The use of distamycin A in human lymphocyte cultures

Summary The effect of the oligopeptide antibiotic distamycin A on human lymphocyte cultures was examined. Distamycin A specifically inhibits the condensation of the Y heterochromatin and induces a fragile site in the chromosome 16 (band q22) in some individuals. The optimal culture conditions under which an undercondensation of the Y heterochromatin and an induction of the fragile site in 16q22 can be achieved by in vitro treatment of lymphocytes were determined. This also permits the use of distamycin A in routine diagnostics of human chromosomes. The use of this technique in the analysis of translocations involving the Y chromosome is presented. The distamycin A-DNA interaction and the different possible explanations for the distamycin A-induced undercondensations of the Y heterochromatin and fragile sites 16q22 are discussed.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.     

A direct demonstration of somatically paired heterochromatin of human chromosomes

Human lymphocyte cultures were treated with different concentrations of 5-azacytidine for various lengths of time. This cytosine analog induces very distinct undercondensation in the heterochromatin of chromosomes 1, 9, 15, 16, and Y if applied in low doses during the last hours of culture. These regions are further distinguished by their intense distamycin A/DAPI-staining and highly methylated DNA. In interphase nuclei, these heterochromatic regions are frequently somatically paired. These somatic pairings are preserved up to the metaphase stage in the 5-azacytidine-treated cultures and are thus susceptible to direct analysis. The specific effect of 5-azacytidine on the heterochromatin of these chromosomes, its conserving effect on somatic pairing, and some of the consequences of the somatic pairing on the development of human chromosome aberrations are discussed.     

The effect of distamycin A on heterochromatin condensation of Drosophila chromosomes

Larval neuroblasts of four species of Drosophila (melanogaster, hydei, virilis and funebris) were treated with distamycin A, a DNA ligand which induces distinct undercondensation in AT-rich heterochromatin. For each species the patterns of undercondensation were correlated with distribution of quinacrine-bright regions and of satellite DNAs. An overlapping of distamycin A-sensitive and quinacrine-bright heterochromatic regions was demonstrated for D. melanogaster and D. virilis, but not for D. hydei and D. funebris. Distamycin A undercondensation is thus a further criterion for resolving heterochromatin into different parts and enables identification of the steps of the condensation process within heterochromatic regions.     

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.     

Discontinuous undercondensation of centromeric heterochromatin in mouse chromosomes: evidence in Hoechst 33258-treated cells.

Mouse chromosomes from the L929 cell line have been treated with Hoechst 33258 to induce undercondensation of centromeric heterochromatin. The morphological changes induced by this fluorochrome were analyzed in electron micrographs of whole-mounted chromosomes. Results show that the condensation inhibition of centromeric heterochromatin caused by Hoechst 33258 is not produced homogeneously and suggest compositional differences within an individual centromere.     

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.     

DA/DAPI-Fluorescent heteromorphism of human Y chromosome

Summary Variation of DA/DAPI intensity in the Yq12 band was observed in five amniotic cell specimens and one blood specimen from the father of one fetus. Three distinct classes of Yq heterochromatin were identified by distamycin A (DA) treatment of the cell cultures and various staining techniques. The heterochromatin in the Yq11.23 sub-band does not under-condense when exposed to DA, and shows pale fluorescence with quinacrine staining, positive C-banding, and bright fluorescence with DA/DAPI technique. This class of heterochromatin was consistently observed in all specimens studied. The other two classes of heterochromatin are in the Yq12 band. Both show undercondensation when exposed to DA, quinacrine-bright fluorescence, and positive C-banding; howover, one class of heterochromatin shows DA/DAPI-bright fluorescence and the other shows pale fluorescence. The size and banding intensity of the two classes of heterochromatin in Yq12 are variable. These results provide cytological evidence of heterogeneity within the Y heterochromatin region containing AT-rich DNA.     

白颊长臂猿染色体的研究

本文对我国云南南部的白须长臂猿（Ｈ．ｌｅｕｃｏｇｅｎｙｓ）染色体的Ｇ带、Ｃ带、晚复制带及Ａｇ－ＮＯＲｓ进行了较为详细的研究。它的２ｎ＝５２,核型公式为４４（Ｍ或ＳＭ）＋６（Ａ）,ＸＹ（Ｍ,Ａ）。Ｃ带表明一些染色体着丝点Ｃ带弱化;有的染色体出现插入的和端位的Ｃ带;Ｘ染色体两臂有端位Ｃ带,Ｙ染色体是Ｃ带阳性和晚复制的。Ａｇ－ＮＯＲｓ的数目,雌体有４个,雄体有５个,Ｙ染色体上具ＮＯＲ。本文对白颊长臂猿与其它长臂猿间的亲缘关系、核型进化的可能途径进行了讨论。     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Demonstration of Y/autosomal translocations using distamycin A

Summary The condensation of the brightly fluorescing Y-heterochromatin is prevented by cultivating leukocytes in a medium containing distamycin A. This technique provides a reliable method for the identification of those Y/autosomal translocations in which Y heterochromatin is involved. A case of a familiar Y/22 translocation and the distamycin A technique are described.     

Differential condensation of human chromosomes 9 and Y

Some differences were observed in the mitotic condensation of regions composing human chromosomes 9 and Y: regions 9p, 9h and Y nf are characterized by an intense condensation by the end of the spiralization interval studied (the length of the repair chromosome 3 varying from 5.4 to 2.9 mkm). At the same time, the condensation of regions 9q-h (region 9q without heterochromatic block) is slowing in the initial spiralization interval (the length of chromosome 3 varying from 16.6 to 5.5 mkm). The Yf-block of Y-chromosome is condensing faster than nf-region. The condensation parameters of Q-heterochromatic blocks are most variable while the euchromatic regions are most stable. The dynamics of 9h and of f-block condensation are independent within one karyotype. Based on the data obtained we doubt the correctness of studies on linear dimensions of the constitutive heterochromatin blocks for the evaluation of its quantity in the karyotype. A possible association of differential mitotic condensation with the chromosome segregation disturbances is discussed.     

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.     

5-Azacytidine-induced undercondensations in human chromosomes

Summary The cytosine analogue 5-azacytidine induces very distinct undercondensations in human chromosomes if applied to lymphocyte cultures. The number of induced undercondensations and their chromosomal localization can be varied by the 5-azacytidine dose and the treatment time. Pulverized chromosomes or undercondensations in the G-band-positive chromosome regions are produced with high doses and long treatment times. If applied in low doses during the last hours of culture, 5-azacytidine induces specific undercondensations in the heterochromatin of chromosomes 1, 9, 15, 16, and Y. Optimum conditions required for inducing the various types of undercondensation in the chromosomes were determined. Various examples of the use 5-azacytidine in the analysis of chromosome rearrangements involving heterochromatic regions are presented.     

Hypersensitivity to mitomycin C cell-killing in Roberts syndrome fibroblasts with, but not without, the heterochromatin abnormality

Roberts syndrome (RS) is a rare genetic disorder, characterized clinically by severe pre- and post-natal growth retardation and symmetric limb reduction deformities. Some patients with RS have a distinctive abnormality of the constitutive heterochromatin (the RS effect) which has been described as a premature separation of the paracentromeric and nucleolar organizing regions of the chromosomes and the distal portion of the long arm of the Y chromosome (German, 1979). These patients [denoted RS(+)] are clinically indistinguishable from the RS(-) patients who lack the cytogenetic marker for Roberts syndrome. Recently, a mutant in Drosophila has been described which has both heterochromatin undercondensation and hypersensitivity to mutagen treatment (Gatti et al., 1983). The authors suggested that the uncondensed heterochromatin may be more accessible to damage by mutagens. Thus, the present study was an investigation of the mutagen sensitivity in Roberts syndrome, to determine whether there is a similar relationship between abnormal heterochromatin structure and mutagen sensitivity. Plating efficiency experiments were performed with RS(+) fibroblasts, RS(-) fibroblasts, RS heterozygous fibroblasts and a large assortment of appropriate control cells. The RS fibroblasts with the heterochromatin abnormality were consistently more sensitive (based on D10 values) to mitomycin C treatment than were any of the other cell strains tested, including RS(-) cells. These results support the hypothesis that mitomycin C sensitivity and abnormal heterochromatin structure in Roberts syndrome are related.     

Preferential somatic pairing between homologous heterochromatic regions of human chromosomes.

The cytidine analog 5-azacytidine (5-azaC) induces an undercondensation of the heterochromatin in human chromosomes 1, 9, 15, 16, and Y when it is added in low concentrations to the late S-phase of growing lymphocyte cultures. In interphase nuclei, these heterochromatic regions are frequently somatically paired. The somatic pairing configurations are preserved up to metaphase stage in the 5-azaC-treated cultures and are thus susceptible to a direct microscopical examination. The statistical analysis of 1,000 somatic pairing configurations from 5-azaC-treated cells showed that the somatic pairing between the heterochromatic regions of homologous chromosomes is preferred over that between nonhomologous chromosomes.     

Inhibition of heterochromatin condensation of human Y chromosome by distamycin-A

Distamycin-A, an oligopeptide antibiotic with a N-methylpyrrole ring system and propionamide side chain, preferentially forms stable bonds with AT rich double stranded DNA. When introduced to cell cultures, it inhibits condensation of the heterochromatic region of the Y chromosome. The frequency of metaphases showing inhibition of heterochromatin condensation of the Y chromosome was found to be dependent on the treatment time and concentration of distamycin-A in the culture medium. When distamycin-A was added to a concentration of 100 micrograms/ml at the start of the culture (72 hours), the frequency of Y heterochromatin decondensation was found to be 48%, 30% and 6% in amniotic fluid, lymphocyte and fibroblast cultures respectively. The highest frequency of metaphases with decondensed Y heterochromatin were observed when distamycin-A treatment was carried out for the last 24 hours prior to harvest, the frequencies being 94%, 72% and 59% in amniotic fluid, lymphocyte and fibroblast cultures respectively. Increase in the concentration of distamycin-A from 25 micrograms/ml to 50 micrograms/ml during the last 24 hours of culture increased the incidence of metaphases with Y heterochromatin decondensation from 51% to 69% in amniotic fluid, 40 to 49% in lymphocyte and 29% to 31% in fibroblast cultures. Highest frequency of metaphases with Y heterochromatin decondensation were observed when the cultures were exposed to distamycin-A at a concentration of 100 micrograms/ml for the last 24 hours of culture.     

Cathepsin L stabilizes the histone modification landscape on the Y chromosome and pericentromeric heterochromatin

Posttranslational histone modifications and histone variants form a unique epigenetic landscape on mammalian chromosomes where the principal epigenetic heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are inversely localized in relation to each other. Trimethylated H3(K9) marks pericentromeric constitutive heterochromatin and the male Y chromosome, while H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a lysosomal and nuclear protease, cathepsin L, causes a global redistribution of epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated H3(K9) decrease dramatically, concomitant with its relocation away from heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin and the Y chromosome. This change is also associated with global relocation of heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from constitutive heterochromatin; however, it does not affect DNA methylation or chromosome segregation, phenotypes commonly associated with impaired histone H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants can dynamically redistribute depending on physiological context but still maintain the essential function(s) of chromosomes. Thus, our data show that cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric heterochromatin and the Y chromosome through a novel mechanism that does not involve DNA methylation or affect heterochromatin structure and operates on both somatic and sex chromosomes.     

Chromatin condensation behaviour of the Y chromosomes in the human testis

The condensation behaviour of the human Y chromosome in germ cells and Sertoli cells of pre- and post-pubertal testes was followed by fluorescence in situ hybridisation using probes for three different regions of the Y chromosome. Patterns of expansion or contraction of signal are taken to reflect degrees of condensation of the related Y chromatin and hence its potential for genetic activity. For probe pHY2.1, which labels the distal non-fluorescent and fluorescent heterochromatin of the Y chromosome (Yq12), an expanded signal seen in gonocytes of the prepubertal testis is superseded by a condensed signal seen in adult germ cells at all but the zygotene stage of meiotic prophase when meiotic pairing takes place. In contrast, Sertoli cells show a condensed signal pre-pubertally but a greatly expanded signal in the adult testis. A totally condensed pHY2.1 signal is found in a chromosomally normal man with Sertoli-cell-only syndrome. It is hypothesised that control over at least some facets of spermatogenesis may not, in the adult, be autonomous to the germ cells, but rather may emanate from the Sertoli cells. Chromatin expansion at zygotene could, however, be important for pairing and crossing over in the XY bivalent, successful synapsis ensuring survival of spermatocytes into the post-meiotic stages.     

Loss of Drosophila Myb interrupts the progression of chromosome condensation

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.