Telomeric Heterochromatin Research Articles

Novel roles for A-type lamins in telomere biology and the DNA damage response pathway

A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.

Silent chromatin at the middle and ends: lessons from yeasts

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

TERRA: telomeric repeat-containing RNA

Telomeres, the physical ends of eukaryotic chromosomes, consist of tandem arrays of short DNA repeats and a large set of specialized proteins. A recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. TERRA functions that are emerging suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation. The accumulation of TERRA at telomeres can also interfere with telomere replication, leading to a sudden loss of telomere tracts. Such a phenotype can be observed upon impairment of the RNA surveillance machinery or in cells from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, in which TERRA is upregulated because of DNA methylation defects in the subtelomeric region. Thus, TERRA may mediate several crucial functions at the telomeres, a region of the genome that had been considered to be transcriptionally silent.

The middle and the end

Welcome to ‘The Middle & The End’, an EMBO Journal focus review series on centromere and telomere biology. The molecular biology of DNA and chromosomes has always been a centre of attention for readers and contributors of The EMBO Journal alike since its inception 27 years ago. Those years have seen amazing progress in our understanding of these notable chromosomal regions, yet even in the light of most recent advances it is clear that a lot more is yet to be deciphered—figuratively speaking, we are still far from the ‘end’ in terms of learning and understanding them, but we may well be in the very ‘middle’ of things by now. Having a middle and an end is a privilege of eukaryotic chromosomes, which—in contrast to the circular DNA of typical prokaryotes—are usually linear, a feature that endows them with characteristic properties as well as problems. For one, ends need to be protected and maintained, despite the intricacies of semi‐conservative replication and in the face of DNA break surveillance mechanisms liable to attempt their repair. The middle, on the other hand, is commonly the place for spindle microtubule attachment and thus important for chromosome segregation. In light of these peculiarities, centromeres and telomeres have become highly specialized compared with the bulk of eukaryotic chromosomes, which fulfills the more intuitive (and most fundamental) role of DNA, to encode genes for proteins or RNAs and to regulate their expression. This specialization of centromeres and telomeres is reflected at numerous levels, starting with DNA sequence through chromatin structure to the recruitment and function of specialized structural and signalling …

TDP-43 localizes in mRNA transcription and processing sites in mammalian neurons

TDP-43 is a RNA/DNA-binding protein structurally related to nuclear hnRNP proteins. Previous biochemical studies have shown that this nuclear protein plays a role in the regulation of gene transcription, alternative splicing and mRNA stability. Despite the ubiquitous distribution of TDP-43, the growing list of TDP-43 proteinopathies is primarily associated with neurodegenerative disorders. Particularly, TDP-43 redistributes to the cytoplasm and forms pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familiar frontotemporal lobar degeneration. Here, we have studied the nuclear compartmentalization of TDP-43 in normal rat neurons by using light and electron microscopy immunocytochemistry with molecular markers for nuclear compartments, a transcription assay with 5'-fluorouridine, and in situ hybridization for telomeric DNA. TDP-43 is concentrated in euchromatin domains, specifically in perichromatin fibrils, nuclear sites of transcription and cotranscriptional splicing. In these structures, TDP-43 colocalizes with 5'-fluorouridine incorporation sites into nascent pre-mRNA. TDP-43 is absent in transcriptionally silent centromeric and telomeric heterochromatin, as well as in the Cajal body, a transcription free nuclear compartment. Furthermore, a weak TDP-43 immunolabeling is found in nuclear speckles of splicing factors. The specific localization of TDP-43 in active sites of transcription and cotranscriptional splicing is consistent with biochemical data indicating a role of TDP-43 in the regulation of transcription and alternative splicing.

Dicer independent small RNAs associate with telomeric heterochromatin

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are approximately 24 nucleotides in length, Dicer-independent, and 2'-O-methylated at the 3' terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll((-/-))) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2((-/-))), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.

Chromatin structural rearrangement during dedifferentiation of protoplasts of Cucumis sativus L.

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere†

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.

Classification of genes, rearrangements and chromosomes according to the chromosome field.

The study of over 700 species, from algae to humans, reveals that specific DNA sequences, have an optimal territory within the centromere-telomere field. These DNA sequences have maintained their territory within the chromosome field for millions of years irrespective of variation in arm length, of change in chromosome type and of species evolution. Some of these DNA sequences have been isolated biochemically or analysed at the molecular level. They include, e.g., the proximal heterochromatic segments, the genes for ribosomal RNA, and the telomeric heterochromatin. Order prevails in the eukaryotic chromosome. This order allows classification of genes, rearrangements and chromosomes on a genetic basis. Genes are classified as centrons, medons and telons. Rearrangements are classified as: conservative, discordant, disruptive, destructive and incompatible. Chromosomes are also classified depending on their length, arm size and number. These chromosome properties and features now acquire an organizatory meaning which they lacked previously. The available molecular information supports the evidence from the field. The study of the split gene reveals that it is the relative position of the DNA sequences which determines their function. On the basis of the chromosome field, predictions can be made concerning gene organization, gene function and chromosome evolution.

Reconstitution of Yeast Silent Chromatin: Multiple Contact Sites and O-AADPR Binding Load SIR Complexes onto Nucleosomes In Vitro

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Sem1p and Ubp6p orchestrate telomeric silencing by modulating histone H2B ubiquitination and H3 acetylation

Histone covalent modifications and 26S proteasome-mediated proteolysis modulate many regulatory events in eukaryotes. In Saccharomyces cerevisiae, heterochromatin mediates transcriptional silencing at telomeres, HM loci and rDNA array. Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing. Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres. Further, the loss of Sem1p and Ubp6p triggers relocation of silencing factors (e.g. Sir proteins) from telomere to HM loci and rDNA array. Such relocation of silencing factors enhances gene silencing at HM loci and rDNA array, but diminishes telomeric silencing. Interestingly, both Sem1p and Ubp6p participate in the proteolytic function of the proteasome. However, we find that the telomeric silencing is not influenced by proteolysis. Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

Cross-Species Bacterial Artificial Chromosome–Fluorescence in Situ Hybridization Painting of the Tomato and Potato Chromosome 6 Reveals Undescribed Chromosomal Rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC-FISH as a unique tool for comparative genetic studies across Solanum species.

Antigenic Variation inPlasmodium falciparum

The persistence of the human malaria parasite Plasmodium falciparum during blood stage proliferation in its host depends on the successive expression of variant molecules at the surface of infected erythrocytes. This variation is mediated by the differential control of a family of surface molecules termed PfEMP1 encoded by approximately 60 var genes. Each individual parasite expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. PfEMP1/var enables parasitized erythrocytes to adhere within the microvasculature, resulting in severe disease. This review highlights key regulatory mechanisms thought to be critical for monoallelic expression of var genes. Antigenic variation is orchestrated by epigenetic factors including monoallelic var transcription at separate spatial domains at the nuclear periphery, differential histone marks on otherwise identical var genes, and var silencing mediated by telomeric heterochromatin. In addition, controversies surrounding var genetic elements in antigenic variation are discussed.

Involvement of Hat1p (Kat1p) Catalytic Activity and Subcellular Localization in Telomeric Silencing

Previous studies have shown that loss of the type B histone acetyltransferase Hat1p leads to defects in telomeric silencing in Saccharomyces cerevisiae. We used this phenotype to explore a number of functional characteristics of this enzyme. To determine whether the enzymatic activity of Hat1p is necessary for its role in telomeric silencing, a structurally conserved glutamic acid residue (Glu-255) that has been proposed to be the enzymes catalytic base was mutated. Surprisingly neither this residue nor any other acidic residues near the enzymes active site were essential for enzymatic activity. This suggests that Hat1p differs from most histone acetyltransferases in that it does not use an acidic amino acid as a catalytic base. The effects of these Hat1p mutants on enzymatic activity correlated with their effects on telomeric silencing indicating that the ability of Hat1p to acetylate substrates is important for its in vivo function. Despite its presumed role in the acetylation of newly synthesized histones in the cytoplasm, Hat1p was found to be a predominantly nuclear protein. This subcellular localization of Hat1p is important for its in vivo function because a construct that prevents its accumulation in the nucleus caused defects in telomeric silencing similar to those seen with a deletion mutant. Therefore, the presence of catalytically active Hat1p in the cytoplasm is not sufficient to support normal telomeric silencing. Hence both enzymatic activity and nuclear localization are necessary characteristics of Hat1p function in telomeric silencing.

Default Pathway of var2csa Switching and Translational Repression in Plasmodium falciparum

Antigenic variation is a subtle process of fundamental importance to the survival of a microbial pathogen. In Plasmodium falciparum malaria, PfEMP1 is the major variable antigen and adhesin expressed at the surface of the infected erythrocyte, which is encoded for by members of a family of 60 var-genes. Peri-nuclear repositioning and epigenetic mechanisms control their mono-allelic expression. The switching of PfEMP1 depends in part on variable transition rates and short-lived immune responses to shared minor epitopes. Here we show var-genes to switch to a common gene that is highly transcribed, but sparsely translated into PfEMP1 and not expressed at the erythrocyte surface. Highly clonal and adhesive P. falciparum, which expressed distinct var-genes and the corresponding PfEMP1s at onset, were propagated without enrichment or panning. The parasites successively and spontaneously switched to transcribe a shared var-gene (var2csa) matched by the loss of PfEMP1 surface expression and host cell-binding. The var2csa gene repositioned in the peri-nuclear area upon activation, away from the telomeric clusters and heterochromatin to transcribe spliced, full-length RNA. Despite abundant transcripts, the level of intracellular PfEMP1 was low suggesting post-transcriptional mechanisms to partake in protein expression. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression.

Identification and description of distinct B chromosomes in Cyphocharax modestus (Characiformes, Curimatidae)

Cytogenetic analyses were performed in Cyphocharax modestus, collected at Paranapanema River and Tiete River (Sao Paulo State, Brazil). A karyotype with 2n = 54 chromosomes was observed in the animals from both Brazilian freshwater river systems. One to four B chromosomes were also detected in individuals from the Paranapanema River, which represents the probable first report of more than a single supernumerary element in a species of the Curimatidae group. C-banding revealed centromeric and telomeric heterochromatin blocks in several chromosomes of the normal karyotype complement of C. modestus. Moreover, while some B chromosomes were characterized by the complete absence of C-bands, others were totally heterochromatic. Although there was a prevalence of B chromosomes in males of C. modestus, at least one supernumerary element was found in males and/or females of several other populations of the species, which suggests that the presence of these chromosomes seems to represent a general trait of C. modestus. A possible origin of the described B chromosomes may be related to the occurrence of a chromosome non-disjunction followed by the loss of euchromatic segments, an event that should have occurred in chromosomes that present conspicuous centromeric heterochromatic blocks and even in chromosomes that lack C-bands in this region, resulting in small supernumerary elements.

Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II

Mammalian telomeres consist of non-coding TTAGGG repeats that are bound by the multi-protein complex 'shelterin', thus protecting chromosome ends from DNA repair mechanisms and degradation. Mammalian telomeric chromatin is enriched for the constitutive heterochromatin marks H3K9me3, H4K20me3 and HP1 (refs 2, 3, 4, 5, 6, 7). Similar to pericentric heterochromatin, telomeric heterochromatin is thought to be fundamental for the maintenance of chromosomal integrity. Here, we report that telomeric repeats are transcribed by DNA-dependent RNA polymerase II, which, in turn, interacts with the TRF1 shelterin protein. Telomeric RNAs (TelRNAs) contain UUAGGG repeats, are polyadenylated and are transcribed from the telomeric C-rich strand. Transcription of mammalian telomeres is regulated by several mechanisms, including developmental status, telomere length, cellular stress, tumour stage and chromatin structure. Using RNA-flourescent in situ hybridization (FISH), we show that TelRNAs are novel structural components of telomeric chromatin. Importantly, we provide evidence that TelRNAs block the activity of telomerase in vitro, suggesting that TelRNAs may regulate telomerase activity at chromosome ends. Our results indicate that TelRNAs are novel components of mammalian telomeres, which are anticipated to be fundamental for understanding telomere biology and telomere-related diseases, such as cancer and ageing.

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3′-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.

Interplay of Chromatin Modifiers on a Short Basic Patch of Histone H4 Tail Defines the Boundary of Telomeric Heterochromatin

Dot1 (Disruptor of telomeric silencing-1) is a histone H3 lysine 79 methyltransferase that contributes to the establishment of heterochromatin boundary and has been linked to transcription elongation. We found that histone H4 N-terminal domain, unlike other histone tails, interacts with Dot1 and is essential for H3 K79 methylation. Furthermore, we show that the heterochromatin protein Sir3 inhibits Dot1-mediated methylation and that this inhibition is dependent on lysine 16 of H4. Sir3 and Dot1 bind the same short basic patch of histone H4 tail, and Sir3 also associates with the residues surrounding H3 K79 in a methylation-sensitive manner. Thus, Sir3 and Dot1 compete for the same molecular target on chromatin. ChIP analyses support a model in which acetylation of H4 lysine 16 displaces Sir3, allowing Dot1 to bind and methylate H3 lysine 79, which in turn further blocks Sir3 binding/spreading. This draws a detailed picture of the succession of molecular events occurring during the establishment of telomeric heterochromatin boundaries.

Sir2 Deacetylates Histone H3 Lysine 56 to Regulate Telomeric Heterochromatin Structure in Yeast

At telomeric heterochromatin in yeast, the Sir protein complex spreads from Rap1 sites to silence adjacent genes. This cascade is believed to occur when Sir2, an NAD(+)-dependent enzyme, deacetylates histone H3 and H4 N termini, in particular histone H4 K16, enabling more Sir protein binding. Lysine 56 of histone H3 is located at the entry-exit points of the DNA superhelix surrounding the nucleosome, where it may control DNA compaction. We have found that K56 substitutions disrupt silencing severely without decreasing Sir protein binding at the telomere. Our in vitro and in vivo data indicate that Sir2 deacetylates K56 directly in telomeric heterochromatin to compact chromatin and prevent access to RNA polymerase and ectopic bacterial dam methylase. Since the spread of Sir proteins is necessary but not sufficient for silencing, we propose that silencing occurs when Sir2 deacetylates H3 K56 to close the nucleosomal entry-exit gates, enabling compaction of heterochromatin.