Silent Loci Research Articles

DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells

Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate tumor metastasis suppressor gene. Our study was to determine whether DNA hypermethylation and histone modification at the NGX6 gene promoter play important roles in silencing NGX6 expression in gastric cancer. NGX6 expression was downregulated in all gastric cancer cells and 76.19 % tissues. In three GC cell lines, hypermethylated NGX6 loci were characterized by histone H3-K9 hypoacetylation and hypermethylation. Trichostatin A treatment could moderately increase H3-K9 acetylation at the silenced loci; however, it had no effect on DNA and H3-K9 methylation and minimal effects on NGX6 expression. In contrast, 5'aza-2'-deoxycytidine treatment could rapidly decrease DNA and H3-K9 methylation at the silenced loci, leading to the reexpression of NGX6. Combined treatment with 5'aza-2'-deoxycytidine and trichostatin A had synergistic effects on the reexpression of NGX6 at the hypermethylation loci. Our current study shows that NGX6 expression is downregulated in GC cancer cells and tissues due to NGX6 promoter methylation and H3-K9 methylation, but not H3-K9 acetylation. Our findings indicate that the downregulation of NGX6 expression contributes to the development and progression of gastric cancer. More studies are needed to determine the precise mechanism of NGX6 in the progression of gastric cancer.

Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1

Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.

How to Choose the Right Mate

How to Choose the Right Mate

Complex structures of transgene rearrangement implicate novel mechanisms of RNA-directed DNA methylation and convergent transcription

An RNAi construct for silencing FtsZ gene functioning on plastid division in higher plants was transformed into tobacco genome. Sequencing of the flanking sequences of the T-DNA insertion site in a positive transgenic plant 2-3, which showed wildtype phenotype, revealed that the construct had rearranged, resulting in convergent transcription of nptII gene by 35S promoter and nos promoter with both transcripts expressing at low level. Although it is possible that the sense and antisense transcripts can form dsRNA, they did not trigger silencing of nptII gene derived from a FtsZ silenced plant 2-12 in the crossed plant 2-3×2-12. Furthermore, although small interfering RNA can trigger DNA methylation of the silencing locus, our investigation revealed that the crossed plant 2-3×2-12 showed partial methylation of the non-silencing transgene locus but full methylation of the non-rearranged silencing locus, suggesting that the efficiency of RNA-directed DNA methylation is associated with its original DNA locus.

Long-range heterochromatin association is mediated by silencing and double-strand DNA break repair proteins

The eukaryotic genome is highly organized in the nucleus, and this organization affects various nuclear processes. However, the molecular details of higher-order organization of chromatin remain obscure. In the present study, we show that the Saccharomyces cerevisiae silenced loci HML and HMR cluster in three-dimensional space throughout the cell cycle and independently of the telomeres. Long-range HML-HMR interactions require the homologous recombination (HR) repair pathway and phosphorylated H2A (γ-H2A). γ-H2A is constitutively present at silenced loci in unperturbed cells, its localization requires heterochromatin, and it is restricted to the silenced domain by the transfer DNA boundary element. SMC proteins and Scc2 localize to the silenced domain, and Scc2 binding requires the presence of γ-H2A. These findings illustrate a novel pathway for heterochromatin organization and suggest a role for HR repair proteins in genomic organization.

266. Gene Targeting at Silent Loci with Adeno-Associated Virus Vectors

266. Gene Targeting at Silent Loci with Adeno-Associated Virus Vectors

CHANGES IN THE GENOME-WIDE LOCALIZATION PATTERN OF SIR3 IN SACCHAROMYCES CEREVISIAE DURING DIFFERENT GROWTH STAGES

In budding yeast, the Sir2, Sir3 and Sir4 proteins form SIR complexes, required for the assembly of silent heterochromatin domains, and which mediate transcription silencing at the telomeres as well as at silent mating type loci. In this study, under fluorescence microscopy, we found most Sir3-GFP expressions in the logarithmic phase cells appeared as multiple punctations as expected. However, some differences in the distribution of fluorescent signals were detected in the diauxic∼early stationary phase cells. To clarify these, we then used ChIP on chip assays to investigate the genome-wide localization of Sir3. In general, Sir3 binds to all 32 telomere proximal regions, the silent mating type loci and also binds to the rDNA region. However, the genome-wide localization patterns of Sir3 are different between these two distinct growth phases. We also confirmed that Sir3 binds to a recently identified secondary binding site, PAU genes, and further identified 349 Sir3-associated cluster regions. These results provide additional support in roles for Sir3 in the modulation of gene expression during physical conditions such as diauxic∼early stationary phase growing. Moreover, they imply that Sir3 may be not only involved in the formation of conventional silent heterochromatin, but also able to associate with some other chromatin regions involved in epigenetic regulation.

Comparative study of the promotion of porcine fetal fibroblast proliferation by overexpression of two transcriptional variants of SIRT6

Mammalian Sirtuin 6 (SIRT6) plays an important role in the transcriptional silencing of silent mating-type loci, telomeres, and rDNA. However, the roles of porcine SIRT6 in cell proliferation are poorly understood, and a better knowledge of these will help improve our understanding of the biological mechanisms of cell growth and development. In this study, a novel variant of porcine SIRT6 (SIRT6 V2) identified by reverse transcription-polymerase chain reaction and BLAST analysis showed a 124-bp deletion compared to wild-type SIRT6 mRNA (SIRT6 V1). Two recombinant plasmids overexpressing SIRT6 V1 and SIRT6 V2 were produced and their roles in the proliferation of porcine fetal fibroblasts (PFFs) were compared. Cells transfected with SIRT6 V1 proliferated significantly faster than those transfected with SIRT6 V2 (P<0.01), though both proliferated significantly faster than cells expressing an empty vector (P<0.01). These results might be caused by altered proportions of α helices and β sheets in the SIRT6 V2 structure relative to SIRT6 V1. These results indicate that overexpression of SIRT6 V1/V2 was involved in promoting PFF proliferation. Deletion of a 124-bp sequence attenuated the effects of SIRT6 on cell proliferation, possibly as a result of changes in the proportions of α helices and β sheets in the protein secondary structure.

Neisseria gonorrhoeae RecQ Helicase HRDC Domains Are Essential for Efficient Binding and Unwinding of the pilE Guanine Quartet Structure Required for Pilin Antigenic Variation

The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.

Mating type specific chromosome conformation in Saccharomyces cerevisiae

Budding yeast switch their mating type by a gene conversion event at the MAT locus which uses either of two silent loci (HML or HMR) on opposite ends of chromosome three as a template. In MATa cells the left arm of Chr. Ill is “activated” which allows for the preferential recombination of HML with the MAT locus. The left arm is otherwise “repressed” for recombination in MATα cells which then prefer to use HMR, on the right arm, as a template for gene conversion. We set out to analyze the potential role of chromosome conformation in this “activation”/”repression” phenomenon observed on the left arm of Chr. III. We used Chromosome Conformation Capture Carbon Copy (5C) to comprehensively analyze the conformation of chromosomes III, V, and XII in the two mating types. Our data reveals that the yeast genome is organized in a unique way compared to other species. We have found that global nuclearorganization such ascentromereclustering, telomere tethering to the periphery, and sequestration of the rDNA array into the nucleolus affect both the specific conformations of each chromosome but also the interactions between these chromosomes. Our analysis indicates that the overall architecture for these 3 chromosomes is very similar between the two mating types. Interestingly, a mating type specific difference in conformation of the left arm of Chr. Ill was identified. Furthermore, the 5C data was used, in conjunction with the Integrative Modeling Platform (IMP), to generate three dimensional models of Chr. III in both mating types. This method provides a more intuitive way of viewing 5C data and reveals that, in general, Chr. Ill has a more crumpled conformation in MATa cells than in MATα. However, this crumpling is most evident on the left arm of the chromosome. Thus the phenomenon of “activation”/”repression” of the left arm of Chr. III which is associated with mating type-specific switching preference is, in fact, associated with a difference in the innate conformation of Chr. Ill between the two mating types. This difference in structure between mating types will be used as a phenotype to analyze the effect of cis and trans acting factors that play a role in switching preference through alteration of chromosome conformation.

Sirtuin 1-mediated Effects of Exercise and Resveratrol on Mitochondrial Biogenesis

The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.

Arabidopsis HIT4 encodes a novel chromocentre-localized protein involved in the heat reactivation of transcriptionally silent loci and is essential for heat tolerance in plants

The Arabidopsis mutant heat-intolerant 4-1 (hit4-1) was isolated from an ethyl methanesulphonate-mutagenized M2 population on the basis of its inability to withstand prolonged heat stress (4 days at 37°C). Further characterization indicated that hit4-1 was impaired specifically in terms of basal but not acquired thermotolerance. Map-based cloning revealed that the HIT4 gene encoded a plant-specific protein for which the molecular function has yet to be studied. To investigate the cellular role of HIT4 and hence elucidate better its protective function in heat tolerance in plants, a GFP-HIT4 reporter construct was created for a protoplast transient expression assay. Results showed that fluorescently tagged HIT4 was localized to the chromocentre, a condensed heterochromatin domain that harbours repetitive elements for which transcription is normally suppressed by transcriptional gene silencing (TGS). DAPI-staining analysis and FISH with a probe that targeted centromeric repeats showed that heat-induced chromocentre decondensation was inhibited in nuclei of hit4-1 subjected to direct heat treatment, but not in those that were allowed to acquire thermotolerance. Moreover, heat reactivation of various TGS loci, regardless of whether they were endogenous or transgenic, or existed as a single copy or as repeats, was found to be attenuated in hit4-1. Meanwhile, the levels of transcripts of heat shock protein genes in response to heat stress were similar in both hit4-1 and wild-type plants. Collectively, these results demonstrated that HIT4 defines a new TGS regulator that acts at the level of heterochromatin organization and is essential for basal thermotolerance in plants.

Generation of an HIV Resistant T-cell Line by Targeted “Stacking” of Restriction Factors

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

Chromatin looping and organization at developmentally regulated gene loci

Developmentally regulated genes are often controlled by distant enhancers, silencers and insulators, to implement their correct transcriptional programs. In recent years, the development of 3C and derived techniques (4C, 5C, HiC, ChIA-PET, etc.) has confirmed that chromatin looping is an important mechanism for the transfer of regulatory information in mammalian cells. At many developmentally regulated gene loci, transcriptional activation is indeed accompanied by the formation of chromatin loops between genes and distant enhancers. Similarly, dynamic looping between insulator elements and changes in local 3D organization may be observed upon variation in transcriptional activity. Chromatin looping also occurs at silent gene loci, where its function remains less understood. In lineage-committed cells, partial 3D configurations are detected at loci that are activated at later stages. However, these partial configurations usually lack promoter-enhancer loops that accompany transcriptional activation, suggesting they have structural functions. Definitive evidence for a repressive role of chromatin looping is still lacking. Chromatin loops have been reported at repressed loci but, alternatively, they may act as a distraction for active loops. Together, these mechanisms allow fine-tuning of regulatory programs, thus providing further diversity in the transcriptional control of developmentally regulated gene loci.

Enforcement of a lifespan‐sustaining distribution of Sir2 between telomeres, mating‐type loci, and rDNA repeats by Rif1

Telomere dysfunction is linked with genome instability and premature aging. Roles for sirtuin proteins at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin histone deacetylase Sir2 to telomeres. Here we show that Rif1 maintains replicative lifespan by ultimately stabilizing another age-related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating-type loci, while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) of rDNA. This is accompanied by a disruption of IGS1 silent chromatin assembly and increases in aberrant recombination within rDNA repeats. Lifespan defects linked with Rif1 deletion are abolished if rDNA repeats are forcibly stabilized via deletion of the replication fork-blocking protein Fob1. In addition, Sir2 overexpression prevents Rif1 deletion from disrupting Sir2 at IGS1 and shortening lifespan. Moreover, subjecting cells lacking Rif1 to caloric restriction increases IGS1 histone deacetylation and lifespan, while uncovering novel genetic interactions between RIF1 and SIR2. Our data indicate that Rif1 maintains lifespan-sustaining levels of Sir2 at rDNA by preventing excessive recruitment of the histone deacetylase to telomeric and silent mating-type loci. As sirtuin histone deacetylases, such as Sir2 or mammalian SIRT6, each operate at multiple age-related loci, we propose that factors limiting the localization of sirtuins to certain age-related loci can promote lifespan-sustaining roles of these sirtuins elsewhere in the genome.

Mechanism for epigenetic variegation of gene expression at yeast telomeric heterochromatin.

Yeast contains heterochromatin at telomeres and the silent mating-type loci (HML/HMR). Genes positioned within the telomeric heterochromatin of Saccharomyces cerevisiae switch stochastically between epigenetically bistable ON and OFF expression states. Important aspects of the mechanism of variegated gene expression, including the chromatin structure of the natural ON state and the mechanism by which it is maintained, are unknown. To address this issue, we developed approaches to select cells in the ON and OFF states. We found by chromatin immunoprecipitation (ChIP) that natural ON telomeres are associated with Rap1 binding and, surprisingly, also contain known characteristics of OFF telomeres, including significant amounts of Sir3 and H4K16 deacetylated nucleosomes. Moreover, we found that H3K79 methylation (H3K79me), H3K4me, and H3K36me, which are depleted from OFF telomeres, are enriched at ON telomeres. We demonstrate in vitro that H3K79me, but not H3K4me or H3K36me, disrupts transcriptional silencing. Importantly, H3K79me does not significantly reduce Sir complex binding in vivo or in vitro. Finally, we show that maintenance of H3K79me at ON telomeres is dependent on transcription. Therefore, although Sir proteins are required for silencing, we propose that epigenetic variegation of telomeric gene expression is due to the bistable enrichment/depletion of H3K79me and not the fluctuation in the amount of Sir protein binding to nucleosomes.

Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo

Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

Schizosaccharomyces pombe reporter strains for relative quantitative assessment of heterochromatin silencing

The fission yeast Schizosaccharomyces pombe has been emerging as an important model organism for studying the formation and repression of heterochromatin. To enable simple and relative quantitative assessment of heterochromatin silencing, we have created bioluminescence-based reporter strains. A green-emitting click beetle luciferase (CBG68) gene was inserted within pericentromeric heterochromatin or at the silent mating-type locus via homologous recombination. In the same strains, a red-emitting click beetle luciferase (CBR) gene is expressed from the euchromatic leu1(+) locus and can be used as a reference in dual-colour assays. Our reporter strains are suitable for performing Chroma-Glo™ assays, which can be carried out directly in the culture medium without prior cell lysis and in a multiwell format. Our reporter system reliably reflects the state of chromatin and can be easily adapted for use in high-throughput screening approaches.

Silencing of the Il2 gene transcription is regulated by epigenetic changes in anergic T cells

Anergy is induced in T cells as a consequence of a partial or suboptimal stimulation. Anergic T cells become unresponsive and fail to proliferate and produce cytokines. We had previously shown that in anergic CD4(+) T cells, Ikaros participates in the transcriptional repression of the Il2 gene by recruiting histone deacetylases that cause core histone deacetylation at the Il2 promoter. Here we show that deacetylation at the Il2 promoter is the initial step in a process that leads to the stable silencing of the Il2 gene transcription in anergic T cells. We have found that anergy-induced deacetylation of the Il2 promoter permits binding of the histone methyl-transferase Suv39H1, which trimethylates lysine-9 of histone H3 (Me3H3-K9). Furthermore, the establishment of the Me3H3-K9 mark allows the recruitment of the heterochromatin protein HP1, allowing the silenced Il2 loci to reposition close to heterochromatin-rich regions. Our results indicate that silencing of Il2 transcription in anergic T cells is attained through a series of epigenetic changes that involve the establishment of repressive marks and the subsequent nuclear repositioning of the Il2 loci, which become juxtaposed to transcriptionally silent regions. This mechanism may account for the stable nature of the inhibition of IL-2 production in anergic cells.