SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span.

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.

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair.

The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor.

SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin.

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.

ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression.

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.

Upregulation of the KIAA1199 gene is associated with cellular mortality.

The microcell-mediated transfer of a normal human chromosome 3 induces replicative senescence in otherwise immortal renal cell carcinoma cells. To identify the genes involved in the chromosome 3-induced cellular mortality, we previously performed a cDNA subtraction experiment using the immortal renal cell carcinoma cells (RCC23) and the mortal counterpart with the transferred chromosome 3 (RCC23+3). We here report the cDNA cloning and characterization of one of the differentially expressed genes, which encodes KIAA1199 protein of unknown function. Northern blot and RT-PCR analyses revealed striking upregulation of KIAA1199 mRNA in mortal RCC23+3 compared with immortal RCC23. However, no significant change in KIAA1199 mRNA expression was observed during replicative aging in vitro (from early passage culture to senescent culture) of mortal human cells including RCC23+3, normal fibroblasts and prostate epithelial cells. Interestingly, an immortal fibroblast cell line and two breast cancer cell lines expressed much lower amounts of KIAA1199 mRNA than their normal counterparts. KIAA1199 mRNA is expressed in a wide range of normal human tissues, with the highest level of expression in brain. The gene is located on chromosome band 15q25, where a brain tumor suppressor gene has been mapped. These findings suggest that KIAA1199 gene may play a role in cellular mortality of normal human cells, which counters cell immortalization and carcinogenesis.

Differential cis-regulation of human versus mouse TERT gene expression in vivo: identification of a human-specific repressive element.

In vivo expression of human telomerase is significantly different from that of mouse telomerase. To assess the basis for this difference, a bacterial artificial chromosome clone containing the entire hTERT (human telomerase reverse transcriptase) gene was introduced in mice. In these transgenic mice, expression of the hTERT transgene was similar to that of endogenous hTERT in humans, rather than endogenous mTERT (mouse telomerase reverse transcriptase). In tissues and cells showing a striking difference in expression levels between hTERT in humans and mTERT in mice (i.e., liver, kidney, lung, uterus, and fibroblasts), expression of the hTERT transgene in transgenic mice was repressed, mimicking hTERT in humans. The transcriptional activity of the hTERT promoter was much lower than that of the mTERT promoter in mouse embryonic fibroblasts or human fibroblasts. Mutational analysis of the hTERT and mTERT promoters revealed that a nonconserved GC-box within the hTERT promoter was responsible for the human-specific repression. These results reveal that a difference in cis-regulation of transcription, rather than transacting transcription factors, is critical to species differences in tissue-specific TERT expression. Our data also suggest that the GC-box-mediated, human-specific mechanism for TERT repression is impaired in human cancers. This study represents a detailed characterization of the functional difference in a gene promoter of mice versus humans and provides not only important insight into species-specific regulation of telomerase and telomeres but also an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression.

Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

Transcriptional activation of hTERT through the NF-kappaB pathway in HTLV-I-transformed cells.

In immortal cells, the existence of a mechanism for the maintenance of telomere length is critical. In most cases this is achieved by the reactivation of telomerase, a cellular reverse transcriptase that prevents telomere shortening. Here we report that the telomerase gene (hTERT) promoter is up-regulated during transmission of human T-cell lymphotropic virus type-I (HTLV-I) to primary T cells in vitro and in ex vivo adult T-cell leukemia/lymphoma (ATLL) samples, but not asymptomatic carriers. Although Tax impaired induction of human telomerase reverse transcriptase (hTERT) mRNA in response to mitogenic stimulation, transduction of Tax into primary lymphocytes was sufficient to activate and maintain telomerase expression and telomere length when cultured in the absence of any exogenous stimulation. Transient transfection assays revealed that Tax stimulates the hTERT promoter through the nuclear factor kappaB (NF-kappaB) pathway. Consistently, Tax mutants inactive for NF-kappaB activation could not activate the hTERT or sustain telomere length in transduced primary lymphocytes. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays suggested that an increased binding of c-Myc and Sp1 is involved in the NF-kappaB-mediated activation of the hTERT promoter. This study establishes the role of Tax in regulation of telomerase expression, which may cooperate with other functions of Tax to promote HTLV-I-associated adult T-cell leukemia.

Regulation of hTERT transcription: a target of cellular and viral mechanisms for immortalization and carcinogenesis.

A hallmark of human cancer cells is immortal cell growth, which is associated with telomere maintenance by telomerase. The transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is a major mechanism that negatively and positively controls telomerase activity in normal and cancer cells, respectively. A growing body of data suggests that various cellular and viral factors and pathways involved in cell senescence, immortalization and carcinogenesis act on the hTERT promoter. The activity of the hTERT promoter is regulated, either directly or through signaling pathways, by oncogene products (e.g., Myc and Ets families) and tumor suppressor proteins (e.g., BRCA1). Endogenous factors involved in the physiological repression of the hTERT gene have also been revealed by chromosome transfer experiments. The integration of viral genomes in the hTERT locus can lead to hTERT activation and telomerase induction. Here, we summarize these findings and pay special attention to recent findings with relevance to the endogenous regulatory mechanisms of hTERT transcription.

The human MYOD1 transgene is suppressed by 5-bromodeoxyuridine in mouse myoblasts.

5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT-PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a transgene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.

Changes in nuclear matrix proteins during the senescence-like phenomenon induced by 5-chlorodeoxyuridine in HeLa cells.

5-Chlorodeoxyuridine (CldU) immediately induces a senescence-like phenomenon in any type of mammalian cells probably due to a change in nuclear matrix structure. We thus examined nuclear matrix proteins in HeLa cells cultured with CldU by high-resolution two-dimensional gel electrophoresis and peptide mass spectrometry. Three proteins were found to be down-regulated and one protein up-regulated by addition of CldU. In addition, one acidic protein accumulated in the nuclear matrix, although not quantitatively changed in the nuclei. Since these alterations were observed within 24h after addition of CldU, these proteins may be involved in an early step of the senescence-like phenomenon.

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells.

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells.

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.

Binding of 5-bromouracil-containing S/MAR DNA to the nuclear matrix.

Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis.