Specific Chromatin Structure Research Articles

Mammalian telomeres and telomerase: why they matter for cancer and aging

Chromosome ends, or telomeres, are formed by a special chromatin structure that protects them from recombination and degradation, thus preventing end-to-end chromosome fusions and other chromosomal aberrations. The functionality of telomeres, and that of the cellular activity that synthesizes them, telomerase, has been shown to impact on both cancer and aging, as well as on the organismal sensitivity to ionizing radiation. This review focuses on the analysis of different mouse models for proteins that are important for telomere function, which have highlighted the importance of telomeres and telomerase for cancer and aging.

Balance between Acetylation and Methylation of Histone H3 Lysine 9 on the E2F-Responsive Dihydrofolate Reductase Promoter

Epigenetic marks that specify silent heterochromatic domains in eucaryotic genomes include methylation of histone H3 lysine 9. Strikingly, active loci in the vicinity of silent domains are sometimes characterized by acetylation of histone H3 lysine 9, suggesting that the balance between these two competitive modifications is important for the establishment of specific chromatin structures. Some euchromatic genes, targeted by the retinoblastoma protein Rb, are also believed to be regulated by histone H3 lysine 9 methylation. Here, we study the dihydrofolate reductase promoter, which is repressed in G0 and at the beginning of G1 by p107 or p130, two Rb-related proteins. We found that these two pocket proteins share with Rb the ability to associate with the histone methyl transferase SUV39H1. SUV39H1 can be recruited to the E2F transcription factor and functions as a transcriptional corepressor. With ChIP assays followed by real-time PCR, we showed that K9 of histone H3 evolves from a hypermethylated state in G0 to a hyperacetylated state at the G1/S transition. Taken together, these results indicate that the temporal regulation of euchromatic promoters may involve controlling the balance between methylation and acetylation of histone H3 lysine 9, a feature previously described for the spatial regulation of chromatin function.

Chromatin proteins are determinants of centromere function.

Recent advances in the identification of molecular components of centromeres have demonstrated a crucial role for chromatin proteins in determining both centromere identity and the stability of kinetochore-microtubule attachments. Although we are far from a complete understanding of the establishment and propagation of centromeres, this review seeks to highlight the contribution of histones, histone deposition factors, histone modifying enzymes, and heterochromatin proteins to the assembly of this sophisticated, highly specialized chromatin structure. First, an overview of DNA sequence elements at centromeric regions will be presented. We will then discuss the contribution of chromatin to kinetochore function in budding yeast, and pericentric heterochromatin domains in other eukaryotic systems. We will conclude with discussion of specialized nucleosomes that direct kinetochore assembly and propagation of centromere-defining chromatin domains.

RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast.

The regulation of higher-order chromosome structure is central to cell division and sexual reproduction. Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis. In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region. In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis. We find that the RNAi machinery is required for the accurate segregation of chromosomes. Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres. Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells. Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase. This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis.

Modifying Gene Expression Programs by Altering Core Promoter Chromatin Architecture

Transcriptional activation of the IFN-β gene in response to virus infection requires the assembly of an enhanceosome, which instructs a recruitment program of chromatin modifiers/remodelers and general transcription factors to the promoter. This program culminates with sliding of a nucleosome blocking the core promoter to a downstream position, a prerequisite for transcriptional activation. We show that delivery of this nucleosome to the same downstream position to create an accessible IFN-β core promoter prior to enhanceosome assembly results in major changes in the gene expression program with regard to the temporal pattern and the signal specificity of the transcriptional response. Thus, the identity of a gene expression program is achieved and maintained by the dynamic interplay between specific enhanceosomes and specific local chromatin structure.

Transcriptional silencing in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Transcriptional silencing is a heritable form of gene inactivation that involves the assembly of large regions of DNA into a specialized chromatin structure that inhibits transcription. This phenomenon is responsible for inhibiting transcription at silent mating-type loci, telomeres and rDNA repeats in both budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe, as well as at centromeres in fission yeast. Although transcriptional silencing in both S.cerevisiae and S.pombe involves modification of chromatin, no apparent amino acid sequence similarities have been reported between the proteins involved in establishment and maintenance of silent chromatin in these two distantly related yeasts. Silencing in S.cerevisiae is mediated by Sir2p-containing complexes, whereas silencing in S.pombe is mediated primarily by Swi6-containing complexes. The Swi6 complexes of S.pombe contain proteins closely related to their counterparts in higher eukaryotes, but have no apparent orthologs in S.cerevisiae. Silencing proteins from both yeasts are also actively involved in other chromosome-related nuclear functions, including DNA repair and the regulation of chromatin structure.

Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae.

Budding yeast centromeres are comprised of approximately 125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes. We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin. The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing. cacDelta hirDelta double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner. Further, cacDelta and hirDelta deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure. Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures.

Methyl CpG-binding proteins and transcriptional repression.

Since its discovery, methylation of DNA in mammalian cells has been correlated with transcriptional repression and with specialized chromatin structures. Recently, considerable progress has been reported in the identification of protein factors with a highly conserved DNA interaction surface, termed the methyl CpG-binding domain or MBD. A subset has been biochemically linked to histone deacetylases, suggesting a molecular mechanism for the functional properties of methylated DNA. Despite several obvious attractions, the connection between MBD proteins and histone deacetylases fails to explain all the existing data. In fact, the biochemistry and DNA-binding properties of most MBD family members have not been adequately described and considerable evidence exists for alternative mechanisms in the repression of methylated loci. Null mutations have been generated in mice for several MBD family members, the phenotypes of the mutant animals raise important questions regarding the functions of the MBD family. Here, I review the biochemistry, DNA-binding properties, and genetics of the MBD proteins that are linked to transcriptional repression, namely, MeCP2, MBD1, MBD2, and MBD3. Several models to account for the functional properties of methylated DNA are presented.

Histone variant H2A.Z is required for early mammalian development

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1° have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

Centromeres

The centromere is the chromosomal site which associates with spindle microtubules and is responsible for equal division of the chromosomes to both daughter cells. Centromeres can often be recognised cytologically as a distinctive narrow region on metaphase chromosomes known as the primary constriction. The terms ‘centromere’ and ‘kinetochore’ are often used synonymously but it is best to consider the centromere as the DNA and chromatin on which the kinetochore is assembled and the kinetochore as the protein machine, including centromeric chromatin, which mediates attachment to the spindle. Centromeres are found near the centre of the chromosome on metacentric chromosomes, but they can also be submetacentric, between the middle and an end; acrocentric, close to an end; and telocentric, at an end. Some organisms such as Caenorhabditis elegans are holocentric and assemble centromeric chromatin and kinetochores along the entire chromosome. Delving further you discover that centromeres are eccentric! The centromere region is also a site of strong cohesion; in higher eukaryotes it is the last point of contact between sister-chromatids at metaphase prior to their separation at anaphase. Cohesion between sister centromeres is intimately associated with kinetochore function as it counteracts forces of microtubule–kinetochore attachment and possibly aids orientation and attachment of sister kinetochores to opposite poles. Centromeres are frequently associated with heterochromatin. Genes are silenced if placed in these regions by accident or design. It is not known why these regions are transcriptionally silent and whether silencing itself is important for centromere function. In fission yeast, at least, it appears that the silent state is a by-product of full kinetochore assembly. Centromeres range in size and complexity from a relatively minute 125 bp in budding yeast, through more complex structures of 40–120 kb in fission yeast, to enormous arrays of relatively simple repeats extending for 250 kb in flies and 5000 kb in humans. Regardless of size, they all assemble kinetochores that segregate chromosomes with awe-inspiring fidelity. The number of microtubules per metaphase kinetochore varies from one in budding yeast, 2–4 in fission yeast and from 4 to over 40 in mammals. DNA sequence is not conserved between centromeres of different organisms. However, all active centromeres have a histone H3-like protein, such as CENP-A, which must contribute to the assembly of a specialised centromere specific chromatin structure. Yes and no. Yes for yeast. But it's more complicated for mammalian centromeres. In humans vast arrays of so-called alphoid satellite repeats are found at centromeres, and mammalian artificial chromosomes have been created that contain alphoid DNA. So, you think, alphoid DNA is the determinant of centromeric activity! But in some dicentric chromosomes, both centromeres contain alphoid DNA, but only one is active, as assayed by the presence of important kinetochore components such as CENP-A and CENP-C. And in certain rare cases centromere activity has been gained by regions which have never before acted as centromeres. These neocentromeres contain no alphoid DNA but have been propagated through up to three human generations. These strange goings-on indicate that centromeres are epigenetically regulated. Presumably there is a meta-stable mark that normally ensures that centromeres and kinetochores do assemble at the same place division after division. Epigenetic phenomena also contribute to centromere function in fission yeast and Drosophila, and probably other organisms. Where can I find out more?

Involvement of histone H4 acetylation in the epigenetic inheritance of different activity states of maternally and paternally derived genomes in the mealybug Planococcus citri.

Modification of histones by acetylation is a well-known mechanism for the establishment and maintenance of specific chromatin structures with different activity states. In Planococcus citri males the paternal genome, early in development, becomes mostly inactive and heterochromatic. As we had not found methylation in the genome of P. citri, we analyzed the acetylation state of histone H4. We report here that, in males, differences in the level of histone H4 acetylation are indeed present in the two genomes of different parental origin; these differences were confirmed by treatment with the histone deacetylase inhibitor Trichostatin A. There is also evidence of acetylation of histone H4 on metaphase chromosomes. Our data therefore suggest a role of histone H4 acetylation in the imprinting of the paternal genome in P. citri males, thus supporting a role of modification of chromatin-related structural proteins in the epigenetic transmission of imprinting.

Effects of cis arrangement of chromatin insulators on enhancer-blocking activity.

Chromatin boundary elements or insulators are believed to regulate gene activity in complex genetic loci by organizing specialized chromatin structures. Here, we report that the enhancer-blocking activity of the Drosophila suHw insulator is sensitive to insulator copy number and position. Two tandem copies of suHw were ineffective in blocking various enhancers from a downstream promoter. Moreover, an enhancer was blocked more effectively from a promoter by two flanking suHw insulators than by a single intervening one. Thus, insulators may modulate enhancer-promoter interactions by interacting with each other and facilitating the formation of chromatin loop domains.

The function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku.

The telomerase enzyme lengthens telomeres, an activity essential for chromosome stability in most eukaryotes. The enzyme is composed of a specialized reverse transcriptase and a template RNA. In Saccharomyces cerevisiae, overexpression of TLC1, the telomerase RNA gene, disrupts telomeric structure. The result is both shortened telomere length and loss of a special chromatin structure that normally silences telomere-proximal genes. Because telomerase function is not required for telomeric silencing, we postulated that the dominant-negative effect caused by overexpression of TLC1 RNA originates in a normal interaction between the RNA and an unknown telomeric factor important for silencing; the overexpressed RNA presumably continues to bind the factor and compromises its function. Here we show that a 48-nt stem-loop structure within the 1.3-kb TLC1 RNA is necessary and sufficient for disrupting telomeric silencing and shortening telomeres. Moreover, this short RNA sequence appears to function through an interaction with the conserved DNA end-binding protein Ku. We propose that, in addition to its roles in telomeric silencing, homologous recombination and non-homologous end-joining (NHEJ), S. cerevisiae Ku also helps to recruit or activate telomerase at the telomere through an interaction with this stem-loop of TLC1 RNA.

A Histone Variant, Htz1p, and a Sir1p-like Protein, Esc2p, Mediate Silencing at HMR

Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.

Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+).

Precisely positioned nucleosomes are not essential for c- fos gene regulation in vivo

Chromatin architecture plays a decisive role in many aspects of transcription regulation. We have tested the role of specific chromatin structures in c- fos gene regulation, using a gene transfer system based on episomes derived from the Epstein–Barr virus (EBV). This system reproduces in several respects the chromatin structure and regulation of the chromosomal c- fos gene. Using this approach, we first demonstrate that the pausing of RNA polymerase II downstream of the transcriptional start site does not require precisely positioned nucleosomes. Indeed, changing the pattern of MNase hypersensitive sites along the transcribed sequence does not perturb RNA polymerase II pausing or the regulation of the c- fos gene. Next, we show that a putative nucleosome positioned between the SIE/SRE elements (−300) and the CRE/TATA elements (−36) is not necessary for activation by a variety of inducers. Accordingly, total or partial deletion of the putative nucleosome sequence does not disturb c- fos regulation while the two regulatory sites flanking the nucleosome sequence remain hypersensitive to MNase. As described in this paper, EBV episomes are useful vectors to critically examine the role of the chromatin structure in gene transcription for human cells.

Forkhead genes in transcriptional silencing, cell morphology and the cell cycle. Overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae.

The SIR1 gene is one of four specialized genes in Saccharomyces cerevisiae required for repressing transcription at the silent mating-type cassettes, HMLalpha and HMRa, by a mechanism known as silencing. Silencing requires the assembly of a specialized chromatin structure analogous to heterochromatin. FKH1 was isolated as a gene that, when expressed in multiple copies, could substitute for the function of SIR1 in silencing HMRa. FKH1 (Forkhead Homologue One) was named for its homology to the forkhead family of eukaryotic transcription factors classified on the basis of a conserved DNA binding domain. Deletion of FKH1 caused a defect in silencing HMRa, indicating that FKH1 has a positive role in silencing. Significantly, deletion of both FKH1 and its closest homologue in yeast, FKH2, caused a form of yeast pseudohyphal growth, indicating that the two genes have redundant functions in controlling yeast cell morphology. By several criteria, fkh1Delta fkh2Delta-induced pseudohyphal growth was distinct from the nutritionally induced form of pseudohyphal growth observed in some strains of S. cerevisiae. Although FKH2 is redundant with FKH1 in controlling pseudohyphal growth, the two genes have different functions in silencing HMRa. High-copy expression of CLB2, a G2/M-phase cyclin, prevented fkh1Delta fkh2Delta-induced pseudohyphal growth and modulated some of the fkhDelta-induced silencing phenotypes. Interestingly, deletions in either FKH1 or FKH2 alone caused subtle but opposite effects on cell-cycle progression and CLB2 mRNA expression, consistent with a role for each of these genes in modulating the cell cycle and having opposing effects on silencing. The differences between Fkh1p and Fkh2p in vivo were not attributable to differences in their DNA binding domains.

Transcriptional Repression Mediated by Polycomb Group Proteins and Other Chromatin-associated Repressors Is Selectively Blocked by Insulators

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

Neocentric activity of rye 5RL chromosome in wheat.

The neocentric activity of a constriction located on the long arm of rye 5R chromosome (5RL) was analysed. It is not observed in normal rye but it is unusually stretched in bivalents involving 5RL telosomes in wheat-ditelosomic 5RL addition lines. In 20% of metaphase I cells, the 5RL bivalent presents the centromeres oriented to one pole and the constrictions oriented towards the opposite pole with a strong tension. In 5% of the cells, the constriction was able to orient the bivalent to the poles without tension in the centromeres. Sister chromatid cohesion, which is one of the distinct features of centromeric function, is persistent at the constriction in delayed 5RL chromosomes at anaphase I. Neither the elongation of the constriction nor the neocentric activity was observed at second meiotic division or mitosis. FISH studies showed that the 5RL constriction lacked detectable quantities of two repetitive DNA sequences, CCS1 and the 180-bp knob repeat, present at cereal centromeres and neocentromeres, respectively. We propose that, under special conditions, such as the wheat background, the normally non-centromeric DNA present at this region of 5RL acquires a specific chromatin structure, differentiated as an elongated constriction, which is able to function as a centromere.

The topological organization of chromosomes 9 and 22 in cell nuclei has a determinative role in the induction of t(9,22) translocations and in the pathogenesis of t(9,22) leukemias

The neighborhood relationships of chromosomes can be of great importance for basic cellular processes such as gene expression or translocation induction. In this study, the topological organization of chromosomes 9 and 22 was investigated in cell nuclei of G0-phase lymphocytes. We found that the territories of both chromosomes are predominantly located in the central region of cell nuclei. In addition to this, chromosomes 9 and 22 were frequently associated in pairs detected as false-positive ABL-BCR fusions. Both effects might substantially increase the probability of interaction between chromosomes. Because of this, exchange aberrations were studied in chromosomes 9 and 22 of human lymphocytes irradiated by neutrons. The rate of aberration induction between these chromosomes was 11 times higher than the expected frequency based on the fractional molecular weight of these chromosomes. We show that the increased rate of exchange between chromosomes 9 and 22 induced by neutrons corresponds to the neighborhood relationships of both chromosomes. Similar topological characteristics of ABL and BCR genes were found in several cell lines: T- and B-lymphocytes. HL60 cells and bone marrow cells. This finding suggests that the specific chromatin structure mentioned might be responsible for the high rate of induction of t(9;22)-positive leukemias in the human population.