Yeast Tdh3 (glyceraldehyde 3-phosphate dehydrogenase) is a Sir2-interacting factor that regulates transcriptional silencing and rDNA recombination

Sir2 is an NAD(+)-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD(+)-binding protein, influences nuclear NAD(+) levels; we speculate that Tdh3 links nuclear Sir2 with NAD(+) from the cytoplasm

The rapid development of a novel kidney-specific digital intervention for self-management of physical activity and emotional well-being during the COVID-19 pandemic and beyond: Kidney Beam

King's College Hospital; Kidney Research UK; Beam; National Institute for Health Research (NIHR) under (NIHR301893), (NIHR301593), and (ICA-CL-2017-03-020)

Physical and functional interaction between Tdh3 and Sir2 in a two-hybrid assay.

<p>(A) <i>TDH3</i> fused to the DNA-binding domain results in the repression of the <i>HIS3</i> reporter gene. Two-hybrid assays were performed as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-James1" target="_blank">[69]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Uetz1" target="_blank">[70]</a> using the complete Sir2 and Tdh3 open reading frames. Rows are labeled with the activation-domain fusions used; pOAD is the vector control. Each column lists the binding-domain fusion used; pOBD is the vector control. Tdh3ΔBD indicates strains that overexpress <i>TDH3</i> from the pOBD vector lacking the Gal4 binding domain. (B) Elevated Tdh3 increases Sir2-dependent repression of a reporter gene. Labels are as described in (A). Sir4Δ730N-AD was included as a positive control for Sir2 interaction. (C) Tdh3 and Sir2 interact in vivo. The activation domain and binding domain fusions from (A) and (B) were assessed in a strain lacking the <i>SIR2, SIR3, and SIR4</i> genes (YSH625).</p

Tdh3 affects nuclear NAD⁺ levels in yeast.

<p>(A) <i>TDH3</i> deletion or overexpression does not affect overall cellular NAD⁺ levels. Left panel: relative NAD⁺ levels are shown for strains lacking the <i>TDH3</i> or <i>NPT1</i> genes, and their matched wild-type strains. Right panel: relative NAD⁺ levels are shown in a strain overexpressing the <i>TDH3</i> gene and in a vector control strain. (B) Tdh3 maintains nuclear NAD⁺ levels. Nuclear NAD⁺ was measured using an NAD<b>⁺</b>-sensitive transcriptional reporter gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Anderson1" target="_blank">[31]</a>. Strains expressed the NAD<b>⁺</b>-dependent transcriptional activator from a <i>LEU2</i>-marked plasmid. Control strains lacked the binding site for the transcriptional activator (no NAD box) or lacked the activator (no NadR-Gal4AD). Serial dilutions of the listed strains were plated on the indicated media. Levels of the NadR-Gal4AD protein were similar in wild type and <i>Δtdh3</i> cells (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871.s003" target="_blank">Figure S3</a>). (C) Tdh3 and Npt1 have a redundant role in promoting cell growth. Doubling times of the indicated single and double mutant strains is shown. (D) Silencing at the telomere in <i>Δnpt1 Δtdh3</i> strains. Expression of the native telomere gene <i>YFR057W</i>, was examined by quantitative RT-PCR in the indicated strains.</p

Strains.

<p>Strains.</p

Co-immunoprecipitation of Tdh3 and Sir2.

<p>A Tdh3-myc fusion protein was immunoprecipitated from yeast cell lysates. The panel on the left shows a western blot probed with anti-myc antibody. Lanes include crude lysate and immunoprecipitated material (IP). Control lysates were made from strains lacking the myc tag on Tdh3. The right panel shows a western blot of the same immunoprecipitated material, probed with an antibody to Sir2. This antibody specifically recognizes Sir2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen-1003871-g001" target="_blank">Figure 1D</a>).</p

Separation of silencing and GAPDH activity in <i>TDH3</i> alleles.

<p>(A) <i>TDH3</i> mutants influence transcriptional silencing at yeast telomeres. For each allele the wild-type amino acid and position is noted, followed by the amino acid replacing it in the mutated allele. A phenotypic assay measuring silencing of a <i>URA3</i> reporter gene was conducted as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen-1003871-g001" target="_blank">Figure 1</a> legend. (B) mRNA levels of <i>YFR057W</i>, a naturally occurring telomere proximal gene, were determined as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen-1003871-g001" target="_blank">Figure 1</a> legend. (C) GAPDH levels of strains bearing <i>TDH3</i> alleles. Levels of glyceraldehyde phosphate dehydrogenase activity were measured in extracts made from the indicated strains, as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Ralser3" target="_blank">[64]</a>.</p

Nuclear localization of Tdh3 influences transcriptional silencing at the telomere.

<p>(A) Tdh3 is localized to both the cytoplasm and nucleus. Cells expressing Tdh3-GFP from the native <i>TDH3</i> locus were visualized by fluorescence microscopy. Size bar: 5 µm. (B) Addition of a nuclear export sequence to Tdh3 reduces silencing at the telomere. The indicated alleles of <i>TDH3</i> were introduced at its endogenous loci in a strain bearing the <i>URA3</i> gene adjacent to a telomere. NES denotes a functional nuclear export sequence; nes denotes a non-functional sequence that differs by two amino acid substitutions <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871-Fritze1" target="_blank">[27]</a>. Expression of <i>URA3</i> was assessed by plating serial dilutions of these strains on the indicated media. (C) Localization of Tdh3-NES-GFP and Tdh3-nes-GFP was examined by cellular fractionation and immunoblotting. Fractions of the indicated strains were probed using an antibody to GFP. Detection of histone H3 was used to monitor the success of fractionation. Fractions included whole cell (WC), nuclear (N), and cytoplasmic (C). Localization of Tdh3-GFP was also examined by fluorescent microscopy (Supplemental <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871.s001" target="_blank">Figure S1A</a>). Addition of the GFP tag to Tdh3 in NES/nes strains did not alter their silencing phenotypes (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003871#pgen.1003871.s001" target="_blank">Figure S1B</a>).</p