5-Azadeoxycytidine induced undercondensation in the giant X chromosomes of Microtus agrestis

Fibroblasts of female Microtus agrestis were treated with 5-azadeoxycytidine (5-aza-dCyd) at a final concentration of 10^–5 M during the last 2 h of culture. This cytidine analogue induces distinct undercondensation of the constitutive heterochromatin in the giant X chromosomes. The undercondensed heterochromatic thread exhibits longitudinal segmentation reminiscent of a chromomere pattern. In the late-replicating X chromosome, 5-aza-dCyd also inhibits condensation of the genetically inactivated euchromatin (facultative heterochromatin). The described effects of 5-aza-dCyd on the X chromosome structure appear to be incorporation independent.     

The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.     

Distribution of 5-methylcytosine-rich regions in the polytene chromosomes of Phaseolus coccineus embryo suspensor as shown by the immunoperoxidase technique

5-Methylcytosine (5-mC) has been visualized in polytene chromosomes of Phaseolus coccineus, scarlet bean using specific antibodies to 5-mC and the immunoperoxidase technique. The results obtained indicate that most heterochromatic regions are methylated, even though the frequency of methylation is highly variable and sometimes low. A preferential binding of anti-5-mC to centromeric heterochromatic blocks was observed. Comparison between anti-5-mC binding and the results of hybridization with highly repetitive DNA and satellite DNA shows, moreover, that centrometric heterochromatic regions hybridize in particular with both DNAs. This finding is consistent with the fact that repetitive DNA and satellite DNA are methylated to a considerably greater extent than main band DNA, in line with many data to be found in the literature. The binding pattern of anti-5-mC that we observed also suggests that methylation does not occur in all classes of repetitive DNA. The high variability of band methylation frequency is discussed in relation to a possible characteristic DNA composition of the band.     

EM autoradiographic studies on polytene nuclei of Drosophila melanogaster

Replication in the chromocentre heterochromatin of salivary gland polytene nuclei of Drosophila melanogaster has been examined by ³H-thymidine EM autoradiography. In vitro pulse labelling of salivary glands from late third instar larvae showed that the chromocentre heterochromatin replicates in synchrony with the euchromatin in the nucleus. Within the chromocentre region, the central compact mass, identified earlier as the alpha heterochromatin, did not incorporate ³H-thymidine at any stage of the S, while the surrounding beta heterochromatin was always labelled in nuclei with labelled euchromatin. In a second set of experiments, growing larvae from just after hatching till late third instar stages, were fed on food containing ³H-thymidine, and at the end of larval life, the incorporation in salivary gland nuclei was examined by EM autoradiography. A grain density analysis of the EM autoradiographs revealed that the alpha heterochromatin does not replicate at all from after hatching till late third instar while the beta heterochromatin replicates as much as the euchromatin. Non-replication of the alpha heterochromatin provides the explanation for the lowered amount of heterochromatin in the polytene nuclei compared to their diploid counterparts. Implications of these observations on the organization of chromocentre heterochromatin in polytene nuclei and its homology to the heterochromatic regions in mitotic chromosomes are discussed.     

Epigenetic characterization of chromatin in cycling cells of pedunculate oak, Quercus robur L.

Cycling cells of Quercus robur have a simple nuclear organization where most of the heterochromatin is visible as DAPI-positive chromocenters, which correspond to DAPI bands at the (peri)centromeric region of each of the 24 chromosomes of the oak complement. Immunofluorescence using 5-mC revealed dispersed distribution of the signal throughout the interphase nucleus/chromosomes without enrichment within DAPI-positive chromocenters/bands, suggesting that DNA methylation was not restricted to constitutive heterochromatin, but was associated with both euchromatic and heterochromatic domains. While H3K9ac exhibited typical euchromatin-specific distribution, the distributional pattern of histone methylation marks H3K9me1, H3K27me2, and H3K4me3 showed some specificity. The H3K9me1 and H3K27me2, both heterochromatin-associated marks, were not restricted to chromocenters, but showed additional dispersed distribution within euchromatin, while H3K27me2 mark also clustered in foci that did not co-localize with chromocenters. Surprisingly, even though H3K4me3 was distributed in the entire chromatin, many chromocenters were enriched with this euchromatin-specific modification. We discuss the distribution of the epigenetic marks in the context of the genome composition and lifestyle of Q. robur.     

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.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

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.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization

The endosperm is a seed tissue unique to flowering plants. Due to its central role in nourishing and protecting the embryo, endosperm development is subject to parental conflicts and adaptive processes, which led to the evolution of parent-of-origin-dependent gene regulation. The role of higher-order chromatin organization in regulating the endosperm genome was long ignored due to technical hindrance. We developed a combination of approaches to analyze nuclear structure and chromatin organization in Arabidopsis thaliana endosperm. Endosperm nuclei showed a less condensed chromatin than other types of nuclei and a peculiar heterochromatin organization, with smaller chromocenters and additional heterochromatic foci interspersed in euchromatin. This is accompanied by a redistribution of the heterochromatin mark H3K9me1 from chromocenters toward euchromatin and interspersed heterochromatin. Thus, endosperm nuclei have a specific nuclear architecture and organization, which we interpret as a relaxed chromocenter-loop model. The analysis of endosperm with altered parental genome dosage indicated that the additional heterochromatin may be predominantly of maternal origin, suggesting differential regulation of maternal and paternal genomes, possibly linked to genome dosage regulation.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

Restriction enzyme banding and in situ nick-translation on different types of hetero- and euchromatin

We studied the role of chromatin accessibility and methylation in the banding patterns produced by means of in situ nick-translation (NT) and restriction enzyme (RE) banding techniques. For these studies we used the X chromosomes of Microtus cabrerae because of their large segment with four different types of constitutive heterochromatin and because in these chromosomes we can also compare active and inactive euchromatin. The results demonstrate that constitutive heterochromatin in the X chromosomes of M. cabrerae is methylated at specific sequences in both active and inactive Xs. They also show that NT-based techniques are suitable for detecting weak differences in chromatin accessibility, such as differences between active and inactive euchromatin, and are able to distinguish methylation only at the accessible sites. Thus, when methylation has to be mapped in situ, additional experiments have to be performed in order to distinguish findings due to differential accessibility. RE banding seems less sensitive to slight differences in chromatin accessibility, and might thus be more suitable than in situ NT-based techniques for methylation mapping. In harmony with these results, HpaII-based RE banding is able to distinguish between active and inactive euchromatin, possibly depending on its methylation status.     

Insect sex chromosomes

A presumptive mechanism of X inactivation has been investigated by using tritiated uridine-induced chromosome aberrations to distinguish active from inactive X chromosome arms in the insect Gryllotalpa fossor. Previous work on therian mammals has shown that constitutive and facultative heterochromatin are less susceptible to breakage by ³H-Urd than euchromatin (active). The present study indicates that, irrespective of the presence of two X chromosomes in females, only one of these is affected as in males and that the total number of aberrations induced by ³H-Urd in both male and female Gryllotalpa is the same. This suggests that in the female only one arm of one X chromosome is active and that a facultative heterochromatinization of the homologous arm of the other X is operative coupled with the presence of constitutive heterochromatin in the second arm of both X chromosomes.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

The replicative status of heterochromatic and euchromatic DNA in two somatic tissues of Dermestes maculatus (Dermestidae: Coleoptera)

Replication of DNA present in euchromatin and heterochromatin remains in step in most nuclei of the testis wall of Dermestes maculatus. However, in a minority class of testis wall nuclei heterochromatic DNA has undergone two or three fewer rounds of replication than DNA located in euchromatin. A similar situation exists in many nuclei of the Malpighian tubule though here there is also the possibility that the euchromatic and heterochromatic DNA components are themselves not completely replicated.     

Scanning Electron Microscopy of Heterochromatin in Chromosome Spreads of Male Germ Cells in Schistocerca gregaria (Acrididae, Orthoptera) after Trypsinization

Chromosome spreads, prepared from testes of the desert locust Schistocerca gregaria, were analyzed using scanning electron microscopy (SEM) after varying periods of preincubation in trypsin. The emphasis of the study was on the appearance of heterochromatin. A trypsin pretreatment of 5 sec resulted in a smooth surface on the chromatin throughout and the heterochromatin was highly electron-emissive. The facultatively heterochromatic X chromosome was clearly visible in interphase spermatogonia and in pachytene and late prophase I spermatocytes. Chromomeres of autosomal bivalents could be recognized in pachytene cells. Centromeric heterochromatin segments were very prominent in autosomes of late prophase I spermatocytes and some chromosomes showed interstitial and telomeric bands. Longer trypsin treatment (10 sec) resulted in a fine globular surface on the chromatin; however, the electron emission of heterochromatic chromosome segments was lower under these conditions. The result of trypsin pretreatment of euchromatin differed only slightly from that of the heterochromatin. Extensive trypsin treatment (20 sec) did not alter further the relative electron emission of heterochromatin and euchromatin, but the regular globular appearance was lost, apparently owing to damage on the chromatin surface. The loss of electron emission from the centromeric heterochromatin of the autosomes and the facultatively heterochromatic X chromosome after extended trypsin treatment suggests a central role of proteins in mediating the heterochromatic status in meiotic chromo somes of the locust. Information obtained using scanning electron microscopy of chromosome spreads is complementary to that obtained by C-banding in that facultative heterochromatin is visualized with particular clarity.     

A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

Restriction enzyme banding and in situ nick-translation on different types of hetero- and euchromatin.

We studied the role of chromatin accessibility and methylation in the banding patterns produced by means of in situ nick-translation (NT) and restriction enzyme (RE) banding techniques. For these studies we used the X chromosomes of Microtus cabrerae because of their large segment with four different types of constitutive heterochromatin and because in these chromosomes we can also compare active and inactive euchromatin. The results demonstrate that constitutive heterochromatin in the X chromosomes of M. cabrerae is methylated at specific sequences in both active and inactive Xs. They also show that NT-based techniques are suitable for detecting weak differences in chromatin accessibility, such as differences between active and inactive euchromatin, and are able to distinguish methylation only at the accessible sites. Thus, when methylation has to be mapped in situ, additional experiments have to be performed in order to distinguish findings due to differential accessibility. RE banding seems less sensitive to slight differences in chromatin accessibility, and might thus be more suitable than in situ NT-based techniques for methylation mapping. In harmony with these results, HpaII-based RE banding is able to distinguish between active and inactive euchromatin, possibly depending on its methylation status.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.