Sex chromatin and nucleolar analyses inRumex acetosa L.

Summary Rumex acetosa (sorrel) is a dioecious plant with a XX/XY₁Y₂ sex chromosome system. Both the Y chromosomes are nearly entirely heterochromatic and it has been hypothesised that they can persist as chromocenters in male interphase nuclei. Using specific antibodies against 5-methylcytosine and histone H4 acetylated at terminal lysine 5, global levels of DNA methylation and histone acetylation were studied on the sex chromosomes and autosomes of both sexes. The heterochromatic Y chromosomes did not display a higher methylation level compared to the autosomes. The only prominent hypermethylation signals were found at two nucleolar organising regions located on the autosome pair V, as confirmed by in situ hybridisation with 25S rDNA probe and staining. Immunoanalysis of DNA methylation on female and male interphase nuclei neither revealed any sex-specific differences. Two active (silverpositive) nucleoli and two likely inactive nucleolar organising regions (displaying prominent methylation signals) were found in both sexes. In a fraction of nuclei isolated from leaf cells, two peripheral bodies strongly positive for 4,6-diamidino-2-phenylindole were observed only in males, never in females. These heterochromatin regions were depleted in histone H4 acetylation at terminal lysine 5 and corresponded, according to in situ hybridisation with a Y-chromosome-specific repetitive probe, to the two Y chromosomes. We conclude that the peripheral condensed bodies observed exclusively in male nuclei represent the constitutive heterochromatin of the Y chromosomes which is characterised by a substantial histone H4 underacetylation.     

Histone acetylation: A possible mechanism for the inheritance of cell memory at mitosis

Immunofluorescent labelling demonstrates that human metaphase chromosomes contain hyperacetylated histone H4. With the exception of the inactive X chromosome in female cells, where the bulk of histone H4 is under-acetylated, H4 hyperacetylation is non-uniformly distributed along the chromosomes and clustered in cytologically resolvable chromatin domains that correspond, in general, with the R-bands of conventional staining. The strongest immunolabelling is often found in T-bands, the subset of intense R-bands having the highest GC content. The majority of mapped genes also occurs in R-band regions, with the highest gene density in T-bands. These observations are consistent with a model in which hyperacetylation of histone H4 marks the position of potentially active gene sequences on metaphase chromosomes. Since acetylation is maintained during mitosis, progeny cells receive an imprint of the histone H4 acetylation pattern that was present on the parental chromosomes before cell division. Histone acetylation could provide a mechanism for propagating cell memory, defined as the maintenance of committed states of gene expression through cell lineages.     

Histone H4 Acetylation and Replication Timing in Chinese Hamster Chromosomes

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, was similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, showed a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions we observe suggest that this link is not absolute or essential.     

The kinetochores of Caenorhabditis elegans

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 m in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18–0.2 m in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 m, and an electron lucent middle layer of 0.03 m. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.     

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

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

Acetylation and deacetylation of histone H4 continue through metaphase with depletion of more-acetylated isoforms and altered site usage

Antibodies specific for acetylated isoforms of histone H4 have been used to compare acetylation of this histone in interphase and metaphase cells. Two rabbit antisera (R5 and R6) were used, each specific for H4 molecules acetylated at one of the four possible acetylation sites, namely Lys-5 (R6) and Lys-12 (R5). Both antisera bound preferentially to the more-acetylated H4 isoforms (H4Ac2-4). To test for continued H4 acetylation in metaphase chromosomes. Chinese hamster ovary cells were blocked in metaphase and treated for one hour with the deacetylase inhibitor sodium butyrate. Isolated chromosomes were assayed for H4 acetylation by antibody labeling and flow cytometry. H4 acetylation was increased several fold by this brief butyrate treatment. The increase was in direct proportion to DNA content, with no evidence for exceptionally high- or low-labeling chromosomes. The results demonstrate that a cycle of H4 acetylation and deacetylation continues within metaphase chromosomes. Immunofluorescence microscopy showed labeling to be distributed throughout the chromosome, but with variable intensity. Western blotting and immunostaining with R5 and R6 showed a net reduction in labeling of H4 from metaphase cells, with major reductions in the more-acetylated isoforms H4Ac3-4. In contrast, labeling of H4Ac1 was reduced to a lesser extent (R6) or increased (R5). This increase indicates more frequent use of the acetylation site at lysine 12 in H4Ac1 from metaphase cells.     

Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae)

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.     

Cytogenetic and immuno-FISH analysis of the 4q subtelomeric region,which is associated with facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortening of a copy-number polymorphic array of 3.3 kb repeats (D4Z4) at one allelic 4q35.2 region. How this contraction of a subtelomeric tandem array causes FSHD is unknown but indirect evidence suggests that a short array has a cis effect on a distant gene or genes. It was hypothesized that the length of the D4Z4 array determines whether or not the array and a large proximal region are heterochromatic and thereby controls gene expression in cis. To test this, we used fluorescence in situ hybridization probes with FSHD and control myoblasts to characterize the distal portion of 4q35.2 with respect to the following: intense staining with the chromatin dye 4,6-diamidino-2-phenylindole; association with constitutively heterochromatic foci; extent of binding of heterochromatin protein 1; histone H3 methylation at lysine 9 and lysine 4; histone H4 lysine 8 acetylation; and replication timing within S-phase. Our results indicate that 4q35.2 does not resemble constitutive heterochromatin in FSHD or control myoblasts. Furthermore, in these analyses, the allelic 4q35.2 regions of FSHD myoblasts did not behave differently than those of control myoblasts. Other models for how D4Z4 array contraction causes long-distance regulation of gene expression in cis need to be tested.Communicated by S. Gerbi     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.     

Karyotypanalyse an Hand des Färbungsmusters der Metaphasechromosomen von Cypripedium debile und Trillium kamtschaticum

The somatic metaphase chromosomes of Cypripedium debile and Trillium kamtschaticum are stained differentially by treatment with an acetic orcein-hydrochloric acid mixture: In Cypripedium, the heterochromatic segments stain densely whereas the euchromatic segments are unstained. In Trillium, in contrast, the heterochromatic segments are unstained and euchromatin is stained. Such an inconsistency in stain patterns is considered to stem from different species-specific reactivity of heterochromatin and euchromatin to hydrochloric acid in the staining medium.—In metaphase chromosomes of Cypripedium stained with fast green (Alfert and Geschwind, 1953), the euchromatic segments are stained positively, whereas the heterochromatin, as well as the chromocenters, are unstained. In Trillium, all heterochromatin, euchromatin and chromocenter are stained homogeneously by this method. These results indicate that the heterochromatic segments and chromocenters in Cypripedium are associated with non-histon type proteins, whereas the euchromatic segments in Cypripedium, as well as two different types of the segments and the chromocenters in Trillium, are all bound to histon type proteins. — From these findings, it is concluded that two types of heterochromatin occur in plant materials, though it remains unsettled whether they correspond to - and -heterochromatin as found in Dipteran giant chromosomes.—Karyotype analysis is made on the basis of the differential staining pattern of chromosomes revealed by means of acetic orcein-hydrochloric procedure.     

Differential underacetylation of histones H2A,H3 and H4 on the inactive X chromosome in human female cells

It has previously been shown that the acetylated forms of histone H4 are depleted or absent in both constitutive, centric heterochromatin and in the facultative heterochromatin of the inactive X chromosome (Xi) in female cells. By immunostaining of metaphase chromosomes from human lymphocytes with antibodies to the acetylated isoforms of histones H2A and H3, we now show that these histones too are underacetylated in both Xi and centric heterochromatin. Xi shows two prominent regions of residual H3 acetylation, one encompassing the pseudoautosomal region at the end of the short arm and one at about Xg22. Both these regions have been shown previously to be sites of residual H4 acetylation. H2A acetylation on Xi is higher overall than that of H3 or H4 and is particularly high around the pseudoautosomal region, but not at Xg22. The results suggest that the acetylated isoforms of H3 and H4 have at least some effects on chromosomal structure and function that are not shared by acetylated H2A.     

Two new chromodomain-containing proteins that associate with heterochromatin in <Emphasis Type="Italic">Sciara coprophila</Emphasis> chromosomes

We report here the molecular and cytological characterization of two proteins, ScoHET1 and ScoHET2 (for Sciara coprophila heterochromatin), which associate to constitutive heterochromatin in the dipteran S. coprophila. Both proteins, ScoHET1 of 37 kDa and ScoHET2 of 44 kDa, display two chromodomain motifs that contain the conserved residues essential for the recognition of methylated histone H3 at lysine 9. We raised antibodies to analyze the chromosomal location of ScoHET1 and ScoHET2 in somatic and germline cells. In S. coprophila polytene chromosomes, both proteins associate to the pericentromeric regions and to the heterochromatic subterminal bands of the chromosomes. In germinal nuclei, ScoHET1 and ScoHET2 proteins distribute to the heterochromatic regions of the regular chromosome complement and are abundantly present along the heterochromatic germline-limited “L” chromosomes. We investigated histone methylation modifications and found that all heterochromatic regions enriched in ScoHET1/ScoHET2 proteins exhibit high levels of di- and tri-methylated histone H3 at lysine 9. Taken together, our results support that the association of ScoHET1/ScoHET2 to heterochromatin is mediated by histone H3K9 methylation. Using 5-methylcytosine antibodies, we proved the cytological detection of DNA methylation in S. coprophila. From our observations in L germline chromosomes, heterochromatin in S. coprophila is highly enriched in DNA 5-methylcytosine residues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae,Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed ZW males are discussed.