Mammalian SIRT6 Represses Invasive Cancer Cell Phenotypes through ATP Citrate Lyase (ACLY)-Dependent Histone Acetylation.

The modulation of dynamic histone acetylation states is key for organizing chromatin structure and modulating gene expression and is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. The mammalian SIRT6 protein, a member of the Class III HDAC Sirtuin family of NAD+-dependent enzymes, plays pivotal roles in aging, metabolism, and cancer biology. Through its site-specific histone deacetylation activity, SIRT6 promotes chromatin silencing and transcriptional regulation of aging-associated, metabolic, and tumor suppressive gene expression programs. ATP citrate lyase (ACLY) is a nucleo-cytoplasmic enzyme that produces acetyl coenzyme A (acetyl-CoA), which is the required acetyl donor for lysine acetylation by HATs. In addition to playing a central role in generating cytosolic acetyl-CoA for de novo lipogenesis, a growing body of work indicates that ACLY also functions in the nucleus where it contributes to the nutrient-sensitive regulation of nuclear acetyl-CoA availability for histone acetylation in cancer cells. In this study, we have identified a novel function of SIRT6 in controlling nuclear levels of ACLY and ACLY-dependent tumor suppressive gene regulation. The inactivation of SIRT6 in cancer cells leads to the accumulation of nuclear ACLY protein and increases nuclear acetyl-CoA pools, which in turn drive locus-specific histone acetylation and the expression of cancer cell adhesion and migration genes that promote tumor invasiveness. Our findings uncover a novel mechanism of SIRT6 in suppressing invasive cancer cell phenotypes and identify acetyl-CoA responsive cell migration and adhesion genes as downstream targets of SIRT6.

Elevated NSD3 histone methylation activity drives squamous cell lung cancer.

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient ß-catenin degradation.

The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scaffolds GSK3ß and CK1 to earmark ß-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of ß-catenin and oncogenesis. However, the molecular mechanism by which APC promotes ß-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple ß-catenin and Axin interacting sites, undergoes liquid-liquid phase separation (LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and ß-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the ß-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of ß-catenin.

Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives.

Mammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis. Sirt6 activation protects against metabolic and aging-related diseases, and Sirt6 inhibition is considered a cancer therapy. Available Sirt6 modulators show insufficient potency and specificity, and even partially contradictory Sirt6 effects were reported for the plant flavone quercetin. To understand Sirt6 modulation by quercetin-based compounds, we analysed their binding and activity effects on Sirt6 and other Sirtuin isoforms and solved crystal structures of compound complexes with Sirt6 and Sirt2. We find that quercetin activates Sirt6 via the isoform-specific binding site for pyrrolo[1,2-a]quinoxalines. Its inhibitory effect on other isoforms is based on an alternative binding site at the active site entrance. Based on these insights, we identified isoquercetin as a ligand that can discriminate both sites and thus activates Sirt6 with increased specificity. Furthermore, we find that quercetin derivatives that inhibit rather than activate Sirt6 exploit the same general Sirt6 binding site as the activators, identifying it as a versatile allosteric site for Sirt6 modulation. Our results thus provide a structural basis for Sirtuin effects of quercetin-related compounds and helpful insights for Sirt6-targeted drug development.

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules.

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein ß-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7.

Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nε-(7-azidoheptanoyl)-l-lysine (AzHeK) by amber codon in Escherichia coli for recombinant expression of a number of AzHeK-containing histone H3 proteins. We assembled in vitro acyl-nucleosomes from these recombinant acyl-H3 histones. All these acyl-nucleosomes contained an azide functionality that allowed quick click labeling with a strained alkyne dye for in-gel fluorescence analysis. Using these acyl-nucleosomes as substrates and click labeling as a detection method, we systematically investigated chromatin deacylation activities of SIRT7, a class III NAD+-dependent histone deacylase with roles in aging and cancer biology. Besides confirming the previously reported histone H3K18 deacylation activity, our results revealed that SIRT7 has an astonishingly high activity to catalyze deacylation of H3K36 and is also catalytically active to deacylate H3K37. We further demonstrated that this H3K36 deacylation activity is nucleosome dependent and can be significantly enhanced when appending the acyl-nucleosome substrate with a short double-stranded DNA that mimics the bridging DNA between nucleosomes in native chromatin. By overexpressing SIRT7 in human cells, we verified that SIRT7 natively removes acetylation from histone H3K36. Moreover, SIRT7-deficient cells exhibited H3K36 hyperacetylation in whole cell extracts, at rDNA sequences in nucleoli, and at select SIRT7 target loci, demonstrating the physiologic importance of SIRT7 in determining endogenous H3K36 acetylation levels. H3K36 acetylation has been detected at active gene promoters, but little is understood about its regulation and functions. Our findings establish H3K36 as a physiologic substrate of SIRT7 and implicate this modification in potential SIRT7 pathways in heterochromatin silencing and genomic stability.

The epigenetic regulator SIRT7 guards against mammalian cellular senescence induced by ribosomal DNA instability.

In the yeast Saccharomyces cerevisiae, genomic instability in rDNA repeat sequences is an underlying cause of cell aging and is suppressed by the chromatin-silencing factor Sir2. In humans, rDNA instability is observed in cancers and premature aging syndromes, but its underlying mechanisms and functional consequences remain unclear. Here, we uncovered a pivotal role of sirtuin 7 (SIRT7), a mammalian Sir2 homolog, in guarding against rDNA instability and show that this function of SIRT7 protects against senescence in primary human cells. We found that, mechanistically, SIRT7 is required for association of SNF2H (also called SMARCA5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A, member 5), a component of the nucleolar heterochromatin-silencing complex NoRC, with rDNA sequences. Defective rDNA-heterochromatin silencing in SIRT7-deficient cells unleashed rDNA instability, with excision and loss of rDNA gene copies, which in turn induced acute senescence. Mounting evidence indicates that accumulation of senescent cells significantly contributes to tissue dysfunction in aging-related pathologies. Our findings identify rDNA instability as a driver of mammalian cellular senescence and implicate SIRT7-dependent heterochromatin silencing in protecting against this process.

SIRT6: Novel Mechanisms and Links to Aging and Disease.

SIRT6, a member of the Sirtuin family of NAD+-dependent enzymes, has established roles in chromatin signaling and genome maintenance. Through these functions, SIRT6 protects against aging-associated pathologies including metabolic disease and cancer, and can promote longevity in mice. Research from the past few years revealed that SIRT6 is a complex enzyme with multiple substrates and catalytic activities, and uncovered novel SIRT6 functions in the maintenance of organismal health span. Here, we review these new discoveries and models of SIRT6 biology in four areas: heterochromatin stabilization and silencing; stem cell biology; cancer initiation and progression; and regulation of metabolic homeostasis. We discuss the possible implications of these findings for therapeutic interventions in aging and aging-related disease processes.

Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules.

Sirtuins are protein deacylases regulating metabolism and stress responses, and are implicated in aging-related diseases. Small molecule activators for the human sirtuins Sirt1-7 are sought as chemical tools and potential therapeutics, such as for cancer. Activators are available for Sirt1 and exploit its unique N-terminus, whereas drug-like activators for Sirt2-7 are lacking. We synthesized and screened pyrrolo[1,2-a]quinoxaline derivatives, yielding the first synthetic Sirt6 activators. Biochemical assays show direct, substrate-independent compound binding to the Sirt6 catalytic core and potent activation of Sirt6-dependent deacetylation of peptide substrates and complete nucleosomes. Crystal structures of Sirt6/activator complexes reveal that the compounds bind to a Sirt6-specific acyl channel pocket and identify key interactions. Our results establish potent Sirt6 activation with small molecules and provide a structural basis for further development of Sirt6 activators as tools and therapeutics.

SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence.

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing has been observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover an essential role of the human SIRT6 enzyme in pericentric transcriptional silencing, and we show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, residue K18 of histone H3 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, depletion of these transcripts through RNA interference rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and regulation of acetylated H3K18 at heterochromatin, and demonstrate the pathogenic role of deregulated pericentric transcription in aging- and cancer-related cellular dysfunction.

Methylation gets into rhythm with NAD(+)-SIRT1.

Circadian regulation of epigenetic chromatin marks drives daily transcriptional oscillation of thousands of genes and is intimately linked to cellular metabolism and bioenergetics. New work links circadian fluctuations in the activity of the SIRT1 deacetylase, a sensor of the cellular energy state, to histone-methylation changes and the circadian expression of clock-controlled genes.

SIRT7 inactivation reverses metastatic phenotypes in epithelial and mesenchymal tumors.

Metastasis is responsible for over 90% of cancer-associated mortality. In epithelial carcinomas, a key process in metastatic progression is the epigenetic reprogramming of an epithelial-to-mesenchymal transition-like (EMT) change towards invasive cellular phenotypes. In non-epithelial cancers, different mechanisms must underlie metastatic change, but relatively little is known about the factors involved. Here, we identify the chromatin regulatory Sirtuin factor SIRT7 as a key regulator of metastatic phenotypes in both epithelial and mesenchymal cancer cells. In epithelial prostate carcinomas, high SIRT7 levels are associated with aggressive cancer phenotypes, metastatic disease, and poor patient prognosis, and depletion of SIRT7 can reprogram these cells to a less aggressive phenotype. Interestingly, SIRT7 is also important for maintaining the invasiveness and metastatic potential of non-epithelial sarcoma cells. Moreover, SIRT7 inactivation dramatically suppresses cancer cell metastasis in vivo, independent of changes in primary tumor growth. Mechanistically, we also uncover a novel link between SIRT7 and its family member SIRT1, providing the first demonstration of direct interaction and functional interplay between two mammalian sirtuins. Together with previous work, our findings highlight the broad role of SIRT7 in maintaining the metastatic cellular phenotype in diverse cancers.

Molecular pathways: emerging roles of mammalian Sirtuin SIRT7 in cancer.

SIRT7 belongs to the Sirtuin family of NAD-dependent enzymes, the members of which play diverse roles in aging, metabolism, and disease biology. Increased SIRT7 expression is observed in human cancers and growing evidence suggests important SIRT7 functions in fundamental cellular programs with an impact on oncogenic transformation and tumor biology. SIRT7 associates with chromatin, where it catalyzes selective deacetylation of lysine 18 on histone H3 (H3K18), an emerging epigenetic biomarker of aggressive tumors and poor clinical outcome in patients with cancer. Through H3K18 deacetylation at specific promoters, SIRT7 controls a tumor-suppressive gene expression program that stabilizes the transformed state of cancer cells. SIRT7 also orchestrates several molecular processes, including rRNA and tRNA synthesis, which ultimately promote the increased ribosome biogenesis necessary for tumor cell growth and proliferation. Remarkably, inactivation of SIRT7 can reverse the transformed phenotype of cancer cells and reduce their tumorigenicity in vivo. These findings place SIRT7 at the crossroads of chromatin signaling, metabolic, and tumor-regulatory pathways. Thus, SIRT7 is a promising pharmacologic target for epigenetic cancer therapy. The development of SIRT7 modulators may allow new therapeutic strategies that control tumor progression by reprogramming the chromatin landscape and biosynthetic machinery of cancer cells.

SIRT7 represses Myc activity to suppress ER stress and prevent fatty liver disease.

Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here, we show that SIRT7, an NAD(+)-dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevent the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7-deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.

Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling.

The chromatin regulatory factor SIRT6 plays pivotal roles in metabolism, tumor suppression, and aging biology. Despite the fundamental roles of SIRT6 in physiology and disease, only a handful of molecular and functional interactions of SIRT6 have been reported. Here, we characterize the SIRT6 interactome and identify 80+ novel SIRT6-interacting proteins. The discovery of these SIRT6-associations considerably expands knowledge of the SIRT6 interaction network, and suggests previously unknown functional interactions of SIRT6 in fundamental cellular processes. These include chromatin remodeling, mitotic chromosome segregation, protein homeostasis, and transcriptional elongation. Extended analysis of the SIRT6 interaction with G3BP1, a master stress response factor, uncovers an unexpected role and mechanism of SIRT6 in regulating stress granule assembly and cellular stress resistance.

SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation.

Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling.

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.