Genomic Silencing Research Articles

Disruption of HDAC/CoREST/REST repressor by dnREST reduces genome silencing and increases virulence of herpes simplex virus

In nonneuronal cells, herpes simplex virus 1 overcomes host defenses, replicates, and ultimately kills the infected cell. Among the host defenses suppressed by the virus is a repressor complex whose key components are histone deacetylase (HDAC) 1 or 2, RE-1 silencing transcription factor (REST), corepressor of REST (CoREST), and lysine-specific demethylase (LSD) 1. In neurons innervating cells at the portal of entry into the body, the virus establishes a "latent" infection in which viral DNA is silenced with the exception of a family of genes. The question posed here is whether the virus hijacks this repressor complex to silence itself in neurons during the latent state. To test this hypothesis, we inserted into the wild-type virus genome a wild-type REST [recombinant (R) 111], a dominant-negative REST (dnREST) lacking the N- and C-terminal repressor domains (R112), or an insertion control consisting of tandem repeats of stop codons (R113). The recombinant virus R112 carrying the dnREST replicated better and was more virulent than the wild-type parent or the other recombinant viruses when administered by the corneal or i.p. routes. Moreover, in contrast to other recombinants, corneal route inoculation by R112 recombinant virus resulted in higher DNA copy numbers, higher levels of infectious virus in eye, trigeminal ganglion, or brain, and virtually complete destruction of trigeminal ganglia in mice that may ultimately succumb to infection. These results support an earlier conclusion that the HDAC/CoREST/REST/LSD1 repressor complex is a significant component of the host innate immunity and are consistent with the hypothesis that HSV-1 hijacks the repressor to silence itself during latent infection.

Hyperphosphorylation of Histone Deacetylase 2 by Alphaherpesvirus US3 Kinases

A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.

DNA Replication Factor C1 Mediates Genomic Stability and Transcriptional Gene Silencing inArabidopsis

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylation-independent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana.

Abstract 5003: Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma

Abstract Deleted in liver cancer 1 (DLC1), which encodes a Rho GTPase activity protein (RhoGAP), is a bona fide tumor suppressor not only in hepatocellular carcinoma, but in a wide spectrum of human cancers. Underexpression of DLC1 is commonly found in human cancers and has been attributed to genomic deletion and epigenetic silencing. Recently, mutation of DLC1 identified from cancer patients is shown to inactivate its RhoGAP activity. However, the regulatory mechanism of the tumor suppressive activities of DLC1 is unclear. Recent study about the regulation of activity and compartmentalization of DLC1 by protein kinases has provided the first evidence that DLC1 activity could be regulated by post-translational modification. Moreover, identification of rat homolog of DLC1, p122RhoGAP, as the substrate of Akt has provided insight into the potential regulatory pathway of DLC1. However, the functional significance Akt phosphorylation in p122RhoGAP and its relevance in human DLC1 have not been investigated. In this study, we aimed to elucidate the molecular mechanism by which Akt phosphorylates DLC1 and the functional impact of this phosphorylation on DLC1 activities. In this study, we have shown that Akt interacts with the C-terminus of DLC1 and only Akt with intact kinase activity can interact and phosphorylate DLC1. Although this newly identified residue is distinct from the reported Akt phosphorylated residue in rat p122RhoGAP, it is well conserved in DLC family. Indeed, another family member, DLC2 is also phosphorylated by Akt. This implicates a common regulatory mechanism of DLC family by Akt signaling pathway. To determine the functional significance of phosphorylation of DLC1 by Akt, site-directed mutagenesis is employed to create phosphomimetic and phosphodefective mutant of DLC1. In vitro functional assays have demonstrated that phosphomimetic DLC1 suppresses growth, colony formation and anchorage independent growth of transformed hepatoblasts. Moreover, using subcutaneous injection and orthotopic tumor implantation models, phosphomimetic DLC1 mutant loses its inhibitory activities in tumorigenesis and metastasis of transformed hepatoblasts in nude mice. In conclusion, our findings have identified a novel post-translational modification by which Akt phosphorylation functionally deregulates the tumor suppressive activities of DLC1. Herein we show that apart from underexpression and mutation of DLC1, the enhanced phosphorylation level of DLC1 could be an indicator of deregulated DLC1 in cancers with normal expression level of DLC1. [This work was supported by the Hong Kong Research Grants Council (HKU7798/07M) and RGC Collaborative Research Grants (HKU 1/06C)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5003.

Sir2-dependent asymmetric segregation of damaged proteins in ubp10 null mutants is independent of genomic silencing

Carbonylation of proteins is an irreversible oxidative damage that increases during both chronological and replicative yeast aging. In the latter, a spatial protein quality control system that relies on Sir2 is responsible for the asymmetrical damage segregation in the mother cells. Proper localization of Sir2 on chromatin depends on the deubiquitinating enzyme Ubp10, whose loss of function deeply affects the recombination and gene-silencing activities specific to Sir2. Here, we have analyzed the effects of SIR2 and UBP10 inactivations on carbonylated protein patterns obtained in two aging models such as stationary phase cells and size-selected old mother ones. In line with the endogenous situation of higher oxidative stress resulting from UBP10 inactivation, an increase of protein carbonylation has been found in the ubp10Δ stationary phase cells compared with sir2Δ ones. Moreover, Calorie Restriction had a salutary effect for both mutants by reducing carbonylated proteins accumulation. Remarkably, in the replicative aging model, whereas SIR2 inactivation resulted in a failure to establish damage asymmetry, the Sir2-dependent damage inheritance is maintained in the ubp10Δ mutant which copes with the increased oxidative damage by retaining it in the mother cells. This indicates that both Ubp10 and a correct association of Sir2 with the silenced chromatin are not necessary in such a process but also suggests that additional Sir2 activities on non-chromatin substrates are involved in the establishment of damage asymmetry.

The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates

The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome- and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2Delta cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

Impact of tumour genotyping on cancer management

Genetic, epigenetic and transcriptomic analyses, hastily conduced for over 20 years, have greatly improved our understanding and classification of tumours and the bulk of accrued information is really overwhelming, since more than 118,000 articles appear in an online search focused on ‘genetic and neoplasms’. The interest of tumour genotyping was originally linked to disclosure of causes of tumours development and evolution, but slowly evidence emerged of how genomic sub-classifications could be established, and of their prognostic and therapeutic interest, particularly in the subset of tailored therapies. The holistic concept of cancer has been subsidized and characterization of genetic features of single cases is gradually emerging as the area of interest both, for research and clinical application. This evolution involved the development of several techniques, such as microarray-based gene expression profiling and comparative genomic hybridization technologies, translational silencing, gene sequencing and transfer, microRNA profiling and epigenetic silencing of tumour suppressor genes. A crucial point of this field of genetic studies on cancer can be referred to the seminal study on breast cancer by the Stanford group [1], which led to the identification of five molecular groups. The related classification is now widely accepted and show definite clinical implications [2]. A similar approach has now been applied to tumours of different sites, particularly of soft tissues, lung, colon, brain and haematopoietic system [3–6]. Time is ripe for affording the question of the present role and impact of molecular genotyping on the clinical management of tumours, and so the present Review Series is aiming to provide the status of the art. Well-known experts will give our update on the impact of tumour genotyping on the management of different tumours, understandably starting from the breast. Further reviews will follow, dedicated to tumour from other sites. Clinical management of cancer patients is still mainly based on a morphological basis, that is histopathological typing and staging supported with additional data from morphology-based procedure as immunohistochemistry and fluorescent in situ hybridization. It is an easy prediction that genetic analysis will not substitute, but complement pathological data and open new prospects. For pathologists, this should represent a challenge and a stimulus [7].

Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis

Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.

Genome-wide analysis of histone H3 lysine 27 trimethylation by ChIP-chip in gastric cancer patients

Trimethylation of histone H3 lysine 27 (H3K27me3) is a posttranslational modification that is highly correlated with genomic silencing. In gastric cancer (GC), global and gene-specific DNA methylation changes have been demonstrated to occur. However, to date, our understanding of the alterations in H3K27me3 in GC is incomplete. This study aimed to investigate the variations in H3K27me3 in CpG island regions between gastric cancerous and matched non-cancerous tissues. H3K27me3 variations were analyzed in eight pairs of GC and adjacent normal tissues, from eight GC patients, using a chromatin immunoprecipitation linked to the microarray (ChIP-chip) approach. ChIP-real time PCR was used to validate the microrray results. In addition, DNA methylation status also was further analyzed by methyl-DNA immunoprecipitation quantitative PCR. One hundred twenty-eight (119 increased and 9 decreased H3K27me3) genes displaying significant H3K27me3 differences were found between GC and adjacent normal tissues. The results of ChIP-real time PCR coincided well with those of microarray. Aberrant DNA methylation can also be found on selected randomly positive genes (MMP15, UNC5B, SHH, AFF3, and RB1). Our study indicates that there are significant alterations of H3K27me3 in gastric cancerous tissues, which may help clarify the molecular mechanisms involved in the pathogenesis of GC. Such novel findings show the significance of H3K27me3 as a potential biomarker or promising target for epigenetic-based GC therapies.

Involvement of SIRT7 in resumption of rDNA transcription at the exit from mitosis

Sirtuins, also designated class III histone deacetylases, are implicated in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing and longevity. The nucleolar Sirtuin7 (SIRT7) was reported to be involved in the regulation of ribosomal gene (rDNA) transcription, but there are no data concerning the regulation of SIRT7 during the cell cycle. Here we have analyzed the behavior of endogenous SIRT7 during mitosis, while rDNA transcription is repressed. SIRT7 remains associated with nucleolar organizer regions, as does the RNA polymerase I machinery. SIRT7 directly interacts with the rDNA transcription factor UBF. Moreover, SIRT7 is phosphorylated via the CDK1-cyclin B pathway during mitosis and dephosphorylated by a phosphatase sensitive to okadaic acid at the exit from mitosis before onset of rDNA transcription. Interestingly, dephosphorylation events induce a conformational modification of the carboxy-terminal region of SIRT7 before the release of mitotic repression of rDNA transcription. As SIRT7 activity is required to resume rDNA transcription in telophase, we propose that this conformational modification regulates onset of rDNA transcription.

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

BackgroundEstablishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.ResultsOur results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.ConclusionThese results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.

Demethylation of H3K27 Regulates Polycomb Recruitment and H2A Ubiquitination

Methylation of histone H3 lysine 27 (H3K27) is a posttranslational modification that is highly correlated with genomic silencing. Here we show that human UTX, a member of the Jumonji C family of proteins, is a di- and trimethyl H3K27 demethylase. UTX occupies the promoters of HOX gene clusters and regulates their transcriptional output by modulating the recruitment of polycomb repressive complex 1 and the monoubiquitination of histone H2A. Moreover, UTX associates with mixed-lineage leukemia (MLL) 2/3 complexes, and during retinoic acid signaling events, the recruitment of the UTX complex to HOX genes results in H3K27 demethylation and a concomitant methylation of H3K4. Our results suggest a concerted mechanism for transcriptional activation in which cycles of H3K4 methylation by MLL2/3 are linked with the demethylation of H3K27 through UTX.

Mitotic Regulation of SIRT2 by Cyclin-dependent Kinase 1-dependent Phosphorylation

Sirtuins are evolutionarily conserved NAD(+)-dependent deacetylases and ADP-ribosyltransferases involved in the regulation of cell division, apoptosis, DNA damage repair, genomic silencing, and longevity. Recent studies have focused on identifying target substrates for human sirtuin enzymatic activity, but little is known about processes that directly regulate their function. Here, we demonstrate that SIRT2 is phosphorylated both in vitro and in vivo on serine 368 by the cell-cycle regulator, cyclin-dependent kinase 1, and dephosphorylated by the phosphatases CDC14A and CDC14B. Overexpression of SIRT2 mediates a delay in cellular proliferation that is dependent on serine 368 phosphorylation. Furthermore, mutation of serine 368 reduces hyperploidy in cells under mitotic stress due to microtubule poisons.

The Transitioning Germ Line

![Figure][1] CREDIT: GETTY IMAGES/VISUALS UNLIMITED Germline constancy is a must for species continuity, but the life of the germ cell itself is full of change. The trek to and from the germ line includes numerous transitions and decisions deftly choreographed for the gametes' unique ability in nature to develop into a complex animal and pass on genetic information. This special section highlights the transitioning germ line and otherwise expands on intriguing aspects of these cells of heredity. In the early embryo, cells decide between becoming soma or germ line. Mechanisms protect germ cells from a somatic fate, just as somatic cells require insulation from the germline differentiation pathway. Strome and Lehmann (p. [392][2]) outline activities in the fly and worm, such as transcriptional quiescence and inheritance of germ cell determinants, and Hayashi et al. (p. [394][3]) tell of the murine system, invoking an inductive signal. Although model systems use different strategies, common themes exist. A new class of molecules reportedly participates in germ cell development. Lin (p. [397][4]) describes the generation and role of germline-specific small RNAs termed piRNAs. Functions as diverse as posttranscriptional regulation and epigenetic programming are suggested. Another epigenetic event—de novo DNA methylation—silences imprinted genes and repeat elements during vertebrate gametogenesis. Schaefer et al. (p. [398][5]) detail this heritable silencing mechanism and its sexual dimorphism. As the germ cell transitions to the mature form, more decisions are made: to enter mitosis or meiosis and to differentiate as sperm or egg, with possible linkage of these decisions as proposed by Kimble and Page (p. 400). Stem cell self-renewal versus differentiation in the Drosophila male and female germ lines is discussed by Fuller and Spradling (p. [402][6]). Switching to vertebrate germline stem cells, Brinster (p. [404][7]) reports methods for the recovery, culture, and transplantation of spermatogonial stem cells; and Daley (p. 409) summarizes advances in embryonic stem cell (ESC) differentiation to a germline fate: procedures with research and clinical applications but not without limitations. In an Editorial, McLaren (p. [339][8]) takes the discussion further, highlighting ethical and social issues surrounding ESC-derived germ cells. Changes continue as the oocyte transitions to the fertilized egg. Stitzel and Seydoux (p. 407) detail the oocyte-to-zygote transition, with associated alterations in protein synthesis, protein and RNA degradation, and organelle remodeling; and Schier (p. 406) discusses zygotic genome silencing and maternal messenger RNA degradation around the maternal-zygotic transition. In News, Travis (p. 390) discusses “urbisexuality” with a biologist pondering the evolution of germ cell specification, and Leslie (p. 388) details how oocyte freezing has become more effective and acceptable despite questions about the long-term health of offspring. Finally, in [ Science 's STKE][9], McClure discusses how poppies reject self-pollen, and Boldajipour and Raz highlight similarities in Drosophila and mouse mechanisms involved in the migration of primordial germ cells and elimination of ectopic cells. The articles here give but a sampling of exciting ongoing research on the seeds of life—themselves products of change. Advances bear fruit in understanding development and its underlying genetics, as well as infertility. [1]: pending:yes [2]: /lookup/doi/10.1126/science.1140846 [3]: /lookup/doi/10.1126/science.1137545 [4]: /lookup/doi/10.1126/science.1137543 [5]: /lookup/doi/10.1126/science.1137544 [6]: /lookup/doi/10.1126/science.1140861 [7]: /lookup/doi/10.1126/science.1137741 [8]: /lookup/doi/10.1126/science.1142267 [9]: http://www.sciencemag.org/sciext/germcells/#section_in-stke

No evidence for parental imprinting of mouse 22q11 gene orthologs

Non-Mendelian factors may influence central nervous system (CNS) phenotypes in patients with 22q11 Deletion Syndrome (22q11DS, also known as DiGeorge or Velocardiofacial Syndrome), and similar mechanisms may operate in mice carrying a deletion of one or more 22q11 gene orthologs. Accordingly, we examined the influence of parent of origin on expression of 25 murine 22q11 orthologs in the developing and mature CNS using single nucleotide polymorphism (SNP)-based analysis in interspecific crosses and quantification of mRNA in a murine model of 22q11DS. We found no evidence for absolute genomic imprinting or silencing. All 25 genes are biallelically expressed in the developing and adult brains. Furthermore, if more subtle forms of allelic biasing are present, they are very small in magnitude and most likely beyond the resolution of currently available quantitative approaches. Given the high degree of similarity of human 22q11 and the orthologous region of mmChr16, genomic imprinting most likely cannot explain apparent parent-of-origin effects in 22q11DS.

Conserved domains control heterochromatin localization and silencing properties of SU(VAR)3–7

The Drosophila protein SU(VAR)3-7 is essential for fly viability, chromosome structure, and heterochromatin formation. We report that searches in silico and in vitro for homologues of SU(VAR)3-7 were successful within, but not outside, the Drosophila genus. Protein sequence homology between the distant sibling species Drosophila melanogaster and Drosophila virilis is low, except for the general organization of the protein and three conserved motives: seven widely spaced zinc fingers in the N-terminal half and the BESS and BoxA motives in the C-terminal half of the protein. We have undertaken a fine functional dissection of SU(VAR)3-7 in vivo using transgenes encoding truncations of the protein. BESS mediates interaction of SU(VAR)3-7 with itself, and BoxA is required for specific heterochromatin association. Both are necessary for the silencing properties of SU(VAR)3-7. The seven zinc fingers, widely spaced over the N-terminal half of SU(VAR)3-7, are required for binding to polytene chromosomes. One finger is necessary and sufficient to determine the appropriate chromatin association of the C-terminal half of the protein. Conferring a function to each of the conserved motives allows us to better understand the mode of action of SU(VAR)3-7 in triggering heterochromatin formation and subsequent genomic silencing.

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, we observe a specific bloating of the male X chromosome, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose.

DNA Methylation, Genomic Silencing, and Links to Nutrition and Cancer

DNA methylation is a heritable epigenetic feature that is associated with transcriptional silencing, X-chromosome inactivation, genetic imprinting, and genomic stability. The addition of the methyl group is catalyzed by a family of DNA methyltransferases whose cosubstrates are DNA and S-adenosylmethionine, the latter being derived from the methionine cycle. Aberrant DNA methylation is linked to numerous pathologies, including cancer. The purpose of this review is to describe DNA methylation and its functions, to examine the relationship between dietary methyl insufficiency and DNA methylation, and to evaluate the associations between DNA methylation and cancer.

DNA Methylation, Genomic Silencing, and Links to Nutrition and Cancer

DNA methylation is a heritable epigenetic feature that is associated with transcriptional silencing, X-chromosome inactivation, genetic imprinting, and genomic stability. The addition of the methyl group is catalyzed by a family of DNA methyltransferases whose co-substrates are DNA and S-adenosylmethionine, the latter being derived from the methionine cycle. Aberrant DNA methylation is linked to numerous pathologies, including cancer. The purpose of this review is to describe DNA methylation and its functions, to examine the relationship between dietary methyl insufficiency and DNA methylation, and to evaluate the associations between DNA methylation and cancer.

Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.