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.     

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

Sequential chromosome banding and in situ hybridization analysis.

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.     

On the chromatin structure of eukaryotic telomeres

Telomeres prevent chromosome fusions and degradation by exonucleases and are implicated in DNA repair, homologous recombination, chromosome pairing and segregation. All these functions of telomeres require the integrity of their chromatin structure, which has been traditionally considered as heterochromatic. In agreement with this idea, different studies have reported that telomeres associate with heterochromatic marks. However, these studies addressed simultaneously the chromatin structures of telomeres and subtelomeric regions or the chromatin structure of telomeres and Interstitial Telomeric Sequences (ITSs). The independent analysis of Arabidopsis telomeres, subtelomeric regions and ITSs has allowed the discovery of euchromatic telomeres. In Arabidopsis, whereas subtelomeric regions and ITSs associate with heterochromatic marks, telomeres exhibit euchromatic features. We think that this scenario could be found in other model systems if the chromatin organizations of telomeres, subtelomeric regions and ITSs are independently analyzed.     

Relation between the SCE points and the DNA replication bands

A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication bands (=euchromatic segments=negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.     

Differentielle Darstellung der Metaphasechromosomen von Cypripedium debile mit Chinacrin- und Giemsa-Färbung

The somatic metaphase chromosomes of C. debile stain differentially with quinacrine and Giemsa respectively. After quinacrine staining the heterochromatic regions show fainter fluorescence than euchromatic regions. With Giemsa the heterochromatic regions are more deeply stained than the euchromatic regions. This is true also for slides which were pre-treated with trypsin or trichloroacetic acid. From these findings it may be supposed, that the heterochromatic regions are G-C rich. Furthermore, differences in protein composition are expected between hetero- and euchromatic regions. The results of differential staining of metaphase chromosomes of C. debile, including those which were published in previous papers, are summarized here.     

Banding pattern analysis of initial structural chromosome alterations induced by n-methyl-n'-nitro-n-nitrosoguanidine in Syrian hamster cells

Cultured secondary Syrian hamster embryo cells exposed to 0.5 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) microgram/ml medium exhibited chromatid type of aberrations consisting of gaps, breaks and exchanges. Although no specific chromosome or chromosome segment was preferentially affected, chromosomes belonging to the larger groups tended to more often involved. G-band analysis demonstrated that 80% of the lesions occurred in negative bands, 9% involved the centromere, 3% were on non-banded heterochromatin, and approximately 8% of the lesions could not be definitely categorized by G-band analysis. Whether the lesions occur at positive bands or at the interface between negative and positive bands is difficult to discern by the G-band resolution. The Y chromosome compared to autosomes of similar size rarely had lesions. X chromosome damage was found in both the euchromatic and heterochromatic arms. However, both sex chromosomes, as well as an autosome (E20) which is heterochromatic on its long arm, were not found joined to the chromatids of other chromosomes, further emphasizing that chromosomes with large heterochromatic areas are isolated in terms of chromatid exchange events. The analysis of MNNG induced chromosome damage indicates that the negative bands are the primary site of damage and points of exchange.     

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.     

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.     

Direct FISH of 5S rDNA on tomato pachytene chromosomes places the gene at the heterochromatic knob immediately adjacent to the centromere of chromosome 1.

We describe a molecular cytogenetic procedure for high resolution physical mapping of DNA markers, an essential step toward construction of an integrated molecular-classical-cytological map. Tomato was selected as material because its pachytene chromosomes are amenable for study and because detailed molecular, classical, and cytological maps are available. Karyotyping of acetocarmine-stained pachytene chromosomes showing detailed cytogenetic landmarks was combined with direct FISH of the 5S rDNA gene. This enabled us to pinpoint the 5S rDNA gene to the first heterochromatic knob immediately adjacent to the centromere in the short arm of chromosome 1. Thus the position of the 5S rDNA gene on the molecular map was related to the position of the 5S rDNA on the cytogenetic map. The results also provide conclusive evidence of the location of a functional gene in the pericentric heterochromatic region, a rare event to date in plants. We conclude that karyotyping of pachytene chromosomes can be combined with FISH to map a DNA sequence to a cytogenetically defined region and to determine the chromatin origin of an expressed gene. Key words : direct fluorescence in situ hybridization, 5S rDNA, pachytene chromosomes, heterochromatic gene.     

Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. Here, we ask how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. We show that the knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, we observed bipolar spindles in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4^th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis.     

A Technique for C-Banding Plant Chromosomes with Wright Stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.     

The Paf1 complex factors Leo1 and Paf1 promote local histone turnover to modulate chromatin states in fission yeast

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1–Paf1 [a subcomplex of the RNA polymerase II‐associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high‐resolution genomewide ChIP (ChIP–exo). Loss of Leo1–Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi‐independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1–Paf1 promotes chromatin state fluctuations by enhancing histone turnover.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

A technique for C-banding plant chromosomes with Wright stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.