Katrin Chua

All Publications

• Elevated NSD3 histone methylation activity drives squamous cell lung cancer. Nature Yuan, G. n., Flores, N. M., Hausmann, S. n., Lofgren, S. M., Kharchenko, V. n., Angulo-Ibanez, M. n., Sengupta, D. n., Lu, X. n., Czaban, I. n., Azhibek, D. n., Vicent, S. n., Fischle, W. n., Jaremko, M. n., Fang, B. n., Wistuba, I. I., Chua, K. F., Roth, J. A., Minna, J. D., Shao, N. Y., Jaremko, Ł. n., Mazur, P. K., Gozani, O. n. 2021

  Abstract

  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.

  View details for DOI 10.1038/s41586-020-03170-y

  View details for PubMedID 33536620

• The epigenetic regulator SIRT7 guards against mammalian cellular senescence induced by ribosomal DNA instability. The Journal of biological chemistry Paredes, S., Angulo-Ibanez, M., Tasselli, L., Carlson, S. M., Zheng, W., Li, T., Chua, K. F. 2018

  Abstract

  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.

  View details for PubMedID 29728458

• SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence NATURE STRUCTURAL & MOLECULAR BIOLOGY Tasselli, L., Xi, Y., Zheng, W., Tennen, R. I., Odrowaz, Z., Simeoni, F., Li, W., Chua, K. F. 2016; 23 (5): 434-440

  Abstract

  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.

  View details for DOI 10.1038/nsmb.3202

  View details for Web of Science ID 000375633100015

  View details for PubMedID 27043296

• CANCER Metabolism in the driver's seat NATURE Tasselli, L., Chua, K. F. 2012; 492 (7429): 362-363

  View details for Web of Science ID 000312488200040

  View details for PubMedID 23257875

• SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation NATURE Barber, M. F., Michishita-Kioi, E., Xi, Y., Tasselli, L., Kioi, M., Moqtaderi, Z., Tennen, R. I., Paredes, S., Young, N. L., Chen, K., Struhl, K., Garcia, B. A., Gozani, O., Li, W., Chua, K. F. 2012; 487 (7405): 114-?

  Abstract

  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.

  View details for DOI 10.1038/nature11043

  View details for Web of Science ID 000305982900061

  View details for PubMedID 22722849

  View details for PubMedCentralID PMC3412143

• SIRT6 is required for maintenance of telomere position effect in human cells NATURE COMMUNICATIONS Tennen, R. I., Bua, D. J., Wright, W. E., Chua, K. F. 2011; 2

  Abstract

  In Saccharomyces cerevisiae, the repressive chromatin environment at telomeres gives rise to telomere position effect (TPE), the epigenetic silencing of telomere-proximal genes. Chromatin-modifying factors that control TPE in yeast have been extensively studied, and, among these, the lifespan regulator and silencing protein Sir2 has a pivotal role. In contrast, the factors that generate and maintain silent telomeric chromatin in human cells remain largely unknown. Here we show that the Sir2 family member SIRT6 is required for maintenance of TPE in human cells. RNAi-mediated depletion of SIRT6 abrogates silencing of both an integrated telomeric transgene and an endogenous telomere-proximal gene. Moreover, enhanced telomeric silencing in response to telomere elongation is associated with increased repressive chromatin marks, and this heterochromatic milieu is lost in SIRT6-deficient cells. Together, these findings establish a new role for SIRT6 in regulating an ageing-associated epigenetic silencing process and provide new mechanistic insight into chromatin silencing at telomeres.

  View details for DOI 10.1038/ncomms1443

  View details for Web of Science ID 000294806500026

  View details for PubMedID 21847107

  View details for PubMedCentralID PMC3528101

• SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span CELL Kawahara, T. L., Michishita, E., Adler, A. S., Damian, M., Berber, E., Lin, M., McCord, R. A., Ongaigui, K. C., Boxer, L. D., Chang, H. Y., Chua, K. F. 2009; 136 (1): 62-74

  Abstract

  Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.

  View details for DOI 10.1016/j.cell.2008.10.052

  View details for PubMedID 19135889

• SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin NATURE Michishita, E., McCord, R. A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T. L., Barrett, J. C., Chang, H. Y., Bohr, V. A., Ried, T., Gozani, O., Chua, K. F. 2008; 452 (7186): 492-U16

  Abstract

  The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

  View details for DOI 10.1038/nature06736

  View details for PubMedID 18337721

• Antibody toolkit to investigate eEF1A methylation dynamics in mRNA translation elongation. The Journal of biological chemistry Mealey-Farr, R., Jeong, J., Park, J., Liu, S., Hausmann, S., Francis, J. W., Angulo Ibanez, M., Cho, J., Chua, K., Mazur, P. K., Gozani, O. 2023: 104747

  Abstract

  Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eEF1A (eukaryotic elongation factor 1A), a fundamental non-ribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases (KMTs) leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry (IHC) applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together our study provides a roadmap for leveraging methyl state and sequence selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.

  View details for DOI 10.1016/j.jbc.2023.104747

  View details for PubMedID 37094697

• Mammalian SIRT6 Represses Invasive Cancer Cell Phenotypes through ATP Citrate Lyase (ACLY)-Dependent Histone Acetylation. Genes Zheng, W., Tasselli, L., Li, T. M., Chua, K. F. 2021; 12 (9)

  Abstract

  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.

  View details for DOI 10.3390/genes12091460

  View details for PubMedID 34573442

• HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies. Cell reports Slaughter, M. J., Shanle, E. K., Khan, A. n., Chua, K. F., Hong, T. n., Boxer, L. D., Allis, C. D., Josefowicz, S. Z., Garcia, B. A., Rothbart, S. B., Strahl, B. D., Davis, I. J. 2021; 34 (3): 108638

  Abstract

  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.

  View details for DOI 10.1016/j.celrep.2020.108638

  View details for PubMedID 33472068

• Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient beta-catenin degradation. Scientific reports Li, T., Ren, J., Husmann, D., Coan, J. P., Gozani, O., Chua, K. F. 2020; 10 (1): 17425

  Abstract

  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 GSK3beta and CK1 to earmark beta-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of beta-catenin and oncogenesis. However, the molecular mechanism by which APC promotes beta-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple beta-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 beta-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the beta-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of beta-catenin.

  View details for DOI 10.1038/s41598-020-74080-2

  View details for PubMedID 33060621

• Chromatin Regulation and Genome Maintenance by Mammalian SIRT6 and SIRT7 Chua, K. WILEY. 2020

  View details for DOI 10.1096/fasebj.2020.34.s1.00152

  View details for Web of Science ID 000546023103121

• Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives. Scientific reports You, W., Zheng, W., Weiss, S., Chua, K. F., Steegborn, C. 2019; 9 (1): 19176

  Abstract

  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.

  View details for DOI 10.1038/s41598-019-55654-1

  View details for PubMedID 31844103

• Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules. Scientific reports Li, T., Coan, J. P., Krajewski, K., Zhang, L., Elias, J. E., Strahl, B. D., Gozani, O., Chua, K. F. 2019; 9 (1): 14226

  Abstract

  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 beta-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

  View details for DOI 10.1038/s41598-019-50817-6

  View details for PubMedID 31578417

• A Click Chemistry Approach Reveals the Chromatin-Dependent Histone H3K36 Deacylase Nature of SIRT7. Journal of the American Chemical Society Wang, W. W., Angulo-Ibanez, M., Lyu, J., Kurra, Y., Tong, Z., Wu, B., Zhang, L., Sharma, V., Zhou, J., Lin, H., Gao, Y. Q., Li, W., Chua, K. F., Liu, W. R. 2019

  Abstract

  Using an engineered pyrrolysyl-tRNA synthetase mutant together with tRNACUAPyl, we have genetically encoded Nepsilon-(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.

  View details for PubMedID 30653310

• CHROMATIN AND NUCLEAR SIGNALING: SIRT7 FUNCTION IN THE NUCLEOLUS AND BEYOND INTRODUCTORY REVIEW ON SIRTUINS IN BIOLOGY, AGING, AND DISEASE Angulo-Ibanez, M., Chua, K. F., Guarente, L., Mostoslavsky, R., Kazantsev, A. 2018: 131–40

  View details for DOI 10.1016/B978-0-12-813499-3.00010-1

  View details for Web of Science ID 000473129200011

• SIRT6: Novel Mechanisms and Links to Aging and Disease. Trends in endocrinology and metabolism Tasselli, L., Zheng, W., Chua, K. F. 2017; 28 (3): 168-185

  Abstract

  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.

  View details for DOI 10.1016/j.tem.2016.10.002

  View details for PubMedID 27836583

  View details for PubMedCentralID PMC5326594

• Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules. Angewandte Chemie (International ed. in English) You, W., Rotili, D., Li, T., Kambach, C., Meleshin, M., Schutkowski, M., Chua, K. F., Mai, A., Steegborn, C. 2017; 56 (4): 1007-1011

  Abstract

  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.

  View details for DOI 10.1002/anie.201610082

  View details for PubMedID 27990725

• SIRT7 clears the way for DNA repair. EMBO journal Paredes, S., Chua, K. F. 2016; 35 (14): 1483-1485

  View details for DOI 10.15252/embj.201694904

  View details for PubMedID 27302089

  View details for PubMedCentralID PMC4946144

• Nuclear DNA damage signalling to mitochondria in ageing NATURE REVIEWS MOLECULAR CELL BIOLOGY Fang, E. F., Scheibye-Knudsen, M., Chua, K. F., Mattson, M. P., Croteau, D. L., Bohr, V. A. 2016; 17 (5): 308-321

  Abstract

  Mitochondrial dysfunction is a hallmark of ageing, and mitochondrial maintenance may lead to increased healthspan. Emerging evidence suggests a crucial role for signalling from the nucleus to mitochondria (NM signalling) in regulating mitochondrial function and ageing. An important initiator of NM signalling is nuclear DNA damage, which accumulates with age and may contribute to the development of age-associated diseases. DNA damage-dependent NM signalling constitutes a network that includes nuclear sirtuins and controls genomic stability and mitochondrial integrity. Pharmacological modulation of NM signalling is a promising novel approach for the prevention and treatment of age-associated diseases.

  View details for DOI 10.1038/nrm.2016.14

  View details for Web of Science ID 000374537000009

  View details for PubMedID 26956196

• SIRT7 inactivation reverses metastatic phenotypes in epithelial and mesenchymal tumors SCIENTIFIC REPORTS Malik, S., Villanova, L., Tanaka, S., Aonuma, M., Roy, N., Berber, E., Pollack, J. R., Michishita-Kioi, E., Chua, K. F. 2015; 5

  View details for DOI 10.1038/srep09841

  View details for Web of Science ID 000353622100001

  View details for PubMedID 25923013

• Methylation gets into rhythm with NAD(+)-SIRT1. Nature structural & molecular biology Tasselli, L., Chua, K. F. 2015; 22 (4): 275-277

  View details for DOI 10.1038/nsmb.3004

  View details for PubMedID 25837871

• Molecular Pathways: Emerging Roles of Mammalian Sirtuin SIRT7 in Cancer. Clinical cancer research Paredes, S., Villanova, L., Chua, K. F. 2014; 20 (7): 1741-1746

  Abstract

  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. Clin Cancer Res; 20(7); 1741-6. ©2014 AACR.

  View details for DOI 10.1158/1078-0432.CCR-13-1547

  View details for PubMedID 24536059

  View details for PubMedCentralID PMC3980586

• SIRT7 Represses Myc Activity to Suppress ER Stress and Prevent Fatty Liver Disease CELL REPORTS Shin, J., He, M., Liu, Y., Paredes, S., Villanova, L., Brown, K., Qiu, X., Nabavi, N., Mohrin, M., Wojnoonski, K., Li, P., Cheng, H., Murphy, A. J., Valenzuela, D. M., Luo, H., Kapahi, P., Krauss, R., Mostoslavsky, R., Yancopoulos, G. D., Alt, F. W., Chua, K. F., Chen, D. 2013; 5 (3): 654-665

  Abstract

  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.

  View details for DOI 10.1016/j.celrep.2013.10.007

  View details for Web of Science ID 000328263400012

  View details for PubMedID 24210820

  View details for PubMedCentralID PMC3888240

• The Role of SIRT6 Protein in Aging and Reprogramming of Human Induced Pluripotent Stem Cells. journal of biological chemistry Sharma, A., Diecke, S., Zhang, W. Y., Lan, F., He, C., Mordwinkin, N. M., Chua, K. F., Wu, J. C. 2013; 288 (25): 18439-18447

  Abstract

  Aging is known to be the single most important risk factor for multiple diseases. Sirtuin-6, or SIRT6, has recently been identified as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging, we demonstrated that human dermal fibroblasts (HDFs) from older subjects were more resistant to reprogramming by classic Yamanaka factors than those from young subjects, but the addition of SIRT6 during reprogramming substantially improved such efficiency in older HDFs. Despite the importance of SIRT6, little is known about the molecular mechanism of its regulation. We show for the first time post-transcriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop, which has implications for the modulation of SIRT6 expression in reprogramming of aging cells.

  View details for DOI 10.1074/jbc.M112.405928

  View details for PubMedID 23653361

• A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation. Molecular cell Moore, K. E., Carlson, S. M., Camp, N. D., Cheung, P., James, R. G., Chua, K. F., Wolf-Yadlin, A., Gozani, O. 2013; 50 (3): 444-456

  Abstract

  Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

  View details for DOI 10.1016/j.molcel.2013.03.005

  View details for PubMedID 23583077

  View details for PubMedCentralID PMC3660009

• Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling. Scientific reports Simeoni, F., Tasselli, L., Tanaka, S., Villanova, L., Hayashi, M., Kubota, K., Isono, F., Garcia, B. A., Michishita-Kioi, E., Chua, K. F. 2013; 3: 3085-?

  View details for DOI 10.1038/srep03085

  View details for PubMedID 24169447

• Finding a Target for Resveratrol CELL Tennen, R. I., Michishita-Kioi, E., Chua, K. F. 2012; 148 (3): 387-389

  Abstract

  Despite resveratrol's well-documented health benefits, its mechanism of action remains controversial. In particular, the direct molecular target of resveratrol has been elusive. Park et al. now show that resveratrol directly inhibits cAMP-dependent phosphodiesterases, triggering a cascade of events that converge on the important energy-sensing metabolic regulators AMPK, SIRT1, and PGC-1α.

  View details for DOI 10.1016/j.cell.2012.01.032

  View details for Web of Science ID 000300225000005

  View details for PubMedID 22304906

• Lysine methylation of the NF-kappa B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappa B signaling NATURE IMMUNOLOGY Levy, D., Kuo, A. J., Chang, Y., Schaefer, U., Kitson, C., Cheung, P., Espejo, A., Zee, B. M., Liu, C. L., Tangsombatvisit, S., Tennen, R. I., Kuo, A. Y., Tanjing, S., Cheung, R., Chua, K. F., Utz, P. J., Shi, X., Prinjha, R. K., Lee, K., Garcia, B. A., Bedford, M. T., Tarakhovsky, A., Cheng, X., Gozani, O. 2011; 12 (1): 29-U47

  Abstract

  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.

  View details for DOI 10.1038/ni.1968

  View details for Web of Science ID 000285465100010

  View details for PubMedID 21131967

  View details for PubMedCentralID PMC3074206

• Chromatin regulation and genome maintenance by mammalian SIRT6 TRENDS IN BIOCHEMICAL SCIENCES Tennen, R. I., Chua, K. F. 2011; 36 (1): 39-46

  Abstract

  Saccharomyces cerevisiae Sir2 is an NAD(+)-dependent histone deacetylase that links chromatin silencing to genomic stability, cellular metabolism and lifespan regulation. In mice, deficiency for the Sir2 family member SIRT6 leads to genomic instability, metabolic defects and degenerative pathologies associated with aging. Until recently, SIRT6 was an orphan enzyme whose catalytic activity and substrates were unclear. However, new mechanistic insights have come from the discovery that SIRT6 is a highly substrate-specific histone deacetylase that promotes proper chromatin function in several physiologic contexts, including telomere and genome stabilization, gene expression and DNA repair. By maintaining both the integrity and the expression of the mammalian genome, SIRT6 thus serves several roles that parallel Sir2 function. In this article, we review recent advances in understanding the mechanisms of SIRT6 action and their implications for human biology and disease.

  View details for Web of Science ID 000286689200005

  View details for PubMedID 20729089

• Functional dissection of SIRT6: Identification of domains that regulate histone deacetylase activity and chromatin localization MECHANISMS OF AGEING AND DEVELOPMENT Tennen, R. I., Berber, E., Chua, K. F. 2010; 131 (3): 185-192

  Abstract

  The mammalian sirtuin SIRT6 is a site-specific histone deacetylase that regulates chromatin structure. SIRT6 is implicated in fundamental biological processes in aging, including maintaining telomere integrity, fine-tuning aging-associated gene expression programs, preventing genomic instability, and maintaining metabolic homeostasis. Despite these important functions, the basic molecular determinants of SIRT6 enzymatic function--including the mechanistic and regulatory roles of specific domains of SIRT6--are not well understood. Sirtuin proteins consist of a conserved central 'sirtuin domain'--thought to comprise an enzymatic core--flanked by variable N- and C-terminal extensions. Here, we report the identification of novel functions for the N- and C-terminal domains of the human SIRT6 protein. We show that the C-terminal extension (CTE) of SIRT6 contributes to proper nuclear localization but is dispensable for enzymatic activity. In contrast, the N-terminal extension (NTE) of SIRT6 is critical for chromatin association and intrinsic catalytic activity. Surprisingly, mutation of a conserved catalytic histidine residue in the core sirtuin domain not only abrogates SIRT6 enzymatic activity but also leads to impaired chromatin association in cells. Together, our observations define important biochemical and cellular roles of specific SIRT6 domains, and provide mechanistic insight into the potential role of these domains as targets for physiologic and pharmacologic modulation.

  View details for DOI 10.1016/j.mad.2010.01.006

  View details for Web of Science ID 000277168500003

  View details for PubMedID 20117128

  View details for PubMedCentralID PMC2846990

• Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6 CELL CYCLE Michishita, E., McCord, R. A., Boxer, L. D., Barber, M. F., Hong, T., Gozani, O., Chua, K. F. 2009; 8 (16): 2664-2666

  View details for Web of Science ID 000268983900036

  View details for PubMedID 19625767

• SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair Aging McCord RA, Michishita E, Hong T, Berber E, Boxer LD, Kusumoto R, Guan S, Shi X, , Gozani O, Burlingame AL, Bohr VA, Chua KF 2009; 1: 109-121
• Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally MOLECULAR AND CELLULAR BIOLOGY Zhang, Y., Kwon, S., Yamaguchi, T., Cubizolles, F., Rousseaux, S., Kneissel, M., Cao, C., Li, N., Cheng, H., Chua, K., Lombard, D., Mizeracki, A., Matthias, G., Alt, F. W., Khochbin, S., Matthias, P. 2008; 28 (5): 1688-1701

  Abstract

  Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.

  View details for DOI 10.1128/MCB.01154-06

  View details for Web of Science ID 000253603100023

  View details for PubMedID 18180281

• ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression NATURE Shi, X., Hong, T., Walter, K. L., Ewalt, M., Michishita, E., Hung, T., Carney, D., Pena, P., Lan, F., Kaadige, M. R., Lacoste, N., Cayrou, C., Davrazou, F., Saha, A., Cairns, B. R., Ayer, D. E., Kutateladze, T. G., Shi, Y., Cote, J., Chua, K. F., Gozani, O. 2006; 442 (7098): 96-99

  Abstract

  Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

  View details for DOI 10.1038/nature04835

  View details for Web of Science ID 000238724500044

  View details for PubMedID 16728974

  View details for PubMedCentralID PMC3089773

• Genomic instability and aging-like phenotype in the absence of mammalian SIRT6 CELL Mostoslavsky, R., Chua, K. F., Lombard, D. B., Pang, W. W., Fischer, M. R., Gellon, L., Liu, P. F., Mostoslavsky, G., Franco, S., Murphy, M. M., Mills, K. D., Patel, P., Hsu, J. T., Hong, A. L., Ford, E., Cheng, H. L., Kennedy, C., Nunez, N., Bronson, R., Frendewey, D., Auerbach, W., Valenzuela, D., Karow, M., Hottiger, M. O., Hursting, S., Barrett, J. C., Guarente, L., Mulligan, R., Demple, B., Yancopoulos, G. D., Alt, F. W. 2006; 124 (2): 315-329

  Abstract

  The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.

  View details for DOI 10.1016/j.cell.2005.11.044

  View details for Web of Science ID 000235068500020

  View details for PubMedID 16439206

• DNA repair, genome stability, and aging CELL Lombard, D. B., Chua, K. F., Mostoslavsky, R., Franco, S., Gostissa, M., Alt, F. W. 2005; 120 (4): 497-512

  Abstract

  Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.

  View details for Web of Science ID 000227271500007

  View details for PubMedID 15734682

• Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase SCIENCE Brunet, A., Sweeney, L. B., Sturgill, J. F., Chua, K. F., Greer, P. L., Lin, Y. X., Tran, H., Ross, S. E., Mostoslavsky, R., Cohen, H. Y., Hu, L. S., Cheng, H. L., Jedrychowski, M. P., Gygi, S. P., Sinclair, D. A., Alt, F. W., Greenberg, M. E. 2004; 303 (5666): 2011-2015

  Abstract

  The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

  View details for DOI 10.1126/science.1094637

  View details for Web of Science ID 000220429800040

  View details for PubMedID 14976264

• Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cheng, H. L., Mostoslavsky, R., Saito, S., Manis, J. P., Gu, Y. S., Patel, P., Bronson, R., Appella, E., Alt, F. W., Chua, K. F. 2003; 100 (19): 10794-10799

  Abstract

  SIRT1 is a mammalian homolog of the Saccharomyces cerevisiae chromatin silencing factor Sir2. Dominant-negative and overexpression studies have implicated a role for SIRT1 in deacetylating the p53 tumor suppressor protein to dampen apoptotic and cellular senescence pathways. To elucidate SIRT1 function in normal cells, we used gene-targeted mutation to generate mice that express either a mutant SIRT1 protein that lacks part of the catalytic domain or has no detectable SIRT1 protein at all. Both types of SIRT1 mutant mice and cells had essentially the same phenotypes. SIRT1 mutant mice were small, and exhibited notable developmental defects of the retina and heart, and only infrequently survived postnatally. Moreover, SIRT1-deficient cells exhibited p53 hyperacetylation after DNA damage and increased ionizing radiation-induced thymocyte apoptosis. In SIRT1-deficient embryonic fibroblasts, however, p53 hyperacetylation after DNA damage was not accompanied by increased p21 protein induction or DNA damage sensitivity. Together, our observations provide direct evidence that endogenous SIRT1 protein regulates p53 acetylation and p53-dependent apoptosis, and show that the function of this enzyme is required for specific developmental processes.

  View details for DOI 10.1073/pnas.1934713100

  View details for Web of Science ID 000185415300041

  View details for PubMedID 12960381

• Histone H2AX: A dosage-dependent suppressor of oncogenic translocations and tumors CELL Bassing, C. H., Suh, H., Ferguson, D. O., Chua, K. F., Manis, J., Eckersdorff, M., Gleason, M., Bronson, R., Lee, C., Alt, F. W. 2003; 114 (3): 359-370

  Abstract

  We employed gene targeting to study H2AX, a histone variant phosphorylated in chromatin surrounding DNA double-strand breaks. Mice deficient for both H2AX and p53 (H(delta/delta)P(-/-)) rapidly developed immature T and B lymphomas and solid tumors. Moreover, H2AX haploinsufficiency caused genomic instability in normal cells and, on a p53-deficient background, early onset of various tumors including more mature B lymphomas. Most H2AX(delta/delta)p53(-/-) or H2AX(+/delta)p53(-/-) B lineage lymphomas harbored chromosome 12 (IgH)/15 (c-myc) translocations with hallmarks of either aberrant V(D)J or class switch recombination. In contrast, H2AX(delta/delta)p53(-/-) thymic lymphomas had clonal translocations that did not involve antigen receptor loci and which likely occurred during cellular expansion. Thus, H2AX helps prevent aberrant repair of both programmed and general DNA breakage and, thereby, functions as a dosage-dependent suppressor of genomic instability and tumors in mice. Notably, H2AX maps to a cytogenetic region frequently altered in human cancers, possibly implicating similar functions in man.

  View details for Web of Science ID 000184679200012

  View details for PubMedID 12914700

• The influence of transcriptional orientation on endogenous switch region function NATURE IMMUNOLOGY Shinkura, R., Tian, M., Smith, M., Chua, K., Fujiwara, Y., Alt, F. W. 2003; 4 (5): 435-441

  Abstract

  Immunoglobulin heavy chain (IgH) class switch recombination (CSR) takes place between large switch (S) regions that precede exons of the constant region. The precise functions of the S region are controversial, although transcription of the S region targets CSR. We have tested the effects of deletion, inversion and replacement of the endogenous 12-kilobase S(gamma1) region on CSR in vivo. Here we show that S(gamma1) is required for CSR, that CSR is effected by a 1-kilobase sequence that generates a G-rich transcript, and that inversion of S(gamma1) or the G-rich sequence decreases CSR. We conclude that S(gamma1) function is dependent on orientation and lacks an absolute requirement for common S region motifs. We propose that single-stranded DNA stabilized by transcription-dependent, higher order structures is a primary substrate of CSR.

  View details for DOI 10.1038/ni918

  View details for Web of Science ID 000182665400010

  View details for PubMedID 12679811

• Transcription-targeted DNA deamination by the AID antibody diversification enzyme NATURE Chaudhuri, J., Tian, M., Khuong, C., Chua, K., Pinaud, E., Alt, F. W. 2003; 422 (6933): 726-730

  Abstract

  Activation-induced cytidine deaminase (AID), which is specific to B lymphocytes, is required for class switch recombination (CSR)--a process mediating isotype switching of immunoglobulin--and somatic hypermutation--the introduction of many point mutations into the immunoglobulin variable region genes. It has been suggested that AID may function as an RNA-editing enzyme or as a cytidine deaminase on DNA. However, the precise enzymatic activity of AID has not been assessed in previous studies. Similarly, although transcription of the target immunoglobulin locus sequences is required for both CSR and somatic hypermutation, the precise role of transcription has remained speculative. Here we use two different assays to demonstrate that AID can deaminate specifically cytidines on single-stranded (ss)DNA but not double-stranded (ds)DNA substrates in vitro. However, dsDNA can be deaminated by AID in vitro when the reaction is coupled to transcription. Moreover, a synthetic dsDNA sequence, which targets CSR in vivo in a manner dependent on transcriptional orientation, was deaminated by AID in vitro with the same transcriptional-orientation-dependence as observed for endogenous CSR. We conclude that transcription targets the DNA deamination activity of AID to dsDNA by generating secondary structures that provide ssDNA substrates.

  View details for DOI 10.1038/nature01574

  View details for Web of Science ID 000182272300043

  View details for PubMedID 12692563

• Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bassing, C. H., Chua, K. F., Sekiguchi, J., Suh, H., Whitlow, S. R., Fleming, J. C., Monroe, B. C., Ciccone, D. N., Yan, C., Vlasakova, K., Livingston, D. M., Ferguson, D. O., Scully, R., Alt, F. W. 2002; 99 (12): 8173-8178

  Abstract

  In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form gamma-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX(Delta)/Delta) mouse embryonic stem (ES) cells. H2AX(Delta)/Delta ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX(Delta)/Delta ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX(Delta)/Delta ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability.

  View details for DOI 10.1073/pnas.122228699

  View details for Web of Science ID 000176217700068

  View details for PubMedID 12034884

• The function of AID in somatic mutation and class switch recombination: upstream or downstream of DNA breaks. journal of experimental medicine Chua, K. F., Alt, F. W., Manis, J. P. 2002; 195 (9): F37-41

  View details for PubMedID 11994429

  View details for PubMedCentralID PMC2193706

• An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing MOLECULAR AND CELLULAR BIOLOGY Chua, K., Reed, R. 2001; 21 (5): 1509-1514

  Abstract

  Specific mechanisms must exist to ensure fidelity in selecting the AG dinucleotide that functions as the 3' splice site during the second transesterification step of splicing. Here we show that the optimal location for this AG is within a narrow distance (19 to 23 nucleotides [nt]) downstream from the branch point sequence (BPS). Contrary to previous expectations, AGs located less than 23 nt from the BPS are always recognized, even when a second AG located more optimally downstream is used in the transesterification reaction. Indeed, the AG closest to the BPS actually dictates the precise location of the AG that engages in the reaction. This mechanism, in which the AG is identified by a general localization step followed by a precise localization step, may be used to achieve fidelity while allowing flexibility in the location of 3' splice sites.

  View details for Web of Science ID 000166942700009

  View details for PubMedID 11238888

• The RNA splicing factor hSlu7 is required for correct 3 ' splice-site choice NATURE Chua, K., Reed, R. 1999; 402 (6758): 207-210

  Abstract

  The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.

  View details for Web of Science ID 000083716400057

  View details for PubMedID 10647016

• Human step II splicing factor hSlu7 functions in restructuring the spliceosome between the catalytic steps of splicing GENES & DEVELOPMENT Chua, K., Reed, R. 1999; 13 (7): 841-850

  Abstract

  The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.

  View details for Web of Science ID 000079692000009

  View details for PubMedID 10197984

• Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells MOLECULAR AND CELLULAR BIOLOGY Seghezzi, W., Chua, K., Shanahan, F., Gozani, O., Reed, R., Lees, E. 1998; 18 (8): 4526-4536

  Abstract

  Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

  View details for Web of Science ID 000074950000013

  View details for PubMedID 9671462

• Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis GENES & DEVELOPMENT Wang, C. Y., Chua, K., Seghezzi, W., Lees, E., Gozani, O., Reed, R. 1998; 12 (10): 1409-1414

  Abstract

  The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

  View details for Web of Science ID 000073793500003

  View details for PubMedID 9585501

• The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC CELL Berglund, J. A., Chua, K., Abovich, N., Reed, R., Rosbash, M. 1997; 89 (5): 781-787

  Abstract

  The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.

  View details for Web of Science ID A1997XB92500015

  View details for PubMedID 9182766