Histone variants and histone modifications in chromatin fractions from heterochromatin-rich Peromyscus cells

In order to investigate the relationship between condensed heterochromatin and histone modification by acetylation, phosphorylation and amino acid variation, chromatin from cultured Peromyscus eremicus cells, containing 35% constitutive heterochromatin, was fractionated into heterochromatin-enriched and heterochromatin-depleted fractions. The constitutive heterochromatin content of these fractions was determined from satellite DNA content. The distribution of phosphorylated and acetylated histones and amino acid variants of histone H2A in these chromatin fractions was examined by gel electrophoresis. Fractionation of histones demonstrated that endogenous histone phosphatase activity was high in chromatin fractions and could not be inhibited sufficiently to allow accurate histone phosphorylation measurements. However, sodium butyrate did inhibit deacetylation activity in the fractions, allowing histone acetylation measurements to be made. It was found that the constitutive heterochromatin content of these fractions was proportional to both their unacetylated H4 content and their more-hydrophobic H2A content. These observations support, by direct measurement, earlier experiments (Exp cell res 111 (1978) 373; 125 (1980) 377; 132 (1981) 201) suggesting that constitutive heterochromatin is enriched in unacetylated arginine-rich histones, and in the more hydrophobic variant of histone H2A.     

Mouse centromeric heterochromatin: Isolation and some characteristics

A method is suggested for isolation of highly purified mouse centromeric heterochromatin. Treatment of mouse liver nuclei with decreasing concentrations of Ca2+ resulted in the gradual unraveling of chromatin in the nucleus and at 0.1 mM Ca2+ electron microscopy revealed several dense particles per nucleus, surrounded by decondensed chromatin. These particles, assumed to represent centromere regions of interphase chromosomes by in situ hybridization with radioactive mouse satellite DNA and by differential staining for centromere heterochromatin, were isolated in preparative amounts and their DNA and protein composition was analyzed. The preparation represented practically pure mouse centromere heterochromatin, since more than 90% of its DNA was satellite DNA.     

Mammalian ChlR1 has a role in heterochromatin organization

The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11^−/− embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1β. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11^−/− embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects.     

Observations on centromeric heterochromatin and satellite DNA in salamanders of the genus Plethodon

DNA from Plethodon cinereus cinereus separates into two fractions on centrifugation to equilibrium in neutral CsCl. The smaller of these fractions has been described as a high-density satellite. It represents about 2% of nuclear DNA from this species, and it has a density of 1.728 g/cm³. It is cytologically localized near the centromeres of all 14 chromosomes of the haploid set. In P. c. cinereus the heavy satellite DNA constitutes about 1/4 of the DNA in centromeric heterochromatin. The nature of the rest of the DNA in centromeric heterochromatin is unknown. The number of heavy satellite sequences clustered around the centromeres in a chromosome from P. c. cinereus is roughly proportional to the size of the chromosome, as determined by in situ hybridization with satellite-complementary RNA, and autoradiography. Likewise the amount of contromeric heterochromatin, as identified by its differential stainability with Giemsa, shows a clear relationship to chromosome size. — The heavy satellite sequences identified in DNA from P. c. cinereus are also present in smaller amounts in other closely related forms of Plethodon. Plethodon cinereus polycentratus and P. richmondi have approximately half as many of these sequences per haploid genome as P. c. cinereus. P. hoffmani and P. nettingi shenandoah have about 1/3 as many of these sequences as P. c. cinereus. P. c. cinereus, P. c. polycentratus, and P. richmondii all have detectable heavy satellites with densities of 1.728 g/cm³. Among these forms, satellite size as determined by optical density measurements, and number of satellite sequences as determined from hybridization studies, vary co-ordinately. P. c. cinereus heavy satellite sequences are not detectable in P. nettingi, P. n. hubrichti, or P. dorsalis. The latter species has a heavy satellite with a density of 1.718 g/cm³, representing about 8% of the genomic DNA, and two light satellites whose properties have not been investigated. The heavy satellite of P. dorsalis is cytologically localized in the centromeric heterochromatin of this species. — These observations are discussed in relation to the function and evolution of highly repetitive DNA sequences in the centromeric heterochromatin of salamanders and other organisms.     

Longitudinal differentiation of the human Yq heterochromatin as revealed by the restriction enzyme TaqI

The restriction endonuclease TaqI cleaves DNA at TCGA sites which are very common in human satellite DNAs. However, this enzyme was not used successfully up to now to digest constitutive heterochromatin of human chromosomes, where those highly repetitive DNAs are preferentially located. In this work, we show that TaqI is able to cut and extract DNA from the major heterochromatic regions on chromosomes 1, 9, 15, and 16 which appear as unstained gaps. Yq heterochromatin displays moderate digestion along its entire length but a middle region can be distinguished which is usually more affected. Complete digestion of Yq heterochromatin can be achieved when this block has been previously undercondensed by treating cell cultures with the cytidine analog, 5-azacytidine. Thus, it may be deduced that some factors related to chromatin organization might be involved in the action of TaqI. These results come to reinforce previous data about heterogeneity of Yq heterochromatin, and allow us to subdivide it into three different regions according to their differential response to TaqI digestion.     

Homologous and non-homologous chromosome associations by interchromosomal chromatin connectives in Ornithogalum virens

Based on interchromosomal chromatin connectives, a statistical analysis of homologous and non-homologous chromosome associations was made on mitotic metaphase chromosomes of Ornithogalum virens. The great majority of connectives involve constitutive heterochromatin, and connections between homologous chromosomes are twice as common as would be expected by chance. It is suggested that constitutive heterochromatin with similar DNA is involved in both homologous and non-homologous chromosome associations.     

Localization of Drosophila nasutoides satellite DNAs in metaphase chromosomes

Metaphase chromosomes of D. nasutoides were hybridized situ with ³H-cRNA synthesized from the four satellites which make up 50–60% of the total DNA of this species. All four satellites were localized in the large, metacentric, heterochromatic chromosome four. They did not, however, appear to hybridize to centromeric or other constitutive heterochromatin, nor did they, with the exception of satellite I, seem to hybridize in the specific regions of chromosome four which, on the basis of C, Q, and H banding and AT contents, were predicted to contain some of these satellites. —Comparison of grain patterns with the results of fluorescent staining indicated that satellite-bearing heterochromatin was not always associated with other fractions of constitutive heterochromatin in interphase nuclei and was, at least partially, decondensed in some larger nuclei.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro.

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.     

Heterochromatin and histone phosphorylation

Histone phosphorylation and nuclear structure have been compared in cultured cell lines of two related species of deer mice, Peromyscus crinitus and Peromyscus eremicus, which differ greatly in their heterochromatin contents but which contain essentially the same euchromatin content. Flow microfluorometry measurements indicated that P. eremicus contained 36% more DNA than did P. crinitus, and C-band chromosome staining indicated that the extra DNA of P. eremicus existed as constitutive heterochromatin. Two striking differences in interphase nuclear structure were observed by electron microscopy. Peromyscus crinitus nuclei contained small clumps of heterochromatin and a loose, amorphous nucleolus, while P. eremicus nuclei contained large, dense clumps of heterochromatin and a densely structured, well defined, nucleolonema form of nucleolus. Incorporation of ³²PO₄ into histones indicated that the steady-state phosphorylation of H1 was identical in P. crinitus and P. eremicus cells. In contrast, the phosphorylation rate of H2a was 58% greater in the highly heterochromatic chromatin of P. eremicus cells than in the lesser heterochromatic chromatin of P. crinitus cells, suggesting an involvement of H2a phosphorylation in heterochromatin structure. It is suggested that the three histone phosphorylations related to cell growth (H1, H2a, and H3) may be associated with different levels of chromatin organization: H1 interphase phosphorylation with some submicroscopic (molecular) level of organization, H2a phosphorylation with a higher level of chromatin organization found in heterochromatin, and H3 and H1 superphosphorylation with the highest level of chromatin organization observed in condensed chromosomes.     

Chromatin fractionation of normal and virus-transformed cells in culture

A simple technique for the obtaining of purified chromatin fractions from mammalian cells in culture is described. The procedure involves the isolation of clean nuclei in 0.30 M sucrose, 1.5 mM MgCl₂, 0.2 mM CaCl₂, 0.01 M Tris HCl pH 7.2, followed by sonication and differential centrifugation to collect specific chromatin fractions. Heterochromatin of SV-40 and Rous sarcoma virus transformed 3T3 cells showed a 6- to 7-fold enrichment in satellite DNA while a 2- to 3-fold enrichment in repetitive DNA was obtained in established and RSV transformed cells of Microtus agrestis. This method will facilitate the search for the site of integration of oncogenic viruses in the chromatin of mammalian cells.     

Rapid,localized amplification of a unique satellite DNA family in the rodent Microtus chrotorrhinus

A novel satellite DNA family (called MSAT-2570) was isolated and characterized from the rodent Microtus chrotorrhinus. With a length of 2,570 bp the repeat unit is among the largest yet reported in mammals and comprises a series of short direct and inverted repeats. These repeat motifs may prevent nucleosome formation or represent an endless source of genetic variation. Restriction enzyme digestion using the two pairs of isoschizomers HpaII/MspI and MboI/Sau3AI demonstrated tissue specific differences in satellite DNA methylation that may reflect variable chromatin conformation or differences in patterns of gene expression. The sex chromosomes of M. chrotorrhinus are unusually large in size among mammals, comprising 15%–20% of the karyotype and containing large blocks of heterochromatin. In situ hybridization of the satellite DNa revealed chromosomal localization predominantly to sex chromosome heterochromatin. A survey of related rodents including three congeneric species also with giant sized sex chromosomes demonstrated that MSAT-2570 is present only in the genome of M. chrotorrhinus. However, another previously reported satellite DNA also isolated from M. chrotorrhinus has been shown to reside on sex chromosome heterochromatin in one of the other three species, indicating that these giant blocks of heterochromatin are complex in structure and comprise multiple, unrelatined satellite DNA families.     

The reaction to c-banding of c-banded constituents during mitotic cycle ofCrepis capillaris

The reaction to C-banding was investigated throughout the mitotic cycle ofCrepis capillaris (2n=6): (1) 18–22 C-bodies or C-bands were found during mid telophase and interphase to prophase and metaphase, and also 12–14 at late anaphase to early telophase in the mitotic cycle. Fewer C-bands in late anaphase to early telophase were due to the absence of minute bands; (2) large and medium sized C-bands were strongly stained by Giemsa, while small and minute bands stained palely. It is suggested that inCrepis capillaris the difference of color in C-banded segments following Giemsa staining is referable to the amount of constitutive heterochromatin rather than to the difference in the condensation and decondensation; (3) the size of C-bodies changed during telophase to interphase and prophase. It is inferred that the extent of C-bodies is regulated by both the length of DNA sequences of constitutive heterochromatin and the amount of proteins combined with C-banded DNA. It was shown that the reaction to C-banding is neither due to the differential condensation of chromatin nor to a higher concentration of DNA in the C-banded regions, in the C-banding mechanism as has been suggested so far at least.     

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae)

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl. endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation. Received: 24 April 1997 / Accepted: 28 July 1997     

Heterochromatin diversity in Cymbidium, and its relationship to differential DNA replication

1. 1. DNA was extracted from aseptical cultures of protocorms of the orchid Cymbidium and analysed by thermal denaturation. The denaturation profiles revealed an AT-rich fraction of about 18% of total DNA.

2. 2. Mitotic chromosomes and diploid and endopolyploid nuclei of in vitro cultured protocorms and root tips were differentially stained with quinacrine (Q), 33258 Hoechst (H) and a novel compound 4′-6-diamidino-2-phenylindole (DAPI) [10], as well as by the Giemsa C-banding technique. The centromeric regions display very bright fluorescence with all three fluorochromes and stain intensely following the Giemsa procedure. It is proposed that the AT-rich fraction of the Cymbidium DNA is located within the centromeric heterochromatin.

3. 3. In interphase nuclei differential Q, H, and DAPI fluorescence both within and between the chromocenters occurs. In nuclei with enlarged chromocenters, i.e. with amplified DNA in heterochromatin [2], the increased size of chromocenters is mainly caused by enlargement of the less brightly fluorescing fractions of the heterochromatin. The proportion of the very brightly fluorescing heterochromatin is similar in all nuclei (about 7%).

4. 4. A comparison of nucleolar size and differential Giemsa staining of the nucleolus organizers showed that there is no disproportional increase of nucleoli and nucleolus organizing heterochromatin during endopolyploidization. That means, there is no indication for amplification of ribosomal DNA.

5. 5. Electron micrographs particularly of heterochromatin-rich nuclei revealed areas of different chromatin density within and between the chromocenters. However, the differences in fiber packaging density are much smaller than the observed differences in fluorescence brightness.

6. 6. The data obtained are interpreted as evidence for differential replication of AT-rich and non-AT-rich heterochromatin. It is suggested that DNA amplification [2, 4] is restricted to a non-AT-rich component which apparently is located neither in the brightly fluorescent centromeric nor in the nucleolus-associated heterochromatin.

     

The repeated DNA sequences of Microtinae : I. Microtus agrestis, Microtus pennsylvanicus and Ellobius lutescens

A comparison has been made of the repeated nucleotide sequences from 3 Microtinae which possess varying amounts of constitutive heterochromatin per cell nucleus. Eight repetitive fractions of DNA, ranging in C_ot values from 10⁻³ to 10⁻³, were obtained by reassociation of sheared, denatured DNA and fractionation on hydroxyapatite. At C_ot values of less than 1, 3 fractions were isolated that amounted to 18.7, 10.0 and 7.4 % of the total DNA of Microtus agrestis, Microtus pennsylvanicus and Ellobius lutescens, respectively, in agreement with the amounts of heterochromatin in these species. At C_ot values higher than 1, the amounts of repeated sequences were more comparable and constituted about 12 to 14 % of the DNA. Thermal denaturation profiles of all the repetitive fractions showed a good deal of order in the reassociated duplexes, with an average hyperchromicity of 20 %. Upon density gradient centrifugation in neutral CsCl, the fractions from M. pennsylvanicus and E. lutescens yielded almost identical patterns and differed significantly from those of M. agrestis. In M. agrestis a fraction of fast-intermediate repetitiveness (reassociating at C_ot values between 10⁻² and 1) was isolated, amounting to about 12 % of the total DNA. This fraction has a base composition comparable to that of total DNA and represents the major component of the constitutive heterochromatin of giant sex chromosomes that have been isolated by the disruption of brain and liver nuclei and differential centrifugation.     

Restrictive distribution of asymmetric C-bands in the chromosome complement of the rhesus (Macaca mulatto)

Lymphocyte chromosomes from a cercopithecoid species, Macaca mulatta, were studied for the occurrence of lateral asymmetry in constitutive heterochromatin. The technique consisted of growing the lymphocytes for one cell cycle in BrdUrd, staining with 33258 Hoechst, exposing them to UV light, treating them with 2 SSC and staining with Giemsa. This procedure revealed asymmetric staining in the region of constitutive heterochromatin of the nucleolar organizer marker chromosome (no. 13 of the complement). In these chromosomes, the darkly staining region was confined at any given point to a single chromatid, while the corresponding region on the sister chromatid was lightly stained. This pattern of asymmetric staining in the constitutive heterochromatic region was not observed in any other chromosome of Macaca mulatta. The lateral asymmetry of constitutive heterochromatin in this species is presumed to reflect the strand bias in the distribution of thymine in the alphoid DNA fractions.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.