Thiamine biosynthesis in Saccharomyces cerevisiae is regulated by the NAD+-dependent histone deacetylase Hst1

Abstract

Genes encoding thiamine biosynthesis enzymes in microorganisms are tightly regulated such that low environmental thiamine concentrations activate transcription and high concentrations are repressive. We have determined that multiple thiamine (THI) genes in Saccharomyces cerevisiae are also regulated by the intracellular NAD(+) concentration via the NAD(+)-dependent histone deacetylase (HDAC) Hst1 and, to a lesser extent, Sir2. Both of these HDACs associate with a distal region of the affected THI gene promoters that does not overlap with a previously defined enhancer region bound by the thiamine-responsive Thi2/Thi3/Pdc2 transcriptional activators. The specificity of histone H3 and/or H4 deacetylation carried out by Hst1 and Sir2 at the distal promoter region depends on the THI gene being tested. Hst1/Sir2-mediated repression of the THI genes occurs at the level of basal expression, thus representing the first set of transcription factors shown to actively repress this gene class. Importantly, lowering the NAD(+) concentration and inhibiting the Hst1/Sum1 HDAC complex elevated the intracellular thiamine concentration due to increased thiamine biosynthesis and transport, implicating NAD(+) in the control of thiamine homeostasis.

FIG. 1.

Effects of elevated nicotinamide and reduced NAD⁺ concentrations on global gene expression. (A) Schematic diagram of NAD⁺ biosynthesis in yeast. NAD⁺ can be synthesized by a de novo pathway starting with tryptophan (Trp) (1) or through the Preiss-Handler NAD⁺ salvage pathway, where nicotinamide (NAM) produced by Sir2 or other sirtuins is deamidated by the nicotinamidase Pnc1 to form nicotinic acid (NA) (2). The nicotinic acid phosphoribosyl transferase Npt1 converts NA into nicotinic acid mononucleotide (NaMN). Alternatively, NA is imported from the growth medium by the nicotinic acid permease (Tna1). Nicotinamide riboside (NR) can be utilized as an NAD⁺ precursor either by phosphorylation by the nicotinamide riboside kinase (Nrk1) to form nicotinamide mononucleotide (NMN) (3) or by degradation into NAM via Pnp1 (a phosphorylase) and Urh1 (a hydrolase) (4). Additional abbreviations: NaAD, deamido-NAD⁺; Qns1, NAD synthetase; Nma1 and Nma2, nicotinate mononucleotide adenylyltransferases. The import mechanism for NAM is uncharacterized. (B) Venn diagrams indicating the overlaps in gene expression changes (both overall and upregulated) between the npt1Δ and pnc1Δ mutants and the WT strain treated with 5 mM NAM. The total number of changes for each strain is also indicated. (C) Venn diagrams showing overlaps in upregulated genes induced by the WT + NAM (left) condition or the npt1Δ mutation (right) compared to previously determined upregulated genes from mutants with small deletions in the N-terminal H3 or H4 tails (45, 46).

FIG. 2.

Thiamine and pyridoxal 5′-phosphate (PLP) biosynthesis genes are upregulated by conditions that inhibit sirtuins. (A) Validation by quantitative RT-PCR analysis of mRNA expression from several thiamine and pyridoxal biosynthesis genes identified from the microarray analysis relative to ACT1 expression. The test gene/ACT1 ratio was normalized to 1.0 for each gene in the WT strain. Values are the averages of the results for the two independent RNA samples used for the microarray analysis. (B) Schematic diagram of thiamine biosynthesis and transport in Saccharomyces cerevisiae. Abbreviations: HMP, 4-amino-5-hydroxymethyl-2-methylpyrimidine; HMP-P, 4-amino-5-hydroxymethyl-2-methylpyrimidine monophosphate; HMP-PP, y-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate; HET, 5-(2-hydroxyethyl)-4-methylthiazole; HET-P, 5-(2-hydroxyethyl)-4-methylthiazole phosphate; PLP, pyridoxal 5′-phosphate; RP, d-ribulose 5-phosphate; TP, thiamine phosphate; TPP, thiamine pyrophosphate; XP, d-xylulose 5-phosphate.

FIG. 3.

Effects of growth media and NAD⁺ concentrations on THI gene expression. (A) Quantitative RT-PCR analysis of THI13, THI4, THI2, and THI73 mRNA levels in SC medium compared to their levels in YPD medium. Expression levels in YPD were normalized to 1.0. (B) THI4 and THI13 expression in the WT strain without or with the addition of 5 mM NAM and in the npt1Δ mutant. Cells were grown in SC medium. (C) THI4 and THI13 expression in the WT strain without or with the addition of 5 mM NAM and in the npt1Δ mutant. Cells were grown in YPD medium. (D) Effects of 10 μM nicotinamide riboside (NR) on the relative intracellular NAD⁺ levels in the WT and npt1Δ strains growing in YPD medium. Results for the WT without NR addition were set to 1. (E) Effects of 10 μM NR on THI4 expression in the WT and npt1Δ mutant strains growing in YPD medium. (F) THI4 expression levels in SC medium containing the indicated concentrations of thiamine. (G) Increased THI4 expression caused by a lack of nicotinic acid (NA) in SC medium containing 100 nM thiamine (the linear range for THI4 expression). In each panel, changes with a P value of <0.05 are indicated by one asterisk, and P values of <0.005 are indicated by two asterisks. Error bars show standard deviations.

FIG. 4.

Repression of THI genes by the SIR and Hst1/Sum1 histone deacetylase complexes. (A to D) Results of quantitative RT-PCR analysis of THI4, THI13, THI71, and THI73 mRNA levels. Expression is relative to the ACT1 RNA level. Results for the WT were normalized to 1.0 for each gene. (E and F) Results of quantitative RT-PCR measurement of THI4 and THI13 mRNA levels in WT and sir2Δ strains with the HML locus either intact or deleted. P values of <0.05 are indicated by an asterisk. P values of <0.005 are indicated by a double asterisk. Error bars show standard deviations.

FIG. 5.

Chromatin IP analysis of Sir2, Hst1, Sum1, and Sir3 association with the THI4 and THI71 genes. (A) Schematic diagram indicating the positions of PCR primers used to detect immunoprecipitated DNA. Base pair positions relative to the transcriptional start site are provided. (B) Association of Myc-tagged Sir2, Hst1, and Sum1 with a region upstream of the traditional THI4 promoter region (position A). The ATS1 promoter is used as a negative control that does not associate with Sir2, Hst1, or Sum1. “Relative IP,” on the y axis, indicates the ratio between the immunoprecipitated DNA's PCR signal and the input DNA's PCR signal. (C) Chromatin IP results showing association of Myc-tagged Sir3 with the same upstream THI4 region (position A). (D) Chromatin IP results showing Myc-tagged Sir2, Hst1, and Sum1 association with the THI71 gene. (E) Chromatin IP results showing that Hst1 association ∼750 bp upstream of the THI71 transcription start site (position A) requires SUM1. (F) Chromatin IP results showing that Myc-tagged Sir2, Hst1, and Sum1 binding to THI4 does not extend close to the transcription start site (position E) or ∼1,150 bp upstream (position D). (G) Binding does extend further upstream from the THI71 start site (position D). In each panel, changes with a P value of <0.05 are indicated by one asterisk, and P values of <0.005 are indicated by two asterisks. Error bars show standard deviations.

FIG. 6.

Effects of SIR2 and HST1 on histone H3 and H4 acetylation at the THI4 and THI71 promoters. (A) Chromatin IP analysis of H3 and H4 acetylation at the THI4 promoter in WT, sir2Δ, hst1Δ, and sir2Δ hst1Δ strains. (B) Chromatin IP analysis of H3 and H4 acetylation at the THI71 promoter. In each panel, changes with a P value of <0.05 relative to results for the WT are indicated by one asterisk, and P values of <0.005 are indicated by two asterisks. Error bars show standard deviations. α, anti.

FIG. 7.

NAD⁺-mediated regulation of thiamine biosynthesis. (A) Intracellular thiamine levels in WT, npt1Δ, hst1Δ, and sum1Δ strains as measured by HPLC. (B) Model of THI gene regulation by the Hst1/Rfm1/Sum1 complex and the SIR complex (Sir2, Sir3, and Sir4). High intracellular NAD⁺ concentrations in cells grown in nutrient-rich YPD medium promote local histone deacetylation by one or both complexes when they are bound to a site upstream of the consensus Thi2 recognition site in the promoter region. High thiamine in the YPD also prevents binding of the Thi2/Thi3 transcriptional activator complex, which, along with Pdc2, activates the THI genes when thiamine concentrations are low. The result is tight repression of basal THI gene expression. The histone deacetylation also generates nicotinamide (NAM) as a by-product of the sirtuin-mediated reaction. In minimal medium, low thiamine levels facilitate binding of Thi2/Thi3/Pdc2 to the THI gene promoter to activate transcription. In parallel, decreased intracellular NAD⁺ levels reduce Hst1 deacetylation activity by the bound Hst1/Rfm1/Sum1 or SIR complex, also promoting increased THI gene expression. Error bars show standard deviations.