 in promoting checkpoint adaptation via deacetylation and inhibition of Rad53.     

A 3-aminobenzamide-resistant labeled protein in [P]NAD-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [³²P]NAD⁺. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [³²P]NAD⁺ was shown to be Mg²⁺-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [α-³²P]ATP, [γ-³²P]ATP and [³²P]orthophosphate, but not from [³H]NAD⁺ or [¹⁴C]NAD⁺. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [³²P]NAD⁺. This phosphorylation appears to occur via an Mg²⁺-dependent conversion of NAD⁺ to AMP with the eventual utilization of the α-phosphate for phosphorylation of the 72 000 molecular weight protein.     

Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis

Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.     

The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target

NAD⁺ metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD⁺ metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD⁺ biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD⁺ synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD⁺ levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD⁺ synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.     

Conceptual Error in Determination of NAD-Malic Enzyme in Extracts Containing NAD-Malic Dehydrogenase

The typical procedure for determining NAD⁺-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be ΔA₃₄₀/Δ time after the endogenous NAD⁺-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD⁺-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD⁺], pH and final [NADH]. For a ΔA₃₄₀ of 0.02, the error is about 80% and for a ΔA₃₄₀ of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

Two Histone Deacetylases,FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn²⁺-dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as Δffhda1 Δffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary-metabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the Δffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.     

Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae

NAD⁺ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD⁺ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD⁺ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD⁺-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD⁺ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD⁺ precursors. Together, our studies provide a molecular basis for how NAD⁺ homeostasis factors confer metabolic flexibility.The pyridine nucleotide NAD⁺ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD⁺ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (1, 2). NAD⁺ also plays an important role in calorie restriction (CR)²-mediated life span extension via regulating NAD⁺-dependent longevity factors (3, 4). CR is the most effective regimen known to extend life span in various species (5, 6). CR also ameliorates many age-related diseases such as cancer and diabetes (5). The Sir2 family proteins are NAD⁺-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (3, 7) and higher eukaryotes (8, 9). By coupling the cleavage of NAD⁺ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (8–10), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging.In eukaryotes, NAD⁺ is generated by de novo synthesis and by salvaging various intermediary precursors (see Fig. 1A). In yeast, the de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (11). Because the de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD⁺ precursors for the nicotinamide (Nam) moiety (11). Yeast cells also salvage Nam from NAD⁺ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD⁺ via Nma1/2 and Qns1 (see Fig. 1A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (12, 13), and Qns1 is a glutamine-dependent NAD⁺ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD⁺ auxotrophic mutants (14–16). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (14, 16). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (15, 16).Open in a separate windowFIGURE 1.Nicotinamide riboside (NmR) is an endogenous metabolite in yeast. A, the current model of the NAD⁺ biosynthesis pathways. Extracellular NmR enters the salvage cycle through Nrk1, Urh1, Pnp1, and Meu1. B, NAD⁺ prototrophic cells release metabolites into growth medium to cross-feed NAD⁺ auxotrophic cells (the npt1Δqpt1Δ and qns1Δ mutants). Micro-colonies of the NAD⁺ auxotrophic mutants become visible after 2-day incubation at 30 °C, which show “gradient” growth patterns descending from the side adjacent to WT. C, Nrk1 is required for NAD⁺ auxotrophic cells to utilize NmR. Anaerobic growth conditions (−O₂) are utilized to block de novo NAD⁺ biosynthesis in the npt1Δ and npt1Δnrk1Δ mutants. D, Nrk1 is required to utilize cross-feeding metabolites. E, cross-feeding activity is modulated by factors in NmR metabolism. Cells defective in NmR utilization (left panel) or transport (middle panel) show increased cross-feeding in spot assays. Overexpressing Nrk1 decreases cross-feeding activity (right panel). The results show growth of the npt1Δqpt1Δ recipient (plated on YPD at a density of ∼9000 cells/cm²) supported by feeder cells (∼2 × 10⁴ cells spotted directly onto the recipient lawn). oe, overexpression.NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (17). For example, NmR protected neurons from axonal degeneration via functioning as a NAD⁺ precursor (18, 19). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (14, 15, 20–22), NmR has been speculated to be an endogenous NAD⁺ precursor (17, 23). Here, we provided direct evidence for endogenous NmR as an integral part of NAD⁺ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD⁺ levels in mammalian cells (24). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

HDAC1 Controls CD8+ T Cell Homeostasis and Antiviral Response

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8⁺ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8⁺CD4^– cells within the CD3/TCRβ^lo population, indicating that HDAC1 is essential for the efficient progression of immature CD8⁺CD4^– cells to the DP stage. Moreover, CD44^hi effector CD8⁺ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44^hi CD8⁺ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44^l°CD62L⁺) HDAC1-null CD8⁺ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8⁺ T cell response upon LCMV infection and impaired expansion of virus-specific CD8⁺ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8⁺ T cell homeostasis and for an efficient in vivo expansion and activation of CD8⁺ T cells in response to LCMV infection.