NAD+-dependent deacetylase Hst1p controls biosynthesis and cellular NAD+ levels in Saccharomyces cerevisiae

Abstract

Nicotine adenine dinucleotide (NAD(+)) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD(+)-dependent protein deacetylases. In the latter two processes, NAD(+) is consumed and converted to ADP-ribose and nicotinamide. NAD(+) levels can be maintained by regeneration of NAD(+) from nicotinamide via a salvage pathway or by de novo synthesis of NAD(+) from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD(+)-dependent deacetylase Hst1p as a sensor of NAD(+) levels and regulator of NAD(+) biosynthesis. Using transcript arrays, we show that low NAD(+) states specifically induce the de novo NAD(+) biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD(+)-dependent deacetylase activity of Hst1p represses de novo NAD(+) biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD(+) biosynthesis genes. The removal of HST1-mediated repression of the NAD(+) de novo biosynthesis pathway leads to increased cellular NAD(+) levels. Transcript array analysis shows that reduction in cellular NAD(+) levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD(+)-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD(+) in comparison to other NAD(+)-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD(+) sensor that monitors and regulates cellular NAD(+) levels.

FIG. 1.

The NAD⁺ salvage pathway is constitutively active, whereas de novo NAD⁺ biosynthesis pathway is highly inducible by low NAD⁺ states. (A) NAD⁺ biosynthesis pathways in yeast. NaMN, NA mononucleotide; NaAD, desamido-NAD⁺. (B) Northern blot analysis of genes required for NA transport (TNA1), de novo NAD⁺ biosynthesis (BNA2), and the NAD⁺ salvage pathway (NPT1 and NMA1) in different strains and culture conditions. Wild-type BY4742 (WT) or isogenic npt1 and hst1 cells were grown in SC medium. -NA, wild-type cells grown in medium lacking NA. PDA1 transcripts were used as a loading control. (C) Northern blot analysis of the high-affinity NA transporter gene TNA1 in different mutants. Cells were grown in SC medium. PDA1 transcripts were used as a loading control. (D) Histone deacetylase activity of Hst1p is required for the repression of TNA1 transcript. Total RNA was isolated from wild-type (BY4742) cells or the isogenic sir2 mutant strains treated with the specific inhibitor of NAD⁺-dependent deacetylases splitomicin (75 μM) for 6 h without or with 40-min pretreatment with cycloheximide (50 μg/ml) in SC medium. PDA1 transcripts were used as a loading control.

FIG. 2.

NPT1 deletion preferentially affects HST1-regulated genes. The Venn diagrams compare genes upregulated 1.6-fold or more relative to wild type in sir2, hst1, hst2, and hst3 cells.

FIG. 3.

Hst1p has lower affinity toward NAD⁺ than Sir2p or Hst1p. Shown are HDA activities of recombinant Hst1p (triangles), Hst2p (circles), and Sir2p (squares). Bacterially expressed and purified GST-Hst1p, GST-Hst2p, and GST-Sir2p proteins were incubated with chemically [³H]acetylated histone H4 peptide (40,000 cpm/reaction mixture) and different concentrations of NAD⁺. NAD⁺-dependent deacetylase activity, normalized for the V_max of each protein, is plotted. The V_max for Sir2p was 375 ± 12 cpm/h, V_max for Hst2p was 331 ± 12 cpm/h, and V_max for Hst1p was 170 ± 4 cpm/h. The NAD⁺ K_m for Sir2p was 29.7 ± 2.7 μM, the NAD⁺ K_m for Hst2p was 15.0 ± 1.9 μM, and the NAD⁺ K_m for Hst1p was 94.2 ± 5.4 μM.

FIG. 4.

(A) Sum1p associated with TNA1 and BNA2 promoters regardless of the NAD⁺ status of the cell. Cell lysates were prepared from formaldehyde cross-linked wild-type (WT) or npt1- or hst1-containing myc epitope-tagged SUM1 or sum1 cells which served as a negative control. DNA immunoprecipitated with anti-myc antibody was analyzed by simultaneous PCR amplification of TNA1 or BNA2 promoter and the SSC1 control gene. One-thirtieth and 1/90 of the immunoprecipitated DNA (IP), 1/50,000 of the input DNA (in), or 1/10 of mock-immunoprecipitated (no antibody used) DNA (m) was used for PCR amplification. (B) Sum1p and Hst1p interact regardless of the NAD⁺ status of the cell. Cell lysates prepared from wild-type, npt1, and sum1 hst1 cells containing plasmids with myc epitope-tagged SUM1 and/or HA epitope-tagged Hst1p were immunoprecipitated using anti-myc antibody. Whole-cell lysates and anti-myc immunoprecipitates were analyzed for the presence of Hst1-HA and Sum1-Myc by immunoblotting. (C) HST1- and SUM1-mediated repression of the TNA1 and BNA2 genes is not associated with global histone H3 and H4 deacetylation. Cell lysates were prepared from formaldehyde cross-linked WT, npt1, hst1, and sum1 cells. DNA immunoprecipitated with anti-acetyl-H3 or anti-acetyl-H4 antibody was analyzed by simultaneous amplification of TNA1, BNA2, or the silent HMR loci and the control SSC1 promoter. One-thirtieth and 1/90 of the immunoprecipitated DNA (IP), 1/50,000 of the input DNA (in), or 1/10 of mock immunoprecipitated (no antibody used) DNA (m) was used for PCR amplification.

FIG. 5.

HST1 deletion increases total cellular NAD⁺ levels through the upregulation of NAD⁺ biosynthesis genes. (A) Relative steady-state NAD⁺ levels in wild-type (WT) and npt1 hst1 mutant cells grown in SC medium. (B) The lack of NAD⁺ precursors in the medium augments the difference in NAD⁺ levels between wild-type cells (white bars) and hst1 mutant cells (gray bars). Relative steady-state NAD⁺ levels in wild-type (wt) and hst1 cells grown in medium with or without added NAD⁺ precursors NA and/or tryptophan are shown. (C) Among mutants in NAD⁺-dependent deacetyalse genes, only hst1 has significantly elevated NAD⁺ levels. Relative steady-state NAD⁺ levels are shown for wild-type and NAD⁺-dependent deacetylase mutants in medium lacking tryptophan. (D) Increased cellular NAD⁺ levels in hst1 mutants depend on an intact NAD⁺ de novo pathway. Relative steady-state NAD⁺ levels in wild-type (WT) and hst1, bna2, and bna2 hst1 mutants in medium lacking tryptophan are shown.