Nucleotide Composition of the Cold-Sensitive Heterochromatic Regions in Paris hainanensis Merrill

Cold-induced decondensation of heterochromatic regions (CSR-bands) in Paris hainanensis(=Daiswa hainanensisMerrill Takht.) (2n= 10; 10 + b) was studied. The comparison of CSR-banding patterns with those obtained by nucleotide-specific staining with fluorochromes DAPI and chromomycin A₃demonstrated that low temperatures induced decondensation only of large AT-rich heterochromatic regions. It is suggested that this is characteristic of all plant species.     

The heterochromatin regions of the mitotic chromosomes in cattle]

Cytochemical and molecular peculiarities of heterochromatic regions of bovine chromosomes have been studied, using specific fluorochrome staining induced decondensation, in situ hybridization, pretreatment of restriction enzymes. The heterochromatin of autosomes demonstrated a strong homogeneity. In chromosome Y two small specific heterochromatic regions were found lacking a long repeated tandem block of nucleotides enriched in GC base pairs and having no tandem block of Bkm repeats (10(4) b.p.). This class repeats are probably interspersed in the bovine genome. A rather seldom character of mammalian karyotypes is the absence of cytochemical heterochromatin in the X chromosome.     

Idoxuridine induction of micronuclei containing the long or short arms of human chromosome 9

PHA-stimulated growth of human lymphocytes in the presence of idoxuridine (IUdR) results in chromosomal decondensation and fragility of large heterochromatic regions. This instability is especially evident in the heterochromatic region of chromosome 9 (9h). A high frequency of micronuclei is seen in all IUdR-treated cultures. By a combination of chromosomal localization of induced aberrations, analysis of metaphases with prematurely condensed micronuclear chromatin, and specific staining of 9h in interphase micronuclei, it can be shown that 80-90% of all micronuclei contains 9h material. This pattern is found whether the heterochromatic region is situated on the long arm or the short arm of chromosome 9. These observations suggest that IUdR-induced micronucleation may be a valuable method for separation of the long and short arms of human chromosome 9.     

Characterization of Drosophila heterochromatin

A number of preliminary experiments have shown that the fluorescence pattern of Hoechst 33258, as opposed to that of quinacrine, varies with the concentration of dye. The metaphase chromosomes of D. melanogaster, D. simulans, D. virilis, D. texana, D. hydei and D. ezoana have therefore been stained with two concentrations of H 33258 (0.05 and 0.5 mug/ml in phosphate buffer at pH 7) and with a single concentration of quinacrine (0.5% in absolute alcohol). The three fluorescence patterns so obtained were shown to be somewhat different in some of the species and the coincide in others. All three stainings gave an excellent longitudinal differentiation of heterochromatin while euchromatin fluoresced homogeneously. Living ganglion cells of the six species mentioned above were treated with quinacrine and H 33258. Quinacrine induced a generalized lengthening and swelling of the chromosomes and H 33258 the decondensation of specific heterochromatic regions. A correlation of the base composition of the satellite DNAs contained in the heterochromatin of the species studied with the relative fluorescence and decondensation patterns showed that: 1) the extremely fluorochrome bright areas and those decondensed are present only in species containing AT rich satellite DNA; 2) the opposite is not true since some AT-rich satellite DNAs are neither fluorochrome bright nor decondensed; 3) there is no good correspondence between Hoechst bright areas and the decondensed ones. AT richness therefore appears to be a necessary but not sufficient condition both for bright fluorescence and decondensation. Some cytological evidence suggests that similarly AT rich satellite DNAs respond differently in fluorescence and decondensation because they are bound to different chromosomal proteins. A combination of the results of fluorescence and decondensation revealed at least 14 types of heterochromatin; 4-7 of which are simultaneously present in the same species. Since closely related species (i.e. D. melanogaster and D. simulans; D. virilis and D. texana) show marked differences in the heterochromatic types they contain, it can be suggested that within the genus Drosophila qualitative variations of heterochromatin have played an important role in speciation.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.     

Early replication banding in <Emphasis Type="Italic">Leporinus</Emphasis> species (Osteichthyes,Characiformes) bearing differentiated sex chromosomes (ZW)

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2′-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.     

Differential reaction of condensed and diffuse chromatin to polyamines. II. Effect of putrescine on the chromatin of mitotic chromosomes]

The response of euchromatin and heterochromatin to putrescine was studied using chinese hamster mitotic chromosomes with heterochromatic segments delayed in condensation due to BUdR treatment. These heterochromatic segments did not react to the condensing effect of putrescine looking during metaphase still more elongated. Additional decondensed segments occurred in chromosomes. This is interpreted as the condensing effect of putrescine on euchromatic segments which accelerates transition of the cells into metaphase with preserved BUdR-induced chromosomal decondensation.     

DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy

The recognition and repair of DNA double-strand breaks (DSBs) occurs in the context of highly structured chromatin. Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic versus heterochromatic regions. While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of γH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Collectively, this TEM approach provides fascinating insights into the dynamic events of the DSB repair process that depend decisively upon the actual chromatin structure around the break.     

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

Heterochromatic regions in corn A- and B- chromosomes

A collection of maize forms from the Vavilov All-Russia Institute of Plant Breeding (VIR) was studied. We compared variation in the following traits: the number and size of heterochromatic knob regions (HKRs) of chromosomes of mother pollen cells at pachytene depending on the presence (B+) or absence (B0) of B-chromosomes; size of B-chromosomes (general, of heterochromatic part); and the frequency of B+ plants in groups of forms contrasting in the HKR number (10-15, 4-10, 2-7, 1-4). It was shown that B chromosomes had statistically significantly different effects on HKR polymorphism, relative heterochromatin "content" in the cell of multiknob and knobless forms, selection of plants for early ear flowering in these forms. The combination of maximum (and minimum) sizes of heterochromatic regions of A- and B-chromosomes was established; the genomic level of control of the trait is suggested. The role of the relationship of polymorphism at heterochromatic regions of A- and B-chromosomes between them and with the systems of maize reproduction is considered as a mechanism of maintenance of optimum plant heterozygosity via adaptive ontogenetic redistribution of heterochromatin among loci, chromosomes, and gametes.     

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ～ 20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces.     

Sequence-directed curvature of repetitive AluI DNA in constitutive heterochromatin of Artemia franciscana.

An Alu I family of repeated DNA sequence 113 bp in length was found to be the major component of the heterochromatin in Artemia franciscana. On the basis of the analysis of cloned oligomeric (monomer to examer) heterchromatic fragments we predicted that the sequence could produce a stable curvature in chromosomal DNA. This prediction was confirmed by polyacrylamide gel electrophoresis analysis and by electron microscope observations. The anomalous mobility of these fragments is reversed when the DNA samples are electrophoresed in the presence of distamycin A. Moreover treatment of living Artemia with this drug produces visible decondensation of heterochromatic masses in the interphase nuclei.     

Electron microscopical analysis of Drosophila polytene chromosomes

Data are presented of electron microscopic (EM) analysis of consecutive developmental stages of Drosophila melanogaster complex puffs, formed as a result of simultaneous decondensation of several bands. EM mapping principles proposed by us permitted more exact determination of the banding patterns of 19 regions in which 31 puffs develop. It is shown that 20 of them develop as a result of synchronous decondensation of two bands, 7 of three and 4 of one band. Three cases of two-band puff formation when one or both bands undergo partial decondensation are described. In the 50CF, 62CE, 63F and 71CF regions puffing zones are located closely adjacent to each other but the decondensation of separate band groups occurs at different puff stages (PS). These data are interpreted as activation of independently regulated DNA sequences. The decondensation of two or three adjacent bands during formation of the majority of the puffs occurs simultaneously in the very first stages of their development. It demonstrates synchronous activation of the material of several bands presumably affected by a common inductor. Bands adjacent to puffing centres also lose their clarity as the puff develops, probably due to "passive" decondensation connected with puff growth. The morphological data obtained suggest a complex genetic organisation of many puffs.     

Epigenetic nature of Arabidopsis thaliana telomeres

The epigenetic features of defined chromosomal domains condition their biochemical and functional properties. Therefore, there is considerable interest in studying the epigenetic marks present at relevant chromosomal loci. Telomeric regions, which include telomeres and subtelomeres, have been traditionally considered heterochromatic. However, whereas the heterochromatic nature of subtelomeres has been widely accepted, the epigenetic status of telomeres remains controversial. Here, we studied the epigenetic features of Arabidopsis (Arabidopsis thaliana) telomeres by analyzing multiple genome-wide ChIP-seq experiments. Our analyses revealed that Arabidopsis telomeres are not significantly enriched either in euchromatic marks like H3K4me2, H3K9ac, and H3K27me3 or in heterochromatic marks such as H3K27me1 and H3K9me2. Thus, telomeric regions in Arabidopsis have a bimodal chromatin organization with telomeres lacking significant levels of canonical euchromatic and heterochromatic marks followed by heterochromatic subtelomeres. Since heterochromatin is known to influence telomere function, the heterochromatic modifications present at Arabidopsis subtelomeres could play a relevant role in telomere biology.

Telomeric regions in Arabidopsis have a bimodal chromatin with telomeres lacking substantial levels of canonical euchromatic and heterochromatic marks followed by heterochromatic subtelomeres.