Associate Professor, Medicine - Endocrinology, Gerontology, & Metabolism
Member, Stanford Cancer Institute
Associate Director, Medical Scientist Training Program (2014 - Present)
Honors & Awards
Sackler Scholar in Psychobiology, Harvard University (1995-1996)
Fellow of the Jane Coffin Childs Memorial Fund For Medical Research, Jane Coffin Childs Memorial Fund For Medical Research (2001-2002)
Pfizer Postdoctoral Fellow in Rheumatology/Immunology, Pfizer (2002-2005)
Paul Beeson Scholar in Aging Research, National Institute on Aging/American Federation for Aging Research (2006-)
Ellison Medical Foundation New Scholar in Aging, Ellison Medical Foundation/AFAR (2008-2012)
Ph.D., Harvard Medical School, Neuroscience/Cell Biology (2001)
M.D., Harvard Medical School, Medicine (2001)
B.A., Harvard University, Biochemistry/Molecular Biology (1991)
Current Research and Scholarly Interests
Our lab is interested in understanding molecular processes that underlie aging and age-associated pathologies in mammals. We focus on a family of genes, the SIRTs, which regulate stress resistance and lifespan in lower organisms such as yeast, worms, and flies. In mammals, we recently uncovered a number of ways in which SIRT factors may contribute to cellular and organismal aging by regulating resistance to various forms of stress. We have now begun to characterize the molecular mechanisms by which these SIRT factors function. In particular, we are interested in how SIRT factors regulate chromatin, the molecular structure in which the DNA of mammalian genomes is packaged, and how such functions may link genome maintenance to stress resistance and aging.
Independent Studies (8)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr)
- Graduate Research
CBIO 399 (Aut, Win, Spr)
- Graduate Research
MED 399 (Aut, Win, Spr)
- Medical Scholars Research
MED 370 (Aut, Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
MED 199 (Aut, Win, Spr)
- Directed Reading in Cancer Biology
Graduate and Fellowship Programs
Molecular Pathways: Emerging Roles of Mammalian Sirtuin SIRT7 in Cancer
CLINICAL CANCER RESEARCH
2014; 20 (7): 1741-1746
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 Web of Science ID 000333900000008
View details for PubMedID 24536059
- CANCER Metabolism in the driver's seat NATURE 2012; 492 (7429): 362-363
SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation
2012; 487 (7405): 114-?
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
SIRT6 is required for maintenance of telomere position effect in human cells
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
SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span
2009; 136 (1): 62-74
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 Web of Science ID 000262318400015
View details for PubMedID 19135889
SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin
2008; 452 (7186): 492-U16
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 Web of Science ID 000254341300036
View details for PubMedID 18337721
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.
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 2015; 22 (4): 275-277
SIRT7 Represses Myc Activity to Suppress ER Stress and Prevent Fatty Liver Disease
2013; 5 (3): 654-665
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
- Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling SCIENTIFIC REPORTS 2013; 3
The Role of SIRT6 Protein in Aging and Reprogramming of Human Induced Pluripotent Stem Cells
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (25): 18439-18447
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 Web of Science ID 000320721900046
View details for PubMedID 23653361
A General Molecular Affinity Strategy for Global Detection and Proteomic Analysis of Lysine Methylation
2013; 50 (3): 444-456
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 Web of Science ID 000319183500015
Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling.
2013; 3: 3085-?
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.
View details for DOI 10.1038/srep03085
View details for PubMedID 24169447
Finding a Target for Resveratrol
2012; 148 (3): 387-389
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-1?.
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
2011; 12 (1): 29-U47
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-?B 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-?B-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-? (PKC-?) 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
Chromatin regulation and genome maintenance by mammalian SIRT6
TRENDS IN BIOCHEMICAL SCIENCES
2011; 36 (1): 39-46
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
2010; 131 (3): 185-192
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
- Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6 CELL CYCLE 2009; 8 (16): 2664-2666
- SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair Aging 2009; 1: 109-121
Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally
MOLECULAR AND CELLULAR BIOLOGY
2008; 28 (5): 1688-1701
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
2006; 442 (7098): 96-99
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
Genomic instability and aging-like phenotype in the absence of mammalian SIRT6
2006; 124 (2): 315-329
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
2005; 120 (4): 497-512
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
2004; 303 (5666): 2011-2015
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
2003; 100 (19): 10794-10799
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
2003; 114 (3): 359-370
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
2003; 4 (5): 435-441
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
2003; 422 (6933): 726-730
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
2002; 99 (12): 8173-8178
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
2002; 195 (9): F37-41
View details for PubMedID 11994429
An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing
MOLECULAR AND CELLULAR BIOLOGY
2001; 21 (5): 1509-1514
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
1999; 402 (6758): 207-210
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
1999; 13 (7): 841-850
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
1998; 18 (8): 4526-4536
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
1998; 12 (10): 1409-1414
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
1997; 89 (5): 781-787
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