Lysine Deacetylation Research Articles

PTU-154 Sirt2 Modulates the Inflammatory Response in Oesophageal Adenocarcinoma

IntroductionGastro-oesophageal reflux disease is the main risk factor for Barrett’s oesophagus (BE), the precursor lesion to oesophageal adenocarcinoma. In BE, GORD leads to chronic inflammation and to NF-κB pathway activation.SIRT2...

Sirtuin deacylases: a molecular link between metabolism and immunity

Lysine deacetylation by the NAD(+)-dependent family of sirtuins has been recognized as an important post-translational modification regulating a wide range of cellular processes. These lysine deacetylases have attracted much interest based on their ability to promote survival in response to stress. Sirtuins require NAD(+) for their enzymatic activity, suggesting that these enzymes may represent molecular links between cell metabolism and several human disorders, including diabetes and cancer. Inflammation represents a pathological situation with clear connections to metabolism and aging in humans, raising the possibility that sirtuins may also play an important role during a normal and/or a pathological immune response. A growing body of data has confirmed the immunomodulatory properties of sirtuins, although often with contrasting and opposing conclusions. These observations will be summarized herein and the possible strategies that may lead to the development of novel therapeutic approaches to treat inflammation briefly discussed.

Sorting out functions of sirtuins in cancer

The sirtuins (SIRT 1-7) comprise a family of NAD⁺-dependent protein-modifying enzymes with activities in lysine deacetylation, adenosinediphospho(ADP)-ribosylation, and/or deacylation. These enzymes are involved in the cell's stress response systems and in regulating gene expression, DNA damage repair, metabolism and survival. Sirtuins have complex roles in both promoting and/or suppressing tumorigenesis. This review presents recent research progress concerning sirtuins and cancer. On one hand, functional loss of sirtuin genes, particularly SIRT1, involved in maintaining genome integrity and DNA repair will promote tumorigenesis because of genomic instability upon their loss. On the other hand, cancer cells tend to require sirtuins for these same processes to allow them to survive, proliferate, repair the otherwise catastrophic genomic events and evolve. The bifurcated roles of SIRT1, and perhaps several other sirtuins, in cancer may be in part a result of the nature of the genes that are involved in the cell's genome maintenance systems. The in-depth understanding of sirtuin functions may have significant implication in designing precise modulation of selective sirtuin members to aid cancer prevention or treatment under defined conditions.

Acute doxorubicin treatment induces proteome lysine deacetylation, with no change in sirtuin expression, in the heart and liver of fasted animals

Doxorubicin (DOX) is an effective chemotherapeutic agent, but with known toxicity which limits its lifetime dosage. In various experimental models, cellular stress has been shown to alter proteome lysine acetylation status, causing deacetylation which was associated with a pro‐apoptotic environment. The effects of DOX on proteome lysine acetylation status and expression of related sirtuins is unknown, thus we aimed to determine these effects in heart and liver of treated animals. Male F344 rats were injected IP with 20 mg/kg of DOX or saline. Once treated, all animals were fasted with no food or water until sacrifice 24 hours later. DOX treatment caused significant deacetylation in heart and liver. DOX did not affect the expression of sirtuin 1 or 3 in either tissue. Western analysis of sirtuin 1 revealed a prominent band of ~40kDa in liver, but not heart. Further, DOX treatment increased the content of this species. It has been reported in chondrocytes that sirtuin 1 can be cleaved into a ~75kDa protein which can bind to cytochrome c and inhibit apoptosis. The ~40 kDa fragment may be the remaining cleaved fragment detected by the antibody used. In conclusion, DOX induces proteome lysine deacetylation, which may be due to other deacetylases. Further, liver, but not heart, may be another tissue type that produces a 75 kDa product of sirtuin 1 as a mechanism to protect against apoptosis and is responsive to DOX treatment.

Sirtuin 1 Is a Key Regulator of the Interleukin-12 p70/Interleukin-23 Balance in Human Dendritic Cells

Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD(+), and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response.

Rif2 Promotes a Telomere Fold-Back Structure through Rpd3L Recruitment in Budding Yeast

Using a genome-wide screening approach, we have established the genetic requirements for proper telomere structure in Saccharomyces cerevisiae. We uncovered 112 genes, many of which have not previously been implicated in telomere function, that are required to form a fold-back structure at chromosome ends. Among other biological processes, lysine deacetylation, through the Rpd3L, Rpd3S, and Hda1 complexes, emerged as being a critical regulator of telomere structure. The telomeric-bound protein, Rif2, was also found to promote a telomere fold-back through the recruitment of Rpd3L to telomeres. In the absence of Rpd3 function, telomeres have an increased susceptibility to nucleolytic degradation, telomere loss, and the initiation of premature senescence, suggesting that an Rpd3-mediated structure may have protective functions. Together these data reveal that multiple genetic pathways may directly or indirectly impinge on telomere structure, thus broadening the potential targets available to manipulate telomere function.

Abstract 141: Histone Deacetylase 1 Regulates Nitric Oxide Production and Nitric Oxide Synthase-3 Acetylation in Endothelial Cells

Previous reports showed that NOS3 is regulated by acetylation through transcriptional mechanisms via histone acetylation or through direct lysine acetylation. Histone deacetylase (HDAC) enzymes and histone acetyltransferases (HATs) modulate acetylation processes. Recent work by our lab, demonstrated increased expression of aortic HDAC1 and HDAC6 while HATs were unchanged in a mouse model of early life stress with endothelial dysfunction. These data suggest a negative correlation between endothelial dysfunction and HDAC expression. The purpose of this study was to test the hypothesis that HDAC1 and 6 regulate endothelial NO production and/or NOS3 acetylation. Initial immunoprecipitation studies with anti-acetyl lysine and anti-NOS3 antibodies demonstrated that NOS3 is basally acetylated in primary bovine aortic endothelial cells (BAECs). Treatment with the HDAC inhibitor, trichostatin A (500 nM) for 1 hr, significantly increased NOS3 acetylation. BAECs were transfected with HDAC1, HDAC6, vector expression plasmids, or untransfected, with nitrite production determined by HPLC and NOS3 acetylation and expression probed by immunoprecipitation and Western blotting. Untransfected and vector transfected control BAECs had similar NO production (357 ± 10 and 344 ± 30 pmol/mg pr/h, respectively, N=6) as well as NOS3 acetylation (7.8 ± 1.6 and 6.8 ±0.3 AU, N=3). HDAC6 transfected BAECs had similar NO production to the control BAECs (272 ± 93 pmol/mg pr/h, N=3) with an increase in NOS3 acetylation (17.4 ± 1.7 AU, N=3). In contrast, HDAC1 overexpression significantly decreased NO production (89 ± 50 pmol/mg pr/h, P< 0.05, N=3) and reduced NOS3 acetylation (3.8 ± 0.5 A.U, N=3), P <0.05). Control transfections, HDAC6, and HDAC1 transfected BAECS all had similar NOS3 expression (10.14 ± 1.8; 9.8 ±1.6; 8.9 ± 1.5; 10.6 ± 1.0 AU, respectively, N=3). Thus, we conclude that HDAC1 regulates NO production via direct lysine deacetylation of NOS3.

Abstract 470: Redox Rgulation of SIRTUIN1 in Endothelium-Dependent Vascular Function

Objective: The DNA repair enzyme/reducing protein Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE/Ref-1) maintains normal endothelial function, as mice heterozygous for APE1/Ref-1 (+/-) have impaired endothelium-dependent vasorelaxation and are hypertensive. APE1/Ref-1 maintains many redox sensitive transcription factors in their reduced active form. The SIRTUIN1 (SIRT1) protein deacetylase also promotes endothelial nitric oxide (NO) production and endothelium-dependent vasorelaxation. Although SIRT1 activity is subject to redox regulation, whether it is governed by APE1/Ref-1 is not known. The purpose of this study is to determine if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function. Methods and results: In endothelial cells APE1/Ref-1 maintains cysteine (thiol) residues in SIRT1 in the reduced (free) form and is obligatory for endothelial SIRT1 activity. In vitro APE1/Ref-1 increases the free thiol content of SIRT1, targeting functionally critical cysteine residues in the catalytic domain of SIRT1 for reduction, and activates SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for activating SIRT1 and for increasing free thiol content of SIRT1. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation and inactivation. Moreover, APE1/Ref-1 stimulates lysine deacetylation of nitric oxide synthase, a target of SIRT1 in endothelial cells. When compared to wild-type mice, APE1/Ref-1(+/-) mice have lower free thiol content of SIRT1, and diminished SIRT1 activity in tissues. Overexpression of SIRT1 in aortas of APE1/Ref-1 (+/-) mice restores endothelium-dependent vasorelaxation and vascular bioavailable NO to levels similar to those observed in wild-type mice. Conclusions: APE1/Ref-1 maintains endothelial SIRT1 in the reduced form, and is required for maintaining SIRT1 activity. This relationship between SIRT1 and APE1/Ref-1 is an important mechanism responsible for APE1/Ref-1-stimulated endothelium-dependent vasorelaxation.

Azalysine analogues as probes for protein lysine deacetylation and demethylation.

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.

Lysine acetylation induced by chronic ethanol consumption impairs dynamin-mediated clathrin-coated vesicle release

The liver is the major site of ethanol metabolism and thus sustains the most injury from chronic alcohol consumption. Ethanol metabolism by the hepatocyte leads to the generation of reactive metabolites and oxygen radicals that can readily adduct DNA, lipids, and proteins. More recently, it has become apparent that ethanol consumption also leads to increased post-translational modifications of the natural repertoire, including lysine hyperacetylation. Previously, we determined that alcohol consumption selectively impairs clathrin-mediated internalization in polarized hepatocytes. However, neither the step at which the block occurs nor the mechanism responsible for the defect have been identified. To identify the specific step at which clathrin-mediated internalization is impaired, we examined the distributions, levels, and assembly of selected components of the clathrin machinery in control and ethanol-treated cells. To determine whether the impairment is caused by ethanol-induced lysine acetylation, we also examined the same coat components in cells treated with trichostatin A (TSA), a deacetylase inhibitor that leads to protein hyperacetylation in the absence of ethanol. We determined that both ethanol and TSA impair internalization at a late stage before vesicle fission. We further determined that this defect is likely the result of decreased dynamin recruitment to the necks of clathrin-coated invaginations resulting in impaired vesicle budding. These results also raise the exciting possibility that agents that promote lysine deacetylation may be effective therapeutics for the treatment of alcoholic liver disease.

SIRT1 Deacetylates the DNA Methyltransferase 1 (DNMT1) Protein and Alters Its Activities

DNA methylation and histone acetylation/deacetylation are distinct biochemical processes that control gene expression. While DNA methylation is a common epigenetic signal that inhibits gene transcription, histone deacetylation similarly represses transcription but can be both an epigenetic and nonepigenetic phenomenon. Here we report that the histone deacetylase SIRT1 regulates the activities of DNMT1, a key enzyme responsible for DNA methylation. In mass spectrometry analysis, 12 new acetylated lysine sites were identified in DNMT1. SIRT1 physically associates with DNMT1 and can deacetylate acetylated DNMT1 in vitro and in vivo. Interestingly, deacetylation of different lysines on DNMT1 has different effects on the functions of DNMT1. For example, deacetylation of Lys1349 and Lys1415 in the catalytic domain of DNMT1 enhances DNMT1's methyltransferase activity, while deacetylation of lysine residues in the GK linker decreases DNMT1's methyltransferase-independent transcriptional repression function. Furthermore, deacetylation of all identified acetylated lysine sites in DNMT1 abrogates its binding to SIRT1 and impairs its capability to regulate cell cycle G(2)/M transition. Finally, inhibition of SIRT1 strengthens the silencing effects of DNMT1 on the expression of tumor suppressor genes ER-α and CDH1 in MDA-MB-231 breast cancer cells. Together, these results suggest that SIRT1-mediated deacetylation of DNMT1 is crucial for DNMT1's multiple effects in gene silencing.

HER2 Interacts With CD44 to Up-regulate CXCR4 via Epigenetic Silencing of microRNA-139 in Gastric Cancer Cells

Human epidermal growth factor receptor 2 (HER2) (neu/ERBB2) is overexpressed on many types of cancer cells, including gastric cancer cells; HER2 overexpression has been associated with metastasis and poor prognosis. We investigated the mechanisms by which HER2 regulates cell migration and invasion. HER2 expression or activity was reduced in gastric cancer cell lines using small interfering RNAs or the monoclonal antibody, trastuzumab. We identified proteins that interact with HER2 or microRNAs (miRNAs) involved in HER2 signaling. We used various software programs to identify miRNAs that regulate factors in the HER2 signaling pathway. We analyzed expression patterns of these miRNAs in gastric cancer cell lines and tumor samples from patients. We found that CD44 binds directly to HER2, which up-regulates the expression of metastasis-associated protein-1, induces deacetylation of histone H3 lysine 9, and suppresses transcription of microRNA139 (miR-139) to inhibit expression of its target gene, C-X-C chemokine receptor type 4 (CXCR4). Knockdown of HER2 and CD44 reduced invasive activity of cultured gastric cancer cells and suppressed tumor growth in nude mice. Lymph node metastasis was associated with high levels of HER2, CD44, and CXCR4, and reduced levels of miR-139 in human metastatic gastric tumors. Cultures of different types of metastatic cancer cells with histone deacetylase inhibitors and/or DNA methyltransferase resulted in up-regulation of miR-139. HER2 interaction with CD44 up-regulates CXCR4 by inhibiting expression of miR-139, at the epigenetic level, in gastric cancer cells. These findings indicate how HER2 signaling might promote gastric tumor progression and metastasis.

SIRT1-mediated acute cardioprotection

Overexpression studies have revealed a role for silent information regulator of transcription 1 (SIRT1) lysine deacetylase in cardioprotection against ischemia-reperfusion injury via long-term transcriptional effects. However, short-term SIRT1-mediated lysine deacetylation, within the context of acute cardioprotection, is poorly understood. In this study, the role of SIRT1 in the acute cardioprotective paradigm of first window ischemic preconditioning (IPC) was studied using SIRT1-deficient (SIRT1(+/-)) and SIRT1-overexpressing (SIRT1(+++)) mice. In wild-type hearts, cytosolic lysine deacetylation was observed during IPC, and overacetylation was observed upon pharmacological SIRT1 inhibition. Consistent with a role for SIRT1 in IPC, SIRT1(+/-) hearts could not be preconditioned and exhibited increased cytosolic lysine acetylation. Furthermore, SIRT1(+++) hearts were endogenously protected against ischemia-reperfusion injury and exhibited decreased cytosolic acetylation. Both of these effects in SIRT1(+++) mice were reversed by pharmacological SIRT1 inhibition on an acute timescale. Several downstream targets of SIRT1 were examined, with data suggesting possible roles for endothelial nitric oxide synthase phosphorylation, NF-κB, and stimulation of autophagy. In conclusion, these data suggest that SIRT1, acting on nontranscriptional targets, is required for cardioprotection by acute IPC and that SIRT1-dependent lysine deacetylation occurs during IPC and may play a role in cardioprotective signaling.

A SIRT1-LSD1 Corepressor Complex Regulates Notch Target Gene Expression and Development

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

Mouse recipient cytoplasts for somatic cell nuclear transfer (SCNT) are routinely prepared by mechanical enucleation (ME), an invasive procedure that requires expensive equipment and considerable micromanipulation skills. Alternatively, oocytes can be enucleated using chemically assisted (AE) or chemically induced (IE) enucleation methods that are technically simple. In this study, we compared the reprogramming potential and developmental capacity of cloned embryos generated by ME, AE, and IE procedures and treated with the histone deacetylase inhibitor valproic acid. A rapid and almost complete deacetylation of histone H3 lysine 14 in the somatic nucleus followed by an equally rapid and complete re-acetylation after activation was observed after the injection of a cumulus cell nucleus into ME and AE cytoplasts. In contrast, histone deacetylation occurred at a much lower level in IE cytoplasts. Despite these differences, the cloned embryos generated from the three types of cytoplasts developed into blastocysts of equivalent total and inner cell mass mean cell numbers, and the rates of blastocyst formation and embryonic stem cell derivation were similar among the three groups. The cloned embryos produced from ME and AE cytoplasts showed an equivalent rate of full-term development, but no offspring could be obtained from the IE group, suggesting a lower reprogramming capacity of IE cytoplasts. Our results demonstrate the usefulness of AE in mouse SCNT procedures, as an alternative to ME. AE can facilitate oocyte enucleation and avoid the need for expensive microscope optics, or for potentially damaging Hoechst staining and u.v. irradiation, normally required in ME procedures.

Deacetylation and methylation at histone H3 lysine 9 (H3K9) coordinate chromosome condensation during cell cycle progression.

Interphasic chromatin condenses into the chromosomes in order to facilitate the correct segregation of genetic information. It has been previously reported that the phosphorylation and methylation of the N-terminal tail of histone H3 are responsible for chromosome condensation. In this study, we demonstrate that the deacetylation and methylation of histone H3 lysine 9 (H3K9) are required for proper chromosome condensation. We confirmed that H3K9ac levels were reduced, whereas H3K9me3 levels were increased in mitotic cells, via immunofluorescence and Western blot analysis. Nocodazole treatment induced G2/M arrest but co-treatment with TSA, an HDAC inhibitor, delayed cell cycle progression. However, the HMTase inhibitor, AdoX, had no effect on nocodazole-induced G2/M arrest, thereby indicating that sequential modifications of H3K9 are required for proper chromosome condensation. The expression of SUV39H1 and SETDB1, H3K9me3-responsible HMTases, are specifically increased along with H3K9me3 in nocodazole-arrested buoyant cells, which suggests that the increased expression of those proteins is an important step in chromosome condensation. H3K9me3 was highly concentrated in the vertical chromosomal axis during prophase and prometaphase. Collectively, the results of this study indicate that sequential modifications at H3K9 are associated with correct chromosome condensation, and that H3K9me3 may be relevant to the condensation of chromosome length.

Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential.

SIRT3 Deacetylates Mitochondrial 3-Hydroxy-3-Methylglutaryl CoA Synthase 2 and Regulates Ketone Body Production

The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3(-/-) (SIRT3KO) mice. HMGCS2 is the rate-limiting step in β-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence ofSIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated invitro when incubated with SIRT3 and invivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that insilico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity.

Aging and exercise affect the level of protein acetylation and SIRT1 activity in cerebellum of male rats

Aging is associated with a gradual decline in cognitive and motor functions, the result of complex biochemical processes including pre- and posttranslational modifications of proteins. Sirtuins are NAD(+) dependent protein deacetylases. These enzymes modulate the aging process by lysine deacetylation, which alters the activity and stability of proteins. Exercise can increase mean life-span and improve quality of life. Data from our laboratories revealed that 4 weeks of treadmill running improves performance in the Morris Maze test for young (4 months, old) but not old (30 months, old) male rats, and the exercise could not prevent the age-associated loss in muscle strength assessed by a gripping test. The positive correlation between protein acetylation and the gripping test suggests that the age-dependent decrease in relative activity of SIRT1 in the cerebellum impairs motor function. Similarly to the acetylation level of total proteins, the acetylation of ά -tubulin is also increased with aging, while the effect of exercise training was not found to be significant. Moreover, the protein content of nicotinamide phosphoribosyltransferase, one of the key enzymes of NAD biosynthesis, decreased in the young exercise group. These data suggest that aging results in decreased specific activity of SIRT1 in cerebellum, which could lead to increased acetylation of protein residues, including ά-tubulin, that interfere with motor function.

Groucho-Mediated Repression May Result from a Histone Deacetylase-Dependent Increase in Nucleosome Density

Groucho (Gro) is a Drosophila melanogaster transcriptional corepressor that directly interacts with the histone deacetylase Rpd3. Although previous studies suggest that this interaction is required for repression of Gro-responsive reporters in cultured cells, the in vivo significance of this interaction and the mechanism by which it leads to repression remain largely unexplored. In this study, we show that Gro is partially dependent on Rpd3 for repression, supporting the idea that Rpd3-mediated repression is one mode of Gro-mediated repression. We demonstrate that Gro colocalizes with Rpd3 to the chromatin of a target gene and that this is accompanied by the deacetylation of specific lysines within the N-terminal tails of histones H3 and H4. Gro overexpression leads to wing patterning defects and ectopic repression in the wing disc of transcription directed by the vestigial quadrant enhancer. These effects are reversed by the histone deacetylase inhibitors TSA and HC-Toxin and by the reduction of Rpd3 gene dosage. Furthermore, repression of the vestigial quadrant enhancer is accompanied by a Gro-mediated increase in nucleosome density, an effect that is reversed by histone deacetylase inhibitors. We propose a model in which Gro-mediated histone deacetylation results in increased nucleosome density leading to transcriptional repression.