Showing papers on "Heterochromatin published in 1998"

Mating-type gene switching in Saccharomyces cerevisiae

Abstract: ▪ Abstract Saccharomyces cerevisiae can change its mating type as often as every generation by a highly choreographed, site-specific recombination event that replaces one MAT allele with different DNA sequences encoding the opposite allele. The study of this process has yielded important insights into the control of cell lineage, the silencing of gene expression, and the formation of heterochromatin, as well as the molecular events of double-strand break-induced recombination. In addition, MAT switching provides a remarkable example of a small locus control region—the Recombination Enhancer—that controls recombination along an entire chromosome arm.

SET domain proteins modulate chromatin domains in eu- and heterochromatin.

Abstract: The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin – a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).

Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation

Abstract: The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.

The Human Polycomb Group Complex Associates with Pericentromeric Heterochromatin to Form a Novel Nuclear Domain

Abstract: The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins— RING1, BMI1, and hPc2—we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG–heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

Regulation of X-Chromosome Inactivation in Development in Mice and Humans

Abstract: Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females. X-chromosome inactivation (XCI) occurs during development, coupled with cell differentiation. In somatic cells, XCI is random, whereas in extraembryonic tissues, XCI is imprinted in that the paternally inherited X chromosome is preferentially inactivated. Inactivation is initiated from an X-linked locus, the X-inactivation center (Xic), and inactivity spreads along the chromosome toward both ends. XCI is established by complex mechanisms, including DNA methylation, heterochromatinization, and late replication. Once established, inactivity is stably maintained in subsequent cell generations. The function of an X-linked regulatory gene, Xist, is critically involved in XCI. The Xist gene maps to the Xic, it is transcribed only from the inactive X chromosome, and the Xist RNA associates with the inactive X chromosome in the nucleus. Investigations with Xist-containing transgenes and with deletions of the Xist gene have shown that the Xist gene is required in cis for XCI. Regulation of XCI is therefore accomplished through regulation of Xist. Transcription of the Xist gene is itself regulated by DNA methylation. Hence, the differential methylation of the Xist gene observed in sperm and eggs and its recognition by protein binding constitute the most likely mechanism regulating imprinted preferential expression of the paternal allele in preimplantation embryos and imprinted paternal XCI in extraembryonic tissues. This article reviews the mechanisms underlying XCI and recent advances elucidating the functions of the Xist gene in mice and humans.

Hypomethylation of pericentromeric DNA in breast adenocarcinomas

Abstract: Drug-induced DNA demethylation in normal human cells and inherited localized hypomethylation in mitogen-stimulated lymphocytes from patients with a rare recessive disease (ICF: immunodeficiency, centromeric region instability, facial anomalies) are associated with karyotypic instability. This chromosomal recombination is targeted to heterochromatin in the vicinity of the centromere (pericentromeric region) of human chromosome 1. Pericentromeric rearrangements in this chromosome as well as overall genomic hypomethylation are frequently observed in many kinds of cancer, including breast adenocarcinoma. We found that almost half of 25 examined breast adenocarcinomas exhibited hypomethylation in satellite 2 DNA, which is located in the long region of heterochromatin adjacent to the centromere of chromosome 1 and is normally highly methylated. One of the 19 examined non-malignant breast tissues displaying fibrocystic changes was similarly hypomethylated in this satellite DNA. We also looked at an opposing type of methylation alteration in these cancers, namely, hypermethylation in a tumorsuppressor gene region that is frequently hypermethylated in breast cancers. We found that increased methylation in the E-cadherin promoter region and decreased methylation in satellite 2 DNA were often present in the same breast cancers. While hypermethylation in certain tumor-suppressor gene regions may favor tumorigenesis by repressing transcription, demethylation of other DNA sequences may predispose to cancer-promoting chromosomal re-arrangements. Int. J. Cancer 77:833‐838, 1998. r 1998 Wiley-Liss, Inc.

Conspiracy of silence among repeated transgenes

Abstract: Summary Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredictable from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of expression in vertebrate transgene arrays might result from interactions between repeats within the array. A recent experiment using transgenic mouse lines demonstrates that reduction in copy number of silenced transgenes within an array leads to a striking increase in expression, (1) demonstrating that silencing is intrinsic to the array, and is not attributable to position effects of nearby sequences. This work calls into question functions that have been attributed to vertebrate locus control regions and boundaries, and draws attention to the notion that repeat-induced gene silencing is a system for protection of eukaryotic genomes against threatening sequence elements. BioEssays 20:532‐535, 1998. r 1998 John Wiley & Sons, Inc. A bold conjecture ‘‘A heterochromatic segment should arise every time that a minute euchromatic region undergoes repeated reduplication in the genotype and the replicas remain adjacent to each other in the chromosome’’ - Pontecorvo, 1944.(2)

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

Abstract: The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1 Hsα . Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci

Abstract: In differentiated cells, two identical genomic sequences can sometimes be found in two distinct states of expression. For example, in female mammals, one of the two X chromosomes is inactivated, whereas the other remains fully active (Latham 1996). Similarly, chromosomal imprinting ensures that a specific locus, when inherited from one parent, is completely inactive, whereas the same locus inherited from the other parent is completely active (Ferguson-Smith 1996). The imprinted state of the locus is inherited through many mitotic divisions and is generally reset only during meiosis. Inappropriate genomic imprinting can cause serious developmental defects, and several human genetic disorders are caused by mutations affecting imprinted genes (Hall 1990; Lalande 1996). Although the molecular mechanisms by which X inactivation and genomic imprinting are initiated and maintained are not well understood, the inactive X chromosome is in a highly condensed heterochromatic state and a similar chromatin state may occur at silenced, imprinted loci (John and Surani 1996). One of the best studied examples of silencing occurs at the HM loci in the budding yeast Saccharomyces cerevisiae. S. cerevisiae has three mating type loci. Mating type genes expressed from the MAT locus normally determine the yeast mating type, either a or α, in haploid cells. Haploid cells normally respond to the mating pheromone of the opposite mating type by arresting in late G1 and forming mating projections (shmoos). In addition, wild-type strains have functional but transcriptionally repressed mating information at the HM loci, HML and HMR. If the HM loci become derepressed in haploid cells, both a and α mating information is expressed and the cells do not arrest growth or form mating projections in response to mating pheromones. Thus, by monitoring the pheromone response of haploid cells one can infer the expression state of the HM loci. The silent state of the HM loci is attributable to a specialized form of chromatin that is the yeast version of metazoan heterochromatin (Grunstein 1995; Braunstein et al. 1996). Genes within the HM loci are inaccessible to DNA modification enzymes, RNA polymerases II and III, and excision repair enzymes (for review, see Fox and Rine 1996). The acetylation state of histones H3 and H4 in the nucleosomes of silent chromatin is different from that of bulk chromatin or of the active MAT locus; at the HM loci, histone H4 is hypoacetylated except on lysine-12 (Braunstein et al. 1996). This is similar to the acetylation pattern conferred on newly synthesized histone H4 by the cytoplasmic histone acetyltransferase Hat1p (Kleff et al. 1995; Parthun et al. 1996). A number of mutations in acetylated lysines in the amino termini of histones H3 and H4 weaken silencing at the HM loci or at telomeres (for review, see Grunstein 1995). Thus, histone acetylation may play an important role in the inheritance of chromatin expression states. The Sir complex proteins (composed of Sir2p, Sir3p, and Sir4p and not including Sir1p) are structural components of yeast heterochromatin that associate with histones (Hecht et al. 1996; Strahl-Bolsinger et al. 1997). Loss of any one of these Sir complex proteins abrogates silencing completely (Rine and Herskowitz 1987). Sir3p and Sir4p interact with one another genetically (Ivy et al. 1986; Marshall et al. 1987) and in two-hybrid screens (Moretti et al. 1994), and all three Sir complex proteins can be isolated in complexes with each other (Moazed et al. 1997; Strahl-Bolsinger et al. 1997). Histones H3 and H4 coprecipitate with Sir3p (Hecht et al. 1996), and mutations in the amino termini of either H3 or H4 that affect silencing in vivo also affect the interaction of H3 and H4 with the Sir complex in vitro (Hecht et al. 1995). The concentration of Sir complex proteins is critical for silencing. Changes in the stoichiometry of Sir complex proteins alters silencing (Ivy et al. 1986; Marshall et al. 1987; Sussel et al. 1993). The Sir complex proteins localize to a number of perinuclear foci that are often associated with silent telomeric DNA (Gotta et al. 1996). These foci are thought to reflect subnuclear domains of high Sir complex concentration in which silent chromatin is localized (Gotta et al. 1996, 1997). The HM loci and telomeres compete for Sir proteins, and the proximity of the HM loci to telomeres contributes to HM silencing (Buck and Shore 1995; Maillet et al. 1996). The DNA sequences at the HM loci differ from the sequences at the MAT locus in that each HM locus is flanked by two silencers, E and I. Each E or I silencer contains an autonomously replicating (ARS) consensus sequence that is bound by the origin recognition complex (ORC) (Bell et al. 1993). In addition, each silencer contains a binding site for the ARS-binding factor 1 (Abf1p) or a binding site for the repressor/activator protein 1 (Rap1p). The E and I silencers, as well as the individual binding sites and the factors that bind them directly, have redundant functions. In most situations, one silencer is sufficient to silence an HM locus and any two of the three individual sites within a silencer are sufficient for HM silencing (Brand et al. 1987; Mahoney and Broach 1989; McNally and Rine 1991). Specific mutations in the sites (or in the factors that bind them) reduce the redundancy of HMR silencing and can reveal the roles of silencing factors such as Rap1p (Sussel and Shore 1991), ORC (Bell et al. 1993; Micklem et al. 1993; Loo et al. 1995a), and Abf1p (Loo et al. 1995b; Fox et al. 1997). The study of situations in which silencing is weakened, but not abrogated, has provided important insights into the mechanisms by which silencing occurs. sir1 mutants exhibit epigenetic silencing of HML. In a subset of the sir1 cells, HML is fully repressed and the repressed state is inherited in most of their progeny; in the remaining sir1 cells, HML is fully derepressed and the derepressed state is inherited (Pillus and Rine 1989). Sir1p interacts physically with both Orc1p and with Sir4p (Triolo and Sternglanz 1996). Sir1p, when tethered to the HML locus in the absence of a silencer, is sufficient to direct silencing (Chien et al. 1993). Deletion of the ORC-binding site also causes defects in the establishment of silencing, which lead to derepression of the HM loci in a subset of the mutant cells (Mahoney et al. 1991; Sussel et al. 1993). Thus, Sir1p contributes to the establishment of silencing in wild-type cells by interacting with ORC and recruiting structural components of silent chromatin, such as Sir4p, to the silent loci. Pillus and Rine (1989) proposed that there are two steps in HM silencing: (1) maintenance of the current state of the silent chromatin, and (2) re-establishment of the repressed state when HML becomes derepressed. Although deletion of SIR1 and mutation of single sites within the HM loci cause defects in the re-establishment of silencing, they do not affect the ability to inherit the repressed chromatin state (Pillus and Rine 1989; Mahoney et al. 1991). Derepression of HMR (by inactivation of a temperature-sensitive Sir3 protein) can be restored only after passage through S phase (Miller and Nasmyth 1984), indicating that the re-establishment of silencing requires passage through S phase. Conversely, Holmes and Broach (1996) demonstrated that if the cis-silencer is excised from the chromosome, the repressed state of the chromatin can be maintained during α-factor arrest, but cannot be inherited efficiently. Taken together, these studies indicate that the establishment, maintenance, and inheritance of silencing all contribute to the formation of fully silenced HM loci. Mammalian chromatin assembly factor I (CAF-I) was identified by its ability to assemble histones into nucleosomes in a DNA replication-dependent manner in vitro (Stillman 1986). CAF-I assembles preferentially histones H3 and H4 with the acetylation pattern of newly synthesized cytoplasmic histones (Smith and Stillman 1991; Kaufman et al. 1995; Verreault et al. 1996). S. cerevisiae CAF-I is encoded by CAC1, CAC2, and CAC3 (Kaufman et al. 1997). CAC1, the largest subunit of CAF-I, is identical to RLF2, a gene that we identified in a screen for mutants defective in telomere-related functions (Enomoto et al. 1994, 1997), and CAC3 is identical to MSI1, a gene identified in high-copy suppressor screens (Ruggieri et al. 1989; Hubbard et al. 1992). All three cac mutant strains display similar phenotypes; cells grow well but are defective in telomeric silencing, the segregation of TEL + CEN plasmids, and Rap1p localization (Enomoto et al. 1997; Kaufman et al. 1997). Similar phenotypes have been observed in strains carrying mutations in either SIR2, SIR3, or SIR4, in strains carrying mutant alleles of histones H3 and H4, and in strains carrying rap1s mutations (Enomoto et al. 1994). Because many of these genes are involved in HM silencing, as well as telomeric silencing, we examined the role of CAF-I in HM silencing. In this paper we show that CAF-I contributes to the maintenance, but not the re-establishment, of silencing at the HM loci. In cac− mutants, we observed a transient loss of α-factor response, at the individual cell level. Cells form mating projections and divide slowly on α-factor, forming clusters of shmooing cells. The formation of shmoo clusters requires α-mating information at HML, indicating that this α-factor response reflects a defect in the maintenance of HML silencing. We have investigated the relationship between the maintenance and the re-establishment of silencing at HML by analyzing the roles of CAF-I, histones, Sir complex proteins, and Sir1p using α-factor confrontation assays.

Chromatin components as part of a putative transcriptional repressing complex

Abstract: The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

Large-scale Chromosomal Movements During Interphase Progression in Drosophila

Abstract: We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only approximately 2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.

The cenpB gene is not essential in mice.

Abstract: Centromere protein B (CENP-B) is a centromeric DNA-binding protein that binds to alpha-satellite DNA at the 17 bp CENP-B box sequence. The binding of CENP-B, along with other proteins, to alpha-satellite DNA sequences at the centromere, is thought to package the DNA into heterochromatin subjacent to the kinetochore of mitotic chromosomes. To determine the importance of CENP-B to kinetochore assembly and function, we generated a mouse null for the cenpB gene. The deletion removed part of the promoter and the entire coding sequence except for the carboxyl-terminal 35 amino acids of the CENP-B polypeptide. Mice heterozygous or homozygous for the cenpB null mutation are viable and healthy, with no apparent defect in growth and morphology. We have established mouse embryo fibroblasts from heterozygous and homozygous cenpB null littermates. Microscopic analysis, using immunofluorescence and electron microscopy of the cultured cells, indicated that the centromere-kinetochore complex was intact and identical to control cells. Mitosis was identical in fibroblasts derived from cenpB wild-type, heterozygous and null animals. Our studies demonstrate that CENP-B is not required for the assembly of heterochromatin or the kinetochore, or for completion of mitosis.

Complex Structure of Knob DNA on Maize Chromosome 9: Retrotransposon Invasion into Heterochromatin

Abstract: The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

Abstract: We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.

Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes.

Abstract: A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34. 8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form "weak points," underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally beta-heterochromatic, acquire a distinct banding pattern, i. e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional alpha-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

Abstract: The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

Structure of the Chromosome VII Centromere Region in Neurospora crassa: Degenerate Transposons and Simple Repeats

Abstract: Centromeres are regions of chromosomes that direct formation of the kinetochore and its subsequent attachment to the spindle, enabling the faithful segregation of the genetic material during cell division. This chromosomal domain, found in all eukaryotes, is functionally conserved but structurally quite divergent between organisms. The overall size and sequence complexity of centromeres generally appears to parallel the developmental complexity of the organism. In Saccharomyces cerevisiae, for example, the centromere is small, consisting of only approximately 200 bp for full function. In higher eukaryotes, however, centromeric regions of the genome not only are much larger (up to 5 Mb in length [see reference 55 for review]) but show no apparent sequence conservation and have been referred to as “regional” centromeres. Despite recent advances in the development of higher eukaryotic experimental systems, relatively little is known about the sequence constituents of centromeres and centric heterochromatin in complex organisms. For Drosophila, the centromere of a minichromosome has been mapped by deletion analysis, and the minimal sequences required for function are now being identified (37, 67). The sequence components necessary for full centromere function appear to include transposable elements of several types as well as low-complexity satellite DNAs (67). The apparent absence of a defined sequence that is responsible for kinetochore formation, as is seen in S. cerevisiae, suggests a redundant, nonsequence-specific initiation event leading to centromere and/or kinetochore formation. There is also evidence in vertebrates for similar redundancy, i.e., Indian muntjac centromeres can be fractionated into multiple individual kinetochore-like units (76). Indirect evidence from humans suggests that megabase arrays of α-satellite or alphoid DNAs, which are a family of A+T-rich 171-bp tandem repeats, are associated with active centromeres (46) and may be sufficient for centromere function (23, 68). Centromere activity can be observed, however, in activated human neocentromeres lacking alphoid repeats (19). The large size of regional centromeres may be important for the additional functions that have been attributed to centromeres of higher eukaryotes, including chromosome adhesion in achiasmate disjunction (32), as well as providing domains of specialized chromatin structure (heterochromatin) in which euchromatic gene transcription and recombination are both repressed. The characteristics of regional centromeric DNA may also reflect an underlying mechanism by which chromatin structure nucleates kinetochore formation. Several regional centromeres display an epigenetic control phenomenon called centromere activation. In the fission yeast Schizosaccharomyces pombe, formation of the centromere into an active state can require multiple cell divisions after introduction of naked minichromosome DNA (66). In other organisms, aberrant chromosomes can form neocentromeres in locations differing from the original centromere, resulting in a mixture of cells containing a chromosome with one site or the other acting as the centromere (1, 6, 69). The regional heterochromatic character of centromeres in complex organisms, however, may result from the accumulation of repeated sequence elements. One consequence of recombinational repression in heterochromatic regions may be the accumulation of mobile genetic elements (13). The prevailing model explaining the observed excess of transposons in centric heterochromatin holds that chromosome rearrangements due to ectopic recombination between similar transposons at different sites in the genome may lead to decreased fitness and selection against such ectopic insertions (12). Alternatively, the insertion of transposons into genic regions may have negative effects on the fitness of individuals in the population, resulting in fewer elements in euchromatic regions of the genome (28). It is possible, however, that repeated sequence elements in some circumstances may have positive effects on fitness rather than just neutral or negative effects. Like centromeric domains, telomeric and subtelomeric regions of most organisms are also regions of specialized chromatin structure and are home to numerous repeated elements (58, 70, 75). The telomerase-elongated simple DNA sequence repeats at the end of the chromosomes of most eukaryotes are essential elements for continued chromosomal end replication and for segregation of the genome. Remarkably, telomeric sequences can function in place of centromeric heterochromatin in Drosophila in the formation of neocentromeres (2, 6, 56, 73). In Drosophila, telomere functions are probably served by the HeT-A and Tart retrotransposons that are found at all chromosomal termini (14, 43). The cell apparently has taken advantage of such elements to serve an essential function. In multicellular fungi, little is known about centromere structure. The chromosomes of the filamentous fungus Neurospora crassa show regions of heavy, intense staining (heterochromatin) in meiosis (44, 64). Centola and Carbon (10) cloned and partially characterized a contiguous set of artificial yeast chromosomes (YACs) containing DNA that spanned the centromere region of linkage group (LG) VII of Neurospora. This region, approximately 450 kb in length, was found to be both A+T rich and recombination deficient. In addition, they identified a centromere-specific repeated DNA sequence. Comparison of the sequence of this centromere-specific clone to homologous DNAs from elsewhere in the genome suggested that they had undergone repeat-induced point mutation (RIP), a process that scans the genome of Neurospora for repeated DNAs during the sexual cycle and induces GC to AT transition mutations, and often DNA methylation, specific to the duplicated sequences (61). In this study, we have further characterized the centromere region of LG VII of Neurospora and have discovered a nested cluster of putative transposable elements and simple sequence repeats. A repeated DNA sequence previously found to map to centromere-linked regions of the Neurospora genome (10) is now shown to be a copia-like element (named Tcen) which is novel in that it is the only known transposon to be shown to map exclusively to centromere regions. In addition, the region contains the degenerate remains of several other transposons, as well as three different low-complexity DNAs organized in a tightly nested arrangement. Although these features have yet to be associated with kinetochore formation, the structural similarity of the Neurospora centromere VII region to the centromere of the Drosophila Dp1187 minichromosome (37, 67) suggests that Neurospora kinetochore-forming regions may be similarly redundant and nonspecific.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

Abstract: pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Terminal heterochromatin and alternative telomeric sequences in Allium cepa

Abstract: The chromosome termini of the onion (Allium cepa) apparently lack Arabidopsis-type telomeric repeats. The terminal Giemsa bands of A. cepa chromosomes contain a 375-bp satellite and (short arm of chromosome 8)/ or (short arm of chromosome 6) rDNA repeats. By means of fluorescence in situ hybridization (FISH) on metaphase chromosomes and on DNA fibres with probes specific for Ty1-copia retroelements and a En/Spm-transposable element-like sequence, respectively, it was demonstrated that the former are rarely and the latter frequently associated with satellite repeats within the terminal heterochromatin. Polymerase chain reaction of polyC-tailed nuclear DNA with anchor primers and single satellite-specific primers yielded amplification products that, after cloning and sequencing, revealed satellite sequences. This supports the idea that satellite repeats represent one class of terminal sequences in A. cepa.

Why is the centromere so cold

Abstract: Chromosomes are known to contain local hot and cold spots that undergo quite different rates of meiotic recombination. The question of why such regional variation in recombination rates exists on chromosomes holds a strange mix of intrigue and frustration for geneticists. One of the human chromosomes that has been carefully studied over the years in this regard is the X chromosome. These studies have concentrated on the evaluation of the recombination rates across various subregions of this chromosome and have, by and large, avoided the more recalcitrant centromere domain. In this issue, Mahtani and Willard (1998) present an analysis of recombination across the centromere itself and provide data indicating a significantly lower exchange rate at the centromere than the average rate of female meiotic recombination on the human X chromosome. In the current climate where much attention is focused on centromere research, this work provides both useful information and a timely reminder for yet another one of the many unique and difficult-to-study properties of the centromere. The notion that the centromere exerts a direct, negative effect on meiotic recombination both within itself and on proximal chromosomal DNA was recognized >60 years ago (Beadle 1932; Mather 1938). This effect, termed the centromere effect, has now been documented in wide-ranging organisms, including Drosophila, Neurospora, Arabidopsis, budding and fission yeasts, tomato, corn, barley, mouse, and human (for references, see Mahtani and Willard 1988; Round et al. 1997). In some of these organisms, the level of recombination suppression at the centromere can be as high as 10to 40-fold that of the rest of the genome (Roberts 1965; Tanksley et al. 1992; Centola and Carbon 1994). The determination of recombination rates within the centromeres involves comparison of physical distances across the centromeres with the genetic distances between pairs of centromereflanking markers. Such an exercise is especially arduous in higher eukaryotes as the centromeres in these organisms contain an abundance of tandemly repeated, heterochromatic DNA sequences that vary greatly in array lengths and, therefore, in the precise physical distances they span, even for different members of the same chromosome. An even greater problem is the severe paucity of well-mapped euchromatic DNA markers near the centromere that can be used to measure genetic distances accurately. Despite these difficulties, Mahtani and Willard (1990, 1998) described the use of pulsed field gel electrophoresis to determine the average long-range physical distance across the centromeres of the human X chromosome. Armed with this information, and using centromere-flanking genetic markers that are sufficiently well mapped, these workers reveal a meiotic exchange rate across the centromere that is at least eightfold lower (colder) than the average estimates on this chromosome. This result therefore provides the first measured value for the level of recombination suppression within the X centromere—a value that closely agrees with that obtained similarly for the centromere of human chromosome 10 in an earlier study (Jackson et al. 1996). So, why is the centromere such a cold spot for meiotic recombination? The answer to this question is far from clear. Recombination suppression is thought to be the result of the more condensed state of centromeric heterochromatin at the time of crossing-over during meiosis compared with euchromatin (Roberts 1965; Khush and Rick 1967, 1968; Rick 1969, 1972). However, evidence indicating that influences other than heterochromatinization may be involved has come from study of the budding yeast, in which a cloned centromere, although lacking any visible form of heterochromatin, shows decreased recombination when it is artificially integrated into new sites in the genome (Lambie and Roeder 1986). Further evidence has come from observation of the persistence of recombination suppression of centromereadjacent euchromatin in Drosophila even when centromeric heterochromatin is deleted (Yamamoto and Miklos 1977). Thus, rather than the heterochromatin, it may simply be the centromere activity itself that exerts the recombination suppression effect. Recent studies have demonstrated that centromere activity can be separated from the heterochromatin. During the past 4–5 years, an increasing number of human marker chromosomes have been reported to contain active centromeres (or neocentromeres) that are devoid of the usual centromeric heterochromatin and are apparently formed in euchromatic regions of chromosomes (for review, see Choo 1997). Studies of dicentric human chromosomes in which one centromere has become inactivated have also indicated that the presence of centromeric heterochromatin does not always correlate with centromere activity. As to which specific aspect of the centromere activity is involved in recombination suppression, the answer is again unknown. A good guess is that it probably has to do with the unique chromatin structure that makes up the active centromere. Increasingly, centromere activity is believed to be linked to some centromere-specific, higher-order chromatin organization (du Sart et al. 1997; Karpen and Allshire 1997; Choo 1998; Williams et al. 1998). This belief is highlighted by the identification of a histone H3-like protein, CENPA, that is centromere specific, associates only with active centromeres (Warburton et al. 1997), and is thought to constitute centromerespecific chromatin (Sullivan et al. 1994). In a recent exciting development, EkE-MAIL choo@cryptic.rch.unimelb.edu.au; FAX 61-3-9348 1391. Insight/Outlook

Heterochromatin protein 1 binds transgene arrays

Abstract: Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation.

Developmental regulation of heterochromatin-mediated gene silencing in Drosophila.

Abstract: The roles of differentiation, mitotic activity and intrinsic promoter strength in the maintenance of heterochromatic silencing were investigated during development using an inducible lacZ gene as an in vivo probe. Heterochromatic silencing is initiated at the onset of gastrulation, approximately 1 hour after heterochromatin is first visible cytologically. A high degree of silencing is maintained in the mitotically active imaginal cells from mid-embryogenesis until early third instar larval stage, and extensive relaxation of silencing is tightly associated with the onset of differentiation. Relaxation of silencing can be triggered in vitro by ecdysone. In contrast, timing and extent of silencing at both the initiation and relaxation stages are insensitive to changes in cell cycle activity, and intrinsic promoter strength also does not influence the extent of silencing by heterochromatin. These data suggest that the silencing activity of heterochromatin is developmentally programmed.