Showing papers on "Heterochromatin published in 2000"

Epigenetic aspects of somaclonal variation in plants.

Abstract: Somaclonal variation is manifested as cytological abnormalities, frequent qualitative and quantitative phenotypic mutation, sequence change, and gene activation and silencing. Activation of quiescent transposable elements and retrotransposons indicate that epigenetic changes occur through the culture process. Epigenetic activation of DNA elements further suggests that epigenetic changes may also be involved in cytogenetic instability through modification of heterochromatin, and as a basis of phenotypic variation through the modulation of gene function. The observation that DNA methylation patterns are highly variable among regenerated plants and their progeny provides evidence that DNA modifications are less stable in culture than in seed-grown plants. Future research will determine the relative importance of epigenetic versus sequence or chromosome variation in conditioning somaclonal variation in plants.

Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.

Abstract: We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.

Genetic and epigenetic interactions in allopolyploid plants

Abstract: Allopolyploid plants are hybrids that contain two copies of the genome from each parent. Whereas wild and cultivated allopolyploids are well adapted, man-made allopolyploids are typically unstable, displaying homeotic transformation and lethality as well as chromosomal rearrangements and changes in the number and distribution of repeated DNA sequences within heterochromatin. Large increases in the length of some chromosomes has been documented in allopolyploid hybrids and could be caused by the activation of dormant retrotransposons, as shown to be the case in marsupial hybrids. Synthetic (man-made) allotetraploids of Arabidopsis exhibit rapid changes in gene regulation, including gene silencing. These regulatory abnormalities could derive from ploidy changes and/or incompatible interactions between parental genomes, although comparison of auto- and allopolyploids suggests that intergenomic incompatibilities play the major role. Models to explain intergenomic incompatibilities incorporate both genetic and epigenetic mechanisms. In one model, the activation of heterochromatic transposons (McClintock's genomic shock) may lead to widespread perturbation of gene expression, perhaps by a silencing interaction between activated transposons and euchromatic genes. Qualitatively similar responses, of lesser intensity, may occur in intraspecific hybrids. Therefore, insight into genome function gained from the study of allopolyploidy may be applicable to hybrids of any type and may even elucidate positive interactions, such as those responsible for hybrid vigor.

Isolation and Characterization of Suv39h2, a Second Histone H3 Methyltransferase Gene That Displays Testis-Specific Expression

Abstract: Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.

Heterochromatic deposition of centromeric histone H3-like proteins

Abstract: Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere–heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the cid upstream region drives expression of H3 and H2B histone–green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results. Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone–green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.

A Bromodomain Protein, MCAP, Associates with Mitotic Chromosomes and Affects G2-to-M Transition

Abstract: We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G(2)/M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.

Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes.

Abstract: New proteins and modules have been invented throughout evolution. Gene “birth dates” in Caenorhabditis elegans range from the origins of cellular life through adaptation to a soil habitat. Possibly half are “metazoan” genes, having arisen sometime between the yeast-metazoan and nematode-chordate separations. These include basement membrane and cell adhesion molecules implicated in tissue organization. By contrast, epithelial surfaces facing the environment have specialized components invented within the nematode lineage. Moreover, interstitial matrices were likely elaborated within the vertebrate lineage. A strategy for concerted evolution of new gene families, as well as conservation of adaptive genes, may underlie the differences between heterochromatin and euchromatin.

Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding

Abstract: Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, we have examined the mechanism by which it is targeted to centromeric foci. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, as only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting was dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain was required only for homodimerization, as targeting was restored when this domain was replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain revealed a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats. Models for the function of Ikaros that are consistent with this targeting mechanism are discussed.

Distinct protein interaction domains and protein spreading in a complex centromere

Abstract: Fission yeast (Schizosaccharomyces pombe) centromeres are composed of large (40‐100 kb) inverted repeats that display heterochromatic features, thus providing a good model for higher eukaryotic centromeres. The association of three proteins that mediate region-specific silencing across centromere 1 has been mapped by quantitative chromatin immunoprecipitation. Swi6 and Chp1 are confined to the flanking outer repeats and Swi6 can spread across at least 3 kb of extraneous chromatin in cen1. In contrast, Mis6 coats the inner repeats and central core. tRNA genes demarcate this transition zone. These analyses clearly define two distinct domains within this complex centromere which interact with different proteins.

DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients

Abstract: The ICF syndrome (immunodeficiency, centromeric region instability, facial anomalies) is a unique DNA methylation deficiency disease diagnosed by an extraordinary collection of chromosomal anomalies specifically in the vicinity of the centromeres of chromosomes 1 and 16 (Chr1 and Chr16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent (qh) heterochromatin, multiradial chromosomes with up to 12 arms, and whole-arm deletions. We demonstrate that lymphoblastoid cell lines from two ICF patients exhibit these Chr1 and Chr16 anomalies in 61% of the cells and continuously generate 1qh or 16qh breaks. No other consistent chromosomal abnormality was seen except for various telomeric associations, which had not been previously noted in ICF cells. Surprisingly, multiradials composed of arms of both Chr1 and Chr16 were favored over homologous associations and cells containing multiradials with 3 or >4 arms almost always displayed losses or gains of Chr1 or Chr16 arms from the metaphase. Our results suggest that decondensation of 1qh and 16qh often leads to unresolved Holliday junctions, chromosome breakage, arm missegregation, and the formation of multiradials that may yield more stable chromosomal abnormalities, such as translocations. These cell lines maintained the abnormal hypomethylation in 1qh and 16qh seen in ICF tissues. The ICF-specific hypomethylation occurs in only a small percentage of the genome, e.g., ICF brain DNA had 7% less 5-methylcytosine than normal brain DNA. The ICF lymphoblastoid cell lines, therefore, retain not only the ICF-specific pattern of chromosome rearrangements, but also of targeted DNA hypomethylation. This hypomethylation of heterochromatic DNA sequences is seen in many cancers and may predispose to chromosome rearrangements in cancer as well as in ICF.

HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Abstract: Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Molecular Determinants for Targeting Heterochromatin Protein 1-Mediated Gene Silencing: Direct Chromoshadow Domain–KAP-1 Corepressor Interaction Is Essential

Abstract: It is clear that many of the effector domains in eukaryotic transcription factors act as protein-protein interfaces which allow the assembly of macromolecular complexes at various sites in the nucleus. The constellation of transcription factors that are arranged at a gene promoter integrate different complexes via coactivators and corepressors, collectively termed cofactors. Cofactors can act directly upon the process of transcription by regulating components of the RNA polymerase complex or through components of chromatin, often leading to changes in gene expression which are stable through cell division. Core histone modification by histone deacetylases and histone acetyltransferases, which can be targeted, respectively, by corepressors and coactivators, has emerged as a common mechanism for influencing gene expression by altering chromatin (58). Another example is the methylation of CpG islands, which is associated with epigenetically imprinted alleles or repressed genes (8). More recent evidence showing that the methyl-CpG-binding protein is present in a complex with histone deacetylase activity (26, 44) suggests a link between these two chromatin modifications that promote gene silencing. Thus, by acting as links between modifiers of chromatin and site-specific transcription factors, corepressors and coactivators can alter the gene expression profile of a cell in a heritable manner. To investigate the mechanistic steps through which transcription repressors down-regulate gene expression, we have focused on the KRAB (Kruppel-associated box) domain as a model. The KRAB domain is a potent repression domain present in nearly one-third of the members of a family of zinc finger transcription factors for which there are an estimated 300 to 700 human genes (7, 36). The family is characterized by an amino-terminal KRAB repression domain linked to multiple arrays of Cys2His2-type zinc fingers, which are responsible for DNA binding (29). Like many other repression domains, the KRAB repression domain retains repressor activity when transferred to a heterologous DNA binding domain (35, 62, 65). KAP-1 is a universal corepressor for the KRAB domain and is the founding member of a small family of cofactors collectively designated the transcriptional intermediary factor 1 (TIF1) family in humans and mice (61). KAP-1 was isolated by affinity chromatography (19) and subsequently by yeast two-hybrid screening (27, 40). It is a 97-kDa nuclear phosphoprotein and contains a number of domains common to the family but also found in other types of transcriptional regulators (see Fig. ​Fig.1A).1A). At the amino terminus is the RBCC multidomain unit, comprised of a RING finger, two B boxes, and a coiled-coil domain. The RBCC domain is essential for binding to the KRAB domain and participates in multimerization of KAP-1 (19, 48). A plant-like homeodomain (PHD) and a bromodomain are tandemly arranged at the carboxy terminus. The central region of KAP-1 is the least conserved among the family members and is generally rich in prolines, glycines, and serines. KAP-1 itself possesses potent repression activity, and this function is contributed by the carboxy terminus, including the PHD and the bromodomain (19, 40, 61). FIG. 1 Schematic representations of the KAP-1 protein and the HP1BD. (A) The KAP-1 protein is shown in linear format with the conserved domains noted by shaded boxes and with amino acid positions indicated. A RING finger, two B boxes, and a coiled-coil region ... The basis for KAP-1 function in transcription repression is not yet fully understood, but the ability of KAP-1 to bind heterochromatin protein 1 (HP1) is very likely to have a role. The interaction between these families of proteins was first uncovered in a yeast two-hybrid screen using TIF1α (32). There are three different HP1 proteins in humans and mice: HP1α, HP1β, and HP1γ (mouse HP1β and HP1γ are also referred to as M31 or MOD1 and M32 or MOD2, respectively [25]). All the proteins share a basic structure of an amino-terminal chromodomain (CD) and a carboxy-terminal chromoshadow domain (CSD) linked by a hinge region (see Fig. ​Fig.2A).2A). The CD is present in numerous proteins, but the CSD has been found only in the HP1 proteins and is thus considered the signature motif for this family (2, 47, 56). FIG. 2 Schematic representations of the HP1 protein, the CD, and the CSD. (A) Diagram of human HP1α depicting the prototypical structure of the HP1 proteins. The relevant domains are noted; NLS, nuclear localization signal. The CSD polypeptide fragment ... Studies of HP1 in Drosophila melanogaster provide one of the best examples of epigenetic mechanisms of gene regulation. Position effect variegation (PEV) in D. melanogaster refers to the process of silencing euchromatic genes that have integrated adjacent to heterochromatin (63). Gene silencing occurs in only a subset of cells, and this state is faithfully inherited by their progeny, leading to variegated or mosaic patterns of expression. The stochastic nature of PEV is hypothesized to be due to the variable spreading of heterochromatin into adjacent regions (55). Genetic screens for modifiers of PEV led to the discovery of the suppressor allele Su(var)2-5, which encoded HP1, a nonhistone chromosomal protein first identified by its localization to heterochromatin in polytene nuclei (14, 23). D. melanogaster HP1 plays a dose-dependent role in PEV and likely contributes to the formation and/or stabilization of heterochromatin (15). More recent studies showing that there is a correlation between overexpression of the CD protein Swi6 and mat locus imprinting in fission yeast and increased expression of an M31 transgene and changes in variegation gene expression in mice indicate that HP1 function has been strongly conserved (17, 43). Position effects are known to play a role in both human and murine genetic defects (6, 28). In many cases, it appears that positively acting elements are removed and/or that negatively acting (silencing) regions are repositioned near genes. Indeed, boundary elements and locus control regions may function as barriers that prevent inappropriate spreading of heterochromatin or association with heterochromatic regions in nuclei (18, 38, 60). Moreover, enhancer function has been postulated to work by keeping genes out of heterochromatic environs (10). Emerging studies of gene regulation by Ikaros have revealed a colocalization of inactive genes with centromeric heterochromatin and M31 in normal cycling lymphocytes (9). Thus, local compartmentalization of genes in the nucleus is emerging as a common mechanism for repressing transcription in a stable manner. However, the mechanisms responsible for sequence-specific targeting of this repression are unclear. Heterochromatin, as defined classically by cytological appearance, is interspersed throughout chromosomes but is abundant near centromeres and telomeres and is frequently composed of repetitive sequences (64). The mammalian HP1 proteins have distinct euchromatic or heterochromatic staining patterns in nuclei, suggesting that their roles have become specialized or that targeting to chromatin has been regulated uniquely (39, 52). KAP-1 is dynamically associated with the euchromatic and heterochromatic regions, suggesting that it links heterochromatin-mediated gene regulation to localization in a specific chromosomal territory (52). This model proposes that KRAB-zinc finger proteins recruit the KAP-1 corepressor to DNA; this complex, in turn, binds to HP1, which may then nucleate local heterochromatin formation, resulting in gene silencing. In order to understand the interaction between KAP-1 and HP1 in more detail, we have reconstituted the complex using recombinant proteins and have comprehensively defined its biochemical properties. We have found that the CSD in HP1 is required for direct binding to KAP-1 and that a stretch of 15 residues in the middle of KAP-1, the HP1BD, is necessary and sufficient for association. We present evidence that the CSD dimerizes and that it binds the KAP-1 HP1BD in a 2:1 stoichiometry, with an apparent Kd of approximately 60 nM. Mutational analysis with the human HP1α CSD and KAP-1 has pinpointed specific residues that are essential for both binding and transcription repression activity in either protein. We also show that interactions with three other HP1 binding proteins are disrupted by the same CSD mutations as those which disrupt KAP-1 binding; however, these domains are not equally able to repress transcription or inhibit HP1-mediated transcription repression.

Heterochromatin Protein 1 Is Required for the Normal Expression of Two Heterochromatin Genes in Drosophila

Abstract: The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1 Here, we show that the Su(var)2-5 lethal period is late third instar Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein We demonstrate that heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes

Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression.

Abstract: Higher-order chromatin is essential for epigenetic gene control and for the structural organization of chromosomes at centromeres and telomeres (21, 26, 35, 36). Despite these important functions, only very few components of higher-order chromatin, particularly for mammalian systems, have been identified. By contrast, genetic screens of Drosophila melanogaster (40) and Schizosaccharomyces pombe (4) characterized a subfamily of ≈30 to 40 loci which, collectively, can be referred to as Su(var) group genes. Since Su(var) genes suppress position effect variegation (PEV), their gene products are implicated in the establishment of repressive chromatin domains. Indeed, the majority of isolated family members encode either heterochromatic proteins or enzymes that can modify the basic unit (DNA and histones) of chromatin (49). For example, several histone deacetylases (11, 16) or protein phosphatase 1 (5) have been classified as Su(var) products. Moreover, Su(var)2–5 (which encodes heterochromatin protein 1 [HP1] [24, 12]), Su(var)3-7 (8, 39), and Su(var)3-9 (47) are all dose-dependent modifiers of PEV, suggesting that subtle differences in the concentration of chromosomal SU(VAR) proteins direct the extension of heterochromatin (21). Su(var) gene function thus supports a simplified model in which modifications at the nucleosomal level, like changes in the acetylation (48) and phosphorylation (20, 50) of core histones, contribute to the definition of repressive chromatin domains. Heterochromatic SU(VAR) proteins would then stabilize such an altered structure and propagate higher-order chromatin. This model predicts that some SU(VAR) proteins will preferably interact with modified histones and/or be able to nucleate extended associations with nuclear chromatin. However, with the exception of the paradigm of SIR proteins (17, 18) in Saccharomyces cerevisiae, which can be regarded as analogues of SU(VAR) in budding yeast, a mechanistic link between histones and heterochromatic SU(VAR) proteins has not been described in multicellular organisms. Moreover, the interdependence of SU(VAR) proteins in regulating the association with heterochromatin is only poorly understood. Although Su(var) genes affect epigenetic control of gene expression, their major function appears to reside in the coregulation of higher-order chromatin at centromeres and telomeres (26). For example, mutations in Su(var)2-5 have recently been shown to result in telomeric fusions (14). Similarly, mutations in swi6 (28) and clr4 (23), the respective Su(var)2-5 and Su(var)3-9 homologues in S. pombe, induce segregation defects and elevated rates of chromosome loss (13)—phenotypes that are accompanied by impaired proliferative potential. Interestingly, accumulation of SWI6p at fission yeast centromeres has been shown to depend on clr4 function (13). Recently, we isolated human (SUV39H1) and murine (Suv39h1) homologues (1) of Su(var)3-9 and clr4. SUV39H1 and Suv39h1 encode novel heterochromatic proteins that accumulate at centromeric positions during mitosis. In addition, SUV39H1 associates with M31 (HP1β) (1), one member of the mammalian SU(VAR)2-5 protein (HP1) family. Su(var)3-9-related genes are of particular interest, since (i) Su(var)3-9 is dominant over most other PEV modifier mutations (47) and (ii) because their products combine two prominent domains of chromatin regulators, i.e., the chromo and SET domains. Interestingly, mutations in either the chromo or SET domain of clr4 have been shown to impair gene silencing, implicating both domains in the modulation of a repressive chromatin structure (23). The 60-amino-acid (aa) chromo domain (3, 27), initially identified in HP1 and POLYCOMB (34), represents a protein-specific interaction surface that resembles an ancient histone-like fold (6) and which directs eu- or heterochromatic associations (30, 37). By contrast, the molecular role of the 130-aa SET domain (47) remains enigmatic. Although SET domain motifs are present in over 140 gene sequences (44) and represent preferred sites for mutations (25), only a few SET domain interactions have been described using yeast two-hybrid and in vitro binding assays (7, 9, 41). However, the SET domain has recently been shown to be a target for dual-specificity phosphatases and their inhibitor Sbf1 (SET binding factor 1), suggesting involvement in phosphorylation-dependent signaling pathways (10). Since SUV39H1 is a phosphoprotein with mitosis-specific isoforms (2), the SET domain could provide a protein module to induce dynamic transitions in chromosomal associations and protein interactions. We have been using a detailed structure-function analysis of mutant SUV39H1 proteins in transfected cells to uncover the functional roles of the chromo and SET domains. Whereas heterochromatin localization is mediated through the N terminus by an M31 interaction surface and the immediately adjacent chromo domain, an isolated C-terminal SET domain appears to be inactive in these assays. However, the SET domain modulates several properties of deregulated SUV39H1. For example, only SUV39H1 proteins with an intact SET domain disperse endogenous M31, abundantly associate with nuclear chromatin, induce growth and chromosome segregation defects, and interfere with the G2-specific distribution of phosphorylated histone H3 at serine 10 (phosH3). Our data indicate a modular nature for SUV39H1 protein function that is largely governed by the SET domain and suggest a possible link between a chromosomal SU(VAR) protein and histone H3.

Patterns of heterochromatin distribution in plant chromosomes.

Abstract: The C-band distribution patterns of 105 angiosperm species were compared to identify general patterns or preferential sites for heterochromatin. The base-specific fluorochrome reaction of heterochromatin for 58 of these species and the role played by the average chromosome size in band distribution were also considered. The results showed that heterochromatin was preferentially located in similar chromosome regions, regardless of the distance from the centromere. This trend results in generalized bands, with heterochromatin distribution being identical in most chromosomes of a karyotype. Such bands very often displayed the same fluorochrome reaction, suggesting possible repeat transfer between non-homologous sites. Chromosome size may also play a role in heterochromatin location, since proximal bands were much more common in small-sized chromosomes.

Spatial Separation of Parental Genomes in Preimplantation Mouse Embryos

Abstract: We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.

HP1γ associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes

Abstract: Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.

Whole-genome methylation scan in ICF syndrome: hypomethylation of non-satellite DNA repeats D4Z4 and NBL2

Abstract: The ICF (immunodeficiency, centromeric instability and facial abnormalities) syndrome is a rare recessive disease characterized by immunodeficiency, extraordinary instability of certain heterochromatin regions and mutations in the gene encoding DNA methyltransferase 3B. In this syndrome, chromosomes 1 and 16 are demethylated in their centromere-adjacent (juxtacentromeric) heterochromatin, the same regions that are highly unstable in mitogen-treated ICF lymphocytes and B cell lines. We investigated the methylation abnormalities in CpG islands of B cell lines from four ICF patients and their unaffected parents. Genomic DNA digested with a CpG methylation-sensitive restriction enzyme was subjected to two-dimensional gel electrophoresis. Most of the restriction fragments were identical in the digests from the patients and controls, indicating that the methylation abnormality in ICF is restricted to a small portion of the genome. However, ICF DNA digests prominently displayed multicopy fragments absent in controls. We cloned and sequenced several of the affected DNA fragments and found that the non-satellite repeats D4Z4 and NBL2 were strongly hypomethylated in all four patients, as compared with their unaffected parents. The high degree of methylation of D4Z4 that we observed in normal cells may be related to the postulated role of this DNA repeat in position effect variegation in facio- scapulohumeral muscular dystrophy and might also pertain to abnormal gene expression in ICF. In addition, our finding of consistent hypomethylation and overexpression of NBL2 repeats in ICF samples suggests derangement of methylation-regulated expression of this sequence in the ICF syndrome.

Histone H4 Acetylation of Euchromatin and Heterochromatin Is Cell Cycle Dependent and Correlated with Replication Rather Than with Transcription

Abstract: Reversible acetylation of nucleosomal histones H3 and H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains. Here, we report that the extent of H4 acetylation within euchromatin and heterochromatic domains is linked with DNA replication rather than with transcriptional activity, whereas H3 acetylation remains fairly constant throughout the cell cycle. Compared with euchromatin, plant nucleolus organizers were more strongly acetylated at H4 during mitosis but less acetylated during S phase, when the nucleolus appeared to be (at least transiently) devoid of nucleosomes. Deposition-related acetylation of lysines 5 and 12 of H4 seems to be conserved in animals and plants and extended to K16 in plants. A possibly species-specific above-average acetylation at lysines 9/18 and 14 of H3 appeared in 4′,6-diamidino-2-phenylindole (DAPI)–stained heterochromatin fractions. These results were obtained by combining immunodetection of all acetylatable isoforms of H3 and H4 on mitotic chromosomes and nuclei in G1, early S, mid-S, late S, and G2 phases of the field bean with identification of specific chromatin domains by fluorescence in situ hybridization or DAPI staining. In addition, the histone acetylation patterns of distinct domains were compared with their replication and transcription patterns.

The fourth chromosome of Drosophila melanogaster: interspersed euchromatic and heterochromatic domains.

Abstract: The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50–75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

A human homologue of yeast anti-silencing factor has histone chaperone activity

Abstract: Background Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Silencing, potent repression of transcription in Saccharomyces cerevisiae, occurs near telomeres and at the silent mating-type loci, as well as at rDNA loci. This type of repression relates to the condensation of chromatin that occurs in the heterochromatin of multicellular organisms. Anti-silencing is a reaction by which silenced loci are de-repressed. Genetic studies revealed that several factors participate in the anti-silencing reaction. However, actions of factors and molecular mechanisms underlying anti-silencing remain unknown. Results Here we report the functional activity of a highly evolutionarily conserved human factor termed CIA (CCG1-interacting factor A), whose budding yeast homologue ASF1 has anti-silencing activity. Using yeast two-hybrid screening, we isolated histone H3 as an interacting factor of CIA. We also showed that CIA binds to histones H3/H4 in vitro, and that the interacting region of histone H3 is located in the C-terminal helices. Considering the functional role of CIA as a histone-interacting protein, we found that CIA forms a nucleosome-like structure with DNA and histones. Conclusions These results show that human CIA, whose yeast homologue ASF1 is an anti-silencing factor, possesses histone chaperone activity. This leads to a better understanding of the relationship between chromatin structural changes and anti-silencing processes.

Yeast heterochromatin is a dynamic structure that requires silencers continuously

Abstract: Transcriptional silencing of the HM loci in yeast requires cis-acting elements, termed silencers, that function during S-phase passage to establish the silent state. To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembled silent chromatin fragments from silencers. DNA rings excised from HMR were initially silent but ultimately reactivated, even in G(1)- or G(2)/M-arrested cells. In contrast, DNA rings bearing HML-derived sequence were stably repressed due to the presence of a protosilencing element. These data show that silencers (or protosilencers) are required continuously for maintenance of silent chromatin. Reactivation of unstably repressed rings was blocked by overexpression of silencing proteins Sir3p and Sir4p, and chromatin immunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin. Importantly, the protein was incorporated even when expressed outside of S phase, during G(1) arrest. That silencing factors can associate with and stabilize preassembled silent chromatin in non-S-phase cells demonstrates that heterochromatin in yeast is dynamic.

Forkhead Genes in Transcriptional Silencing, Cell Morphology and the Cell Cycle: Overlapping and Distinct Functions for FKH1 and FKH2 in Saccharomyces cerevisiae

Abstract: 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.

Conservation of Heterochromatin Protein 1 Function

Abstract: Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.