Histone Deacetylase SIRT1 Research Articles

Nicotinamide Augments the Anti-Inflammatory Properties of Resveratrol through PARP1 Activation

Resveratrol (RSV) and nicotinamide (NAM) have garnered considerable attention due to their anti-inflammatory and anti-aging properties. NAM is a transient inhibitor of class III histone deacetylase SIRTs (silent mating type information regulation 2 homologs) and SIRT1 is an inhibitor of poly-ADP-ribose polymerase-1 (PARP1). The debate on the relationship between RSV and SIRT1 has precluded the use of RSV as a therapeutic drug. Recent work demonstrated that RSV facilitates tyrosyl-tRNA synthetase (TyrRS)-dependent activation of PARP1. Moreover, treatment with NAM is sufficient to facilitate the nuclear localization of TyrRS that activates PARP1. RSV and NAM have emerged as potent agonists of PARP1 through inhibition of SIRT1. In this study, we evaluated the effects of RSV and NAM on pro-inflammatory macrophages. Our results demonstrate that treatment with either RSV or NAM attenuates the expression of pro-inflammatory markers. Strikingly, the combination of RSV with NAM, exerts additive effects on PARP1 activation. Consistently, treatment with PARP1 inhibitor antagonized the anti-inflammatory effect of both RSV and NAM. For the first time, we report the ability of NAM to augment PARP1 activation, induced by RSV, and its associated anti-inflammatory effects mediated through the induction of BCL6 with the concomitant down regulation of COX-2.

Abstract SY20-01: Linking epigenetics, metabolism, and cancer: Lessons from SIRT6

Abstract In recent years, chromatin regulators have emerged as key modulators in cancer. These factors could work as both tumor suppressors and oncogenes, providing plasticity to cancer cells as they adapt to overcome the host responses. In recent years, we discovered that the mammalian histone deacetylase SIRT6 is a key chromatin factor, modulating expression of metabolic, developmental, and ribosomal protein genes. In recent studies, we identified a loss of function mutation in SIRT6 behind a human syndrome of perinatal lethality. In the context of cancer, we found SIRT6 to act as a robust tumor suppressor, by modulating glucose metabolism. As Otto Warburg described decades ago, cancer cells exhibit glycolytic metabolism, where pyruvate, instead of contributing to ATP production in the mitochondria, is converted to lactate even under normoxia conditions. We found SIRT6 as the first chromatin factor in charge of suppressing the Warburg effect in colon cancer. At the cellular level, SIRT6 inactivation leads to increased cellular glucose uptake, higher lactate production and decreased mitochondrial activity. Our results indicate that SIRT6 directly regulates expression of several key glycolytic and ribosomal genes, co-repressing Hif1a and Myc, respectively, and acting as a histone H3 lysine9 (H3K9) and lysine 56 (H3K56) deacetylase to specifically inhibit transcriptional elongation in these genes. Strikingly, we determined in new studies that such glycolytic switch provides an advantage even at the early initiating cancer stem cells stage, in what we identified as the cell-of-origin for the Warburg effect. In addition, we found SIRT6 to act as a robust tumor suppressor in the context of pancreatic cancer. However, in this case, SIRT6 did not influence metabolism, but rather silenced expression of the developmental gene Lin28b, in this way protecting against aggressive undifferentiated pancreatic adenocarcinoma. Our studies highlight the important role epigenetic factors, such as SIRT6, play in protecting against tumor progression by providing “epigenetic plasticity”, inhibiting adaptive responses in transformed cells.

Estrogen-SIRT1 Axis Plays a Pivotal Role in Protecting Arteries Against Menopause-Induced Senescence and Atherosclerosis.

Aim: Menopause causes arterial senescence and atherosclerotic development through decrease of estrogen. Recently, histone deacetylase SIRT1 has been reported to have protective effects against arterial senescence and atherosclerosis. However, the relationship between estrogen and SIRT1 in the context of menopause-induced arterial senescence is not well understood. The present study aims to investigate whether SIRT1 is involved in the etiology of menopause-induced arterial senescence and atherosclerotic development.Methods: Twelve-week old female apolipoprotein E-knockout (ApoE-KO) mice underwent ovariectomy (OVX) or sham surgery.Results: SIRT1 expression and endothelial nitric oxide synthase (eNOS) activation in the aorta were significantly lower in OVX mice than they were in sham mice (OVX vs. sham, n = 5 per group). Senescence-associated β-galactosidase activity, protein expression of Ac-p53 and PAI-1, and aortic atherosclerosis lesions were significantly greater in OVX mice than they were in sham mice. Administration of 17β-estradiol (E2) for eight weeks to OVX mice restored aortic SIRT1 expression, activated eNOS, and retarded OVX-induced arterial senescence and atherosclerotic development (E2 vs. control, n = 5 per group). The effects of E2 on SIRT1 upregulation, anti-senescence and anti-atherosclerosis were attenuated by administration of a SIRT1 inhibitor, sirtinol. In vitro experiment using human endothelial cells demonstrated that E2 also increased SIRT1 expression and retarded oxidized low density lipoprotein-induced premature senescence, which were also abolished by sirtinol. These results suggested that estrogen modulated arterial senescence and atherosclerosis through SIRT1. Additionally a selective estrogen receptor modulator (SERM), bazedoxifene, also augmented SIRT1 and inhibited arterial senescence and atherosclerotic development (SERM vs. control, n = 3 per group).Conclusions: Downregulation of SIRT1 causes OVX-induced arterial senescence and atherosclerosis in ApoE-KO mice. Administration of estrogen or SERM enables OVX mice to restore these alterations by SIRT1 induction.

Ferritinophagy is required for the induction of ferroptosis by the bromodomain protein BRD4 inhibitor (+)-JQ1 in cancer cells

(+)-JQ1 is an inhibitor of the tumor-driver bromodomain protein BRD4 and produces satisfactory effects because it efficiently increases apoptosis. Ferroptosis is an oxidative cell death program differing from apoptosis. Ferroptosis is characterized by high levels of iron and reactive oxygen species and has been confirmed to suppress tumor growth. In this study, BRD4 expression in cancer and its influence on the prognosis of cancer patients were analyzed using data from public databases. In addition, the effect of the BRD4 inhibitor (+)-JQ1 on ferroptosis was investigated via a series of in vitro assays. A nude mouse model was used to evaluate the function of (+)-JQ1 in ferroptosis in vivo. The potential mechanisms by which (+)-JQ1 regulates ferroptosis were explored. The results showed that BRD4 expression levels were higher in cancer tissues than in normal tissues and were related to poor prognosis in cancer patients. Furthermore, ferroptosis was induced under (+)-JQ1 treatment and BRD4 knockdown, indicating that (+)-JQ1 induces ferroptosis via BRD4 inhibition. Moreover, the anticancer effect of (+)-JQ1 was enhanced by ferroptosis inducers. Further studies confirmed that (+)-JQ1 induced ferroptosis via ferritinophagy, which featured autophagy enhancement by (+)-JQ1 and increased iron levels. Subsequently, the reactive oxygen species levels were increased by iron via the Fenton reaction, leading to ferroptosis. In addition, expression of the ferroptosis-associated genes GPX4, SLC7A11, and SLC3A2 was downregulated under (+)-JQ1 treatment and BRD4 knockdown, indicating that (+)-JQ1 may regulate ferroptosis by controlling the expression of ferroptosis-associated genes regulated by BRD4. Finally, (+)-JQ1 regulated ferritinophagy and the expression of ferroptosis-associated genes via epigenetic inhibition of BRD4 by suppressing the expression of the histone methyltransferase G9a or enhancing the expression of the histone deacetylase SIRT1. In summary, the BRD4 inhibitor (+)-JQ1 induces ferroptosis via ferritinophagy or the regulation of ferroptosis-associated genes through epigenetic repression of BRD4.

Inhibition of Src Reduces Cortactin Phosphorylation and Transendothelial Migration of Acute Lymphoblastic Leukemia B cells

B‐cell acute lymphoblastic leukemia (B‐ALL) is the most common leukemia in children and adults. B‐ALL cells have the ability to extravasate and infiltrate bone marrow and organs such as lungs, testis and central nervous system (CNS). Dynamic actin cytoskeletal remodeling is needed to accomplish transendothelial migration. Cortactin is an actin‐binding protein which accumulates in lamellipodia and invadopodia and regulates neutrophil transendothelial migration during inflammation. Recently, we showed that cortactin is overexpressed in B‐cells of B‐ALL patients, and significantly correlates with bone marrow colonization, organ infiltration, drug resistance and disease relapse. Cortactin is a target for several posttranslational modifications such as phosphorylation and acetylation, being first discovered as a substrate of Src kinase and recently as substrate of the histone deacetylases SIRT1 and HDAC6. Phosphorylation of cortactin is thought to lead to lamellipodia and invadopodia protusion, whereas acetylation diminishes the affinity of cortactin to F‐actin, decreases cell migration capability, and may lead to nuclear translocation. However, it remains to be explored whether these modifications are required for the aggressiveness of leukemic B‐ALL cells. Thus, in this study, we analyzed the importance of such modifications for transendothelial migration and bone marrow organoid colonization. A MTT (3‐(4, 5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide) assay was performed to determine effects of certain pharmacological inhibitors of posttranslational modifications on cell viability. To this end, REH cells were incubated with the indicated pharmacological compounds for 24 hours at different concentrations (50 to 150 mM). The IC50 value for PP2 (Src inhibitor) was 190.82 mM, for EX‐527 (SIRT1 inhibitor) was 190.94 mM, for Tubacin (HDAC6 inhibitor) was 41.39 mM, and for Tubastatin‐A (HDAC6 inhibitor) was 94.09 mM. Importantly, after 20 min of treatment of only REH cells with PP2 at 20 μM, REH cells transmigrated significantly less across CXCL12‐stimulated HUVEC monolayers, which may be due to a reduction of phosphorylated cortactin that impairs cytoskeletal remodeling needed for migration of the cells. Taken together, these results suggest that cortactin phosphorylation may be needed by leukemic B cells to achieve transmigration and may reveal cortactin as a potential new therapeutic target for high risk B‐ALL patients to prevent disease relapse.Support or Funding InformationCONACyTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular docking analysis of imine stilbene analogs and evaluation of their anti-aging activity using yeast and mammalian cell models

The NAD+-dependent histone deacetylase SIRT1 was shown to be associated with aging and longevity. A stilbene, resveratrol (RV) was shown to exert anti-aging activity by stimulating the SIRT1 activity. However, the utility of RV is limited by its low bioavailability and structural instability. It is thus envisaged to test imine stilbene (IMS) analogs of RV for their potential anti-aging activity. In the present study, molecular docking analysis of five IMS analogs (3a, 3b, 3c, 3d and 3e) against the SIRT1 protein has been carried out. All the five IMS analogs displayed enhanced binding affinity towards SIRT1; three out of five IMS analogs (3a, 3 b, 3e) showed significantly higher affinity with lower binding energies (−9.58, −9.54, and −9.82 kcal mol−1) than RV (−8.11 kcal mol−1). Further, experimental validation of anti-aging activity was performed by measuring the chronological life span in vitro using yeast and cellular replicative senescence (CRS) in mammalian cell line models. All IMS analogs extended the chronological life span in yeast as compared to untreated cells as well as RV treated cells. Enhanced anti-aging activity was also observed in an analogous mammalian cell line model upon treatment with either RV or IMS analogs. The results thus suggest that most of the IMS analogs tested may serve as potent drug lead molecules with anti-aging activity.

Induction of a senescent like phenotype and loss of gap junctional intercellular communication by carbon nanoparticle exposure of lung epithelial cells.

Inhalation of combustion-derived particles is associated with the development of age-related diseases like chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. In both diseases senescence of lung epithelial cells has been observed. Employing an in vitro system of repetitive exposure to pure carbon nanoparticles we asked whether this kind of particles are able to induce a senescent like phenotype, which might be accompanied by a loss of functionality at the level of gap junctional intercellular communication. Non-cytotoxic doses of carbon nanoparticles but not of bigger carbon particles led to an irreversible reduction of the proliferative capacity accompanied by the accumulation of the cell cycle blocking proteins p21 and p16 as well as a loss of both redox sensitive histone deacetylase SIRT1 and connexin-43. Gap junction intercellular communication detected by microinjection of fluorescent lucifer yellow was dramatically decreased after exposure. This loss of functionality was associated with a reduction of Connexin 43 at the plasma membrane. As the experimental system was chosen to study the effects of pure carbon nanoparticles in the absence of inflammatory cells, the data indicate that cumulative long-term exposure of the lung epithelium to low doses of combustion-derived nanoparticles might contribute to epithelial senescence and age-associated diseases of the airways.

P53 cooperates with SIRT6 to regulate cardiolipin de novo biosynthesis

The tumor suppressor p53 has critical roles in regulating lipid metabolism, but whether and how p53 regulates cardiolipin (CL) de novo biosynthesis is unknown. Here, we report that p53 physically interacts with histone deacetylase SIRT6 in vitro and in vivo, and this interaction increases following palmitic acid (PA) treatment. In response to PA, p53 and SIRT6 localize to chromatin in a p53-dependent manner. Chromatin p53 and SIRT6 bind the promoters of CDP-diacylglycerol synthase 1 and 2 (CDS1 and CDS2), two enzymes required to catalyze CL de novo biosynthesis. Here, SIRT6 serves as a co-activator of p53 and effectively recruits RNA polymerase II to the CDS1 and CDS2 promoters to enhance CL de novo biosynthesis. Our findings reveal a novel, cooperative model executed by p53 and SIRT6 to maintain lipid homeostasis.

Histone deacetylase SIRT1 mediates C5b-9-induced cell cycle in oligodendrocytes

Histone deacetylase SIRT1 mediates C5b-9-induced cell cycle in oligodendrocytes

SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling

While multiple mechanisms of BRAFV600-mutant melanoma resistance to targeted MAPK signaling inhibitors (MAPKi) have been reported, the epigenetic regulation of this process remains undetermined. Here, using a CRISPR–Cas9 screen targeting chromatin regulators, we discover that haploinsufficiency of the histone deacetylase SIRT6 allows melanoma cell persistence in the presence of MAPKi. Haploinsufficiency, but not complete loss of SIRT6 promotes IGFBP2 expression via increased chromatin accessibility, H3K56 acetylation at the IGFBP2 locus, and consequent activation of the IGF-1 receptor (IGF-1R) and downstream AKT signaling. Combining a clinically applicable IGF-1Ri with BRAFi overcomes resistance of SIRT6 haploinsufficient melanoma cells in vitro and in vivo. Using matched melanoma samples derived from patients receiving dabrafenib + trametinib, we identify IGFBP2 as a potential biomarker for MAPKi resistance. Our study has not only identified an epigenetic mechanism of drug resistance, but also provides insights into a combinatorial therapy that may overcome resistance to standard-of-care therapy for BRAFV600-mutant melanoma patients.

Abstract 4461: Sirt6 deletion slows mouse mammary tumorigenesis

Abstract Studies show that the NAD+ dependent histone deacetylase Sirt6 exerts tumor suppressive effects in different tissues and organs by mechanisms that include the dampening of the Warburg effect and of Myc activity. In breast cancer (BC), Sirt6 was recently shown to blunt PI3K signaling and to reduce cancer stem cell like capacity in tumors with mutated PI3K. Here we sought define the effects of Sirt6 overexpression and of Sirt6 silencing in breast cancer cell lines, as well as the impact of a heterozygous Sirt6 deletion on polyoma middle T antigen (PyMT)-induced mouse mammary tumorigenesis, with the expectation of an accelerated mammary tumor development in PyMT+/-;Sirt6+/- as compared to PyMT+/-;Sirt6+/+ mice. SIRT6 was overexpressed in either wild type or catalytically inactive (H133Y) form, or silenced in BC cell lines and in non-tumorigenic MCF10A and HMLE cells, monitoring glycolytic enzyme activity (iHK, PFK, PK, LDH), lactate production, oxygen consumption rate, complex I, III, IV, and ATP synthase activity, and matrix metalloproteinase 9 (MMP9) expression. In vivo, we monitored the growth of MDA-MB-231 xenografts in which SIRT6 was silenced vs. control tumors. In addition, we crossed Sirt6+/- mice with MMTV-PyMT+/- mice (a kind gift of Dr. Thorsten Berger, University Health Network, Toronto, Canada) and comparatively monitored tumor latency and overall survival in PyMT+/-;Sirt6+/- vs. PyMT+/-;Sirt6+/+ mice. In cultured breast cancer cell lines, overexpression of catalytically active SIRT6 (but not of a catalytically inactive enzyme) reduced the activity of glycolytic enzymes as well as lactate production, while boosting OXPHOS and the ATP/AMP ratio. Opposite effects were obtained by SIRT6 silencing. Curiously, no effect of SIRT6 overexpression or of its silencing on glycolysis or OXPHOS was observed in non-tumorigenic mammary epithelial cells. SIRT6 was found to regulate MMP9 expression. Namely, in MDA-MB-231, SIRT6 overexpression increased, while SIRT6 silencing reduced MMP production. In vivo, subcutaneous xenografts of SIRT6-silenced MDA-MB-231 cells were found to grow slower than control cells. PyMT+/-;Sirt6+/- mice exhibited a markedly increased tumor latency and an increased overall survival as compared to the control PyMT+/-;Sirt6+/+ mice. The anticancer effect of Sirt6 heterozygous deletion did not reflect reduced glucose levels in Sirt6+/- mice, as the latter had normal blood glucose levels. In conclusion, our data show that reducing Sirt6 levels has antitumor effects in mouse breast cancer models, which would not be anticipated based on the available knowledge of Sirt6's biological role. In addition, by virtue of its ability to regulate MMP9 expression, SIRT6 could be a potential target for countering metastasis. Future studies should assess which molecular features predict the potential benefit of SIRT6 inhibition in breast cancer and test the anticancer activity of SIRT6 inhibitors. Citation Format: Pamela Becherini, Irene Caffa, Silvia Ravera, Patrizia Damonte, Francesco Piacente, Michele Cea, Antonia Cagnetta, Fiammetta Monacelli, Raul Mostoslavsky, Santina Bruzzone, Alberto Ballestrero, Patrizio Odetti, Alessio Nencioni. Sirt6 deletion slows mouse mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4461.

Abstract 4992: Sirt6 loss increases stemness and defense against oxidative stress in tumor-propagating cells, promoting tumor growth and maintenance in squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the US. As such, a better understanding in HNSCC carcinogenic process and contributing molecular mechanisms may pinpoint to new therapeutic interventions for the patients. Notably, despite extensive genetic studies, little is known on the metabolic features of HNSCC. Metabolic reprogramming to aerobic glycolysis has recently emerged as a key event during carcinogenesis. Our lab has shown that the histone deacetylase Sirt6 inhibits HIF-1α- and c-Myc-dependent transcription, both of which are critical in metabolic reprogramming of cells, thereby acting as a tumor suppressor in the context of colon cancer. Based on these results, we aimed to assess whether Sirt6 loss facilitates squamous cell carcinogenesis, specifically affecting glucose metabolism and stemness of cancer cells. In an in vivo model of skin squamous cell carcinogenesis with Sirt6 deletion (K14-cre+; Sirt6fl/fl), tumor onset was faster in Sirt6fl/fl mice compared to WT animals, and those tumors from Sirt6fl/fl mice were bigger. Sirt6 loss also positively regulates tumor maintenance without continuous proliferative cues, suggesting that Sirt6 deletion could impact on the tumor propagating cells (TPCs). Strikingly, loss of Sirt6 increased the percentage of TPCs (a6high/CD34+), most of which were highly glycolytic. Administration of a glycolytic inhibitor (dichloroacetate) in vivo effectively inhibited tumor growth and maintenance, resulting in much fewer percentages of TPCs. Our results indicate that enhanced glucose metabolism is crucial in maintaining and expanding TPCs. In human HNSCC cell lines, Sirt6 negatively regulates glucose metabolism and cell growth in vitro and ex vivo, nicely recapitulating the in vivo observations. Glucose isotope tracing experiments in vitro suggest that pentose phosphate pathway and generation of antioxidant molecules are main metabolic pathways affected by Sirt6. Transcriptomic analysis of TPCs from in vivo skin tumors revealed critical gene expression signatures, where TPCs exhibit decreased differentiation, increased stem-like, and enhanced antioxidative/redox markers. Sirt6 loss even further up-regulated stemness genes and the genes that generate antioxidant molecules, providing molecular mechanisms to explain a more aggressive tumorigenic phenotype in Sirt6-deleted tumors. Overall, our studies indicate that high glycolytic flux (which can contribute to the pentose phosphate pathway and antioxidant molecules) is crucial in TPCs. Our studies clearly demonstrate that Sirt6 acts as a tumor suppressor by regulating glucose metabolism and stemness of tumor cells. This study has further therapeutic implication that targeting antioxidant defense pathways could effectively eradicate TPCs, critical population maintaining a whole tumor. Citation Format: Jee-Eun Choi, Carlos Sebastian, Caroline Lewis, Murat Cetinbas, Gregory Wojtkiewicz, Ruslan Sadreyev, Leif W. Ellisen, Salvador Aznar-Benitah, Raul Mostoslavsky. Sirt6 loss increases stemness and defense against oxidative stress in tumor-propagating cells, promoting tumor growth and maintenance in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4992.

JAK1-mediated Sirt1 phosphorylation functions as a negative feedback of the JAK1-STAT3 pathway

The type III NAD-dependent histone deacetylase Sirt1 plays important roles in a variety of pathobiological functions through targeting either the acetylated histones or transcription factors. However, the molecular mechanisms underlying how the Sirt1 functions are regulated remain vague. Herein we identified that the Janus kinase 1 (JAK1) interacts with Sirt1 and catalyzes its phosphorylation at the tyrosine residues of 280 and 301, both of which are highly conserved and located in the histone deacetylase catalytic domain of Sirt1. IL-6 stimulation enhanced Sirt1 interaction with JAK1 and JAK1-mediated Sirt1 phosphorylation. Interestingly, JAK1-mediated Sirt1 phosphorylation did not alter Sirt1 deacetylase catalytic activity, but instead it is required for Sirt1 interaction with the downstream transcription factor STAT3. JAK1-mediated phosphorylation enhanced Sirt1 suppression of STAT3 acetylation and transcriptional activity. As a consequence, Sirt1 activation attenuates IL-6 activity in protecting cancer cells from chemotherapeutic drug-induced apoptosis. Our studies identify JAK1 as a previously unappreciated tyrosine kinase of Sirt1 and reveal a novel negative feedback of the JAK1-STAT3 pathway.

Sirt1-mediated metabolic modulation of Aicda, class-switch DNA recombination and somatic hypermutation

Abstract Maturation of the antibody response entails Ig somatic hypermutation (SHM) and class switch DNA recombination (CSR). SHM and CSR perfect the antigen-binding activity and diversify the effector functions of antibodies. By showing that histone deacetylase (HDAC) inhibitors selective for class I/II HDACs dampened SHM/CSR by decreasing activation induced cytidine deaminase (AID) expression through upregulation of miRNAs that target Aicda, we have outlined an important role of Zn-dependent HDACs in the modulation of the antibody response. Here we have shown that the NAD+-dependent Class III HDAC Sirt1 also plays an important role in the regulation of SHM and CSR. B cells undergoing CSR/SHM profoundly downregulated Sirt1. Sirt1 deletion in AID-expressing B cells of AicdacreSirt1fl/fl mice, led to increased CSR and SHM in response to NP-CGG, as a result of upregulated AID expression, without altering Prdm1 expression. Sirt1-deficient B cells reduced DNA methylation in the Aicda promoter and hyperacetylated p65, thus activating the canonical NF-kB pathway, which mediates Aicda activation, in absence of any change in DNA methylation of the Prdm1 promoter or histone acetylation of the Aicda or Prdm1 promoter. Increased glucose concentration, which reduced NAD+ Sirt1 co-factor, led to hyperacetylated p65 and Aicda upregulation. Conversely, the NAD+ Sirt1 co-factor or SRT1720 small molecule, a specific Sirt1 activator, dampened Aicda expression and CSR once added to B cells. Thus, our findings outline an important B cell-intrinsic role of Sirt1 in the modulation of the antibody response. Such a role is independent from and may be in addition to the histone deacetylation activity of this Class III HDAC in immune cells other than B lymphocytes.

Epigenetic effects of metformin: From molecular mechanisms to clinical implications.

There is a growing body of evidence that links epigenetic modifications to type 2 diabetes. Researchers have more recently investigated effects of commonly used medications, including those prescribed for diabetes, on epigenetic processes. This work reviews the influence of the widely used antidiabetic drug metformin on epigenomics, microRNA levels and subsequent gene expression, and potential clinical implications. Metformin may influence the activity of numerous epigenetic modifying enzymes, mostly by modulating the activation of AMP-activated protein kinase (AMPK). Activated AMPK can phosphorylate numerous substrates, including epigenetic enzymes such as histone acetyltransferases (HATs), class II histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), usually resulting in their inhibition; however, HAT1 activity may be increased. Metformin has also been reported to decrease expression of multiple histone methyltransferases, to increase the activity of the class III HDAC SIRT1 and to decrease the influence of DNMT inhibitors. There is evidence that these alterations influence the epigenome and gene expression, and may contribute to the antidiabetic properties of metformin and, potentially, may protect against cancer, cardiovascular disease, cognitive decline and aging. The expression levels of numerous microRNAs are also reportedly influenced by metformin treatment and may confer antidiabetic and anticancer activities. However, as the reported effects of metformin on epigenetic enzymes act to both increase and decrease histone acetylation, histone and DNA methylation, and gene expression, a significant degree of uncertainty exists concerning the overall effect of metformin on the epigenome, on gene expression, and on the subsequent effect on the health of metformin users.

Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology.

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress, CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits SIRT1 activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2 functions as a tumor suppressor and therefore its role in cancer formation and progression is still unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological activities in unstressed cells.

Histone deacetylase SIRT6 inhibits glioma cell growth through down-regulating NOTCH3 expression.

Gliomas are the most common brain tumors of the central nervous system. In this study, we investigated the molecular mechanisms and biological function of SIRT6 in human gliomas. The expression levels of SIRT6 in glioma tissues and cells were analyzed by qRT-PCR and western blot analysis. CCK8 and clonogenicity assays were performed to detect the cell proliferation. Furthermore, the migration and invasion of glioma cells were examined by transwell assays. It was found that the expression of SIRT6 was significantly lower in human glioma tissues or cell lines compared with the normal brain tissue or NHA. Up-regulated SIRT6 significantly decreased cell proliferation, migration and invasion of U87 and U251 cells. By contrast, knockdown of SIRT6 dramatically increased cell proliferation, migration and invasion of U87 and U251 cells. Moreover, over expression of NOTCH3 significantly increased the cell proliferation, migration, and invasion of U87 and U251 cells. However, these effects were abolished after overexpression of SIRT6. These results suggest that SIRT6 may suppress cell proliferation, migration, and invasion via inhibition of the NOTCH3 signaling pathway in glioma.

An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality.

It has been well established that histone and DNA modifications are critical to maintaining the equilibrium between pluripotency and differentiation during early embryogenesis. Mutations in key regulators of DNA methylation have shown that the balance between gene regulation and function is critical during neural development in early years of life. However, there have been no identified cases linking epigenetic regulators to aberrant human development and fetal demise. Here, we demonstrate that a homozygous inactivating mutation in the histone deacetylase SIRT6 results in severe congenital anomalies and perinatal lethality in four affected fetuses. In vitro, the amino acid change at Asp63 to a histidine results in virtually complete loss of H3K9 deacetylase and demyristoylase functions. Functionally, SIRT6 D63H mouse embryonic stem cells (mESCs) fail to repress pluripotent gene expression, direct targets of SIRT6, and exhibit an even more severe phenotype than Sirt6-deficient ESCs when differentiated into embryoid bodies (EBs). When terminally differentiated toward cardiomyocyte lineage, D63H mutant mESCs maintain expression of pluripotent genes and fail to form functional cardiomyocyte foci. Last, human induced pluripotent stem cells (iPSCs) derived from D63H homozygous fetuses fail to differentiate into EBs, functional cardiomyocytes, and neural progenitor cells due to a failure to repress pluripotent genes. Altogether, our study described a germline mutation in SIRT6 as a cause for fetal demise, defining SIRT6 as a key factor in human development and identifying the first mutation in a chromatin factor behind a human syndrome of perinatal lethality.

SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells.

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro‐inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF‐κB nuclear accumulation, SIRT1 deacetylase activity, IL‐8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF‐κB binding to the IL‐8 gene promoter thus increasing IL‐8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF‐κB. FoxO3 siRNA treatment increased IL‐8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF‐κB activity and inducing pro‐inflammatory responses.

Epigenetic Mechanisms of Alcohol Neuroadaptation: Insights from Drosophila.

Alcohol addiction is a serious condition perpetuated by enduring physiological and behavioral adaptations. An important component of these adaptations is the long-term rearrangement of neuronal gene expression in the brain of the addicted individual. Epigenetic histone modifications have recently surfaced as important modulators of the transcriptional adaptation to alcohol as these are thought to represent a form of transcriptional memory that is directly imprinted on the chromosome. Some histone modifications affect transcription by modulating the accessibility of the underlying DNA, whereas others have been proposed to serve as marks read by transcription factors as a “histone code” that helps to specify the expression level of a gene. Although the effects of some epigenetic modifications on the transcriptional activity of genes are well known, the mechanisms by which alcohol consumption produces this rearrangement and leads to lasting changes in behavior remain unresolved. Recent advances using the Drosophila model system have started to unravel the epigenetic modulators underlying functional alcohol neuroadaptations. In this review, we discuss the role of 3 different histone modification systems in Drosophila, which have a direct impact on key alcohol neuroadaptations associated with the addictive process. These systems involve the histone deacetylase Sirt1, the histone acetyltransferase CREB-binding protein (CBP), and a subset of the Drosophila JmjC-Domain histone demethylase family.