Reversible modulation of SIRT1 activity in a mouse strain

<div><p>The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4–5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues <i>in vivo</i>. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2–4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.</p></div

The SIRT1-ER protein is located in the nucleus in the presence and absence of 4OHT.

<p>MEFs derived from wild type (<i>sirt1</i>^<i>+/+</i>) or homozygous mutant (<i>sirt1</i>^<i>ER/ER</i>) embryos were cultured, treated for 24 hr with 0 or 100 nM 4OHT and fixed for immunofluorescence. Antibodies to SIRT1 react with protein in both wild type and mutant cells while antibodies to the murine estrogen receptor α (ERα) reacted with the nuclei from the <i>sirt1</i>^<i>ER/ER</i> cells. Cell nuclei were counterstained with DAPI.</p

Creation of the <i>sirt1</i>^<i>ER</i> allele by homologous recombination.

<p>A. The <i>sirt1</i> gene is depicted with its 9 exons indicated as boxes above the line. A replacement vector was constructed from a cloned genomic fragment (pKJ209). The region coding for amino acids 281–599 of the mutated murine ERα [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173002#pone.0173002.ref029" target="_blank">29</a>] was fused in frame downstream of the SIRT1 coding region and all but 130 bp of the 3’UTR of the <i>sirt1</i> gene was removed. A selectable <i>pgk-puro</i> gene was inserted downstream between 2 <i>loxP</i> sites. This insertion vector was electroporated into R1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173002#pone.0173002.ref028" target="_blank">28</a>] ES cells and clones carrying the correctly targeted insertion were identified by Southern blots of BamH1 digested cellular DNA probed with the region identified. B. Northern blot of RNA isolated from the parental embryonic stem cells (<i>sirt1</i>^<i>+/+</i>) and a subclone carrying the correctly inserted DNA fragment (<i>sirt1</i>^<i>+/ER</i>). The blot was probed with the <i>sirt1</i> cDNA and the <i>puro</i> gene. Bands corresponding to the native SIRT1 mRNA and the mRNA encoding SIRT1-ER are shown in the upper panel. Cells were treated for 24 hr with 30 nM 4-hydroxytamoxifen (4OHT) with little effect on the abundance of the mRNAs. C. Western blot of protein isolated from <i>sirt1</i>^<i>+/+</i> and <i>sirt1</i>^<i>+/ER</i> ES cells after treatment with 4OHT for 24 hr. The locations of the SIRT1 and the SIRT1-ER proteins are shown. The abundance of the SIRT1-ER fusion protein was increased about 3–4 fold following 4OHT treatment.</p

Evidence for SIRT1-ER deacetylase activity in cultured cells.

<p>A. <i>sirt1</i>^<i>+/ER</i> embryonic stem cells were transfected with the knockout (KO) insertion vector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173002#pone.0173002.ref017" target="_blank">17</a>] and clones screened for those with the KO inserted into the wild type allele. A clone of these sirt1^ER/- cells was isolated and treated for 24 hr with 4OHT (30 nM) and then irradiated with ultraviolet radiation (30J/m²), incubated for 18 hr and harvested for immunoblotting. Blots were probed with antibodies reactive with acetylated-p53 (Ac-K379) and total p53 protein as indicated. ES cells that are <i>sirt1</i>^<i>-/-</i> and <i>sirt1</i>^<i>+/+</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173002#pone.0173002.ref060" target="_blank">60</a>] were irradiated in parallel to show the effect on acetylated p53 of the absence and presence of SIRT1 activity respectively. The intensity of the acetyl-p53 signal is plotted on the bar graph. B. <i>sirt1</i>^<i>ER/ER</i> MEFs were treated or untreated for 24 hr with 4OHT before 1 μM doxorubicin was added for an additional 24 hr. Immunoblots were probed for SIRT1, acetyl-p53 and total p53 as indicated. The bar graphs show the intensity of the acetyl-p53 signal (upper graph) and of SIRT1-ER (lower graph).</p

4 month test mating to sirt1^+/+.

<p>4 month test mating to sirt1^+/+.</p

Characteristics of mice bearing induced mutations of the <i>sirt1</i> gene.

<p>Characteristics of mice bearing induced mutations of the <i>sirt1</i> gene.</p

<i>sirt1</i>^<i>ER/ER</i> mice have craniofacial abnormalities similar to those present in <i>sirt1</i>^<i>-/-</i> animals.

<p>A. Photographs of <i>sirt1</i>^<i>+/+</i> and <i>sirt1</i>^<i>ER/ER</i> littermates (on the left) and <i>sirt1</i>^<i>+/+</i> and <i>sirt1</i>^<i>-/-</i> littermates (on the right). The <i>sirt1</i>^<i>-/-</i> and <i>sirt1</i>^<i>ER/ER</i> animals have a shortened snout compared to their <i>sirt1</i>^<i>+/+</i> littermates. <i>sirt1</i>^<i>-/-</i> animals are also smaller than their normal littermates. B. MRI images of the heads of animals of various genotypes show that the interorbital distance is reduced in <i>sirt1</i>^<i>-/-</i> and <i>sirt1</i>^<i>ER/ER</i> mice. The <i>sirt1</i>^<i>+/+</i> image is colored blue while the images from the other genotypes were rendered red before superimposition. Panel A is from <i>sirt1</i>^<i>Y/Y</i>, B from <i>sirt1</i>^<i>+/-</i>, C from <i>sirt1</i>^<i>-/-</i>, and D from <i>sirt1</i>^<i>ER/ER</i>. Quantitation of interorbital distance shown in the bottom bar graph was measured by taking the ratio of the green over the blue lines. Measurements were made from 3 MRI images of each genotype from male animals of 3–4 months of age. * indicates P<0.05.</p