Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.     

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.     

Histone modifications related to chromosome silencing and elimination during male meiosis in Bengalese finch

We report here that a germline-restricted chromosome (GRC) is regularly present in males and females of the Bengalese finch (Lonchura domestica). While the GRC is euchromatic in oocytes, in spermatocytes this chromosome is cytologically seen as entirely heterochromatic and presumably inactive. The GRC is observed in the cytoplasm of secondary spermatocytes, indicating that its elimination from the nucleus occurs during the first meiotic division. By immunofluorescence on microspreads, we investigated the presence of histone H3 modifications throughout male meiosis, as well as in postmeiotic stages. We found that the GRC is highly enriched in di- and trimethylated histone H3 at lysine 9 during prophase I, in agreement with the presumed inactive state of this chromosome. At metaphase I, dimethylated histone H3 is no longer detectable on the GRC and its chromatin is more faintly stained with DAPI. The condensed GRC is underphosphorylated at serine 10 compared to the regular chromosomes during metaphase I, being phosphorylated later at this site after the first meiotic division. From these results, we proposed that trimethylation of histone H3 at lysine 9 on the GRC chromatin increases during metaphase I. This hypermethylated state at lysine 9 may preclude the phosphorylation of the adjacent serine 10 residue, providing an example of cross-talk of histone H3 modifications as described in experimental systems. The differential underphosphorylation of the GRC chromatin before elimination is interpreted as a cytologically detectable byproduct of deficient activity of Aurora B kinase, which is responsible for the phosphorylation of H3 at serine 10 during mitosis and meiosis.     

Histones H1 and H4 of surface-spread meiotic chromosomes

The chromatin conformation of somatic and meiotic chromosomes is, at least in part, a function of electrostatic nucleosome interactions that are mediated by transient acetylation of the histone H4 N-terminal domain and phosphorylation of histone H1. The distribution of those histones in the chromatin of meiotic chromosomes is reported here. Antibodies to testis-specific histone 1, H1t, detect H1t in the chromatin of mouse meiotic prophase chromosomes only after synapsis and synaptonemal complex (SC) assembly is completed and before core separation is initiated. The H1t protein is evenly distributed over euchromatin, heterochromatin and the SC. Antibodies to acetylated lysine residues 5, 12 or 16 of histone H4, indicate that the euchromatin is more acetylated than the centromeric heterochromatin. The pattern is most pronounced for acetylated residue 5 and least for 16. Antibodies to phosphorylated H1 epitopes do not react with chromatin but, instead, recognize the chromosome cores and SCs. Possibly these are not phosphorylated histone H1 epitopes, but SC proteins with similar potentially phosphorylatable sequences such as KTPTK of the synaptic protein Syn1.     

Chromosomal phylogeny of certain shrews of the genera Crocidura and Suncus (Insectivora)

High-resolution chromosome analysis of eight Palaearctic and Oriental species of white-toothed shrews reveals almost complete chromosomal homology between the karyotypes studied, and extensive G-band homology is demonstrated even between species of the genera Crocidura and Suncus . Robertsonian translocations, tandem fusions, fissions, whole-arm reciprocal translocations, centromeric shifts, heterochromatin additions, and inversions are identified as the main mechanisms of chromosomal evolution. The evolutionary relationships of the Eurasian crocidurines under study are reconstructed and a hypothetical ancestral karyotype with 44 chromosomes is proposed.     

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.     

Transmission of chromosomal abnormalities: participation of chromosomally unbalanced gametes in fertilization and early development of unbalanced embryos in the Chinese hamster

Using 14 Chinese hamster stocks with various reciprocal translocations, chromosomally unbalanced gametes were produced and used to investigate the participation of the unbalanced gametes in fertilization and the development of unbalanced embryos. The selection of chromosomally abnormal gametes during fertilization was investigated by the chromosomal analysis of meiotic cells in heterozygotes for the 14 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from meiotic metaphase II (MII) scoring, the frequencies of male pronuclei having commonly a deficiency of chromosome 1 (q14-->q42) or chromosome 3 (p23-->q31) in one-cell embryos decreased significantly. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same ones, as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results suggest that gametes with nullisomies as well as disomies for any chromosomal segments may mostly participate in fertilization, whereas some sperm nullisomic for the specific segments of chromosomes 1 and 3 may fail to fertilize. On the other hand, the zygotic selection of chromosomal imbalance was investigated by direct analyses of pre-implantation embryos from crosses between chromosomally normal females and male heterozygotes from the 14 stocks with various reciprocal translocations. The chromosomal and morphological analysis revealed that some embryos were arrested in development at the two-cell stage and their common abnormality was partial monosomy for chromosome 1 or 2. Embryos with partial monosomy including chromosomes 1, 3 and 4 showed arrested development at four-eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments, such as chromosomes 1p, 2q, 5q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between the mouse and the Chinese hamster chromosomes relating to the developmental abnormalities at early stages was partially confirmed.     

Contribution of chromosomal imbalance to sperm selection and pre-implantation loss in translocation-heterozygous Chinese hamsters

Chinese hamster stocks with various structurally abnormal chromosomes have been produced by X irradiation. Among these stocks, 18 with various reciprocal translocations were used to investigate the participation of unbalanced gametes in fertilization and the development of unbalanced embryos. Among males as well as females heterozygous for the same translocation, there is no difference in the frequency of each disjunctional class. The participation of chromosomally unbalanced gametes in fertilization was investigated by chromosomal analysis of meiotic cells in heterozygotes for the 18 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from MII scoring, the frequencies of male pronuclei having a common deficiency of chromosome 1 (1q17-->1q42) or chromosome 3 (3p23-->3q31) decreased significantly in one-cell embryos. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same abnormalities as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results revealed clearly that most gametes with nullisomies as well as disomies for any chromosomal segments may participate in fertilization, whereas only male gametes nullisomic for certain segments of chromosomes 1 and 3 failed to participate in fertilization. The zygotic selection of chromosomal imbalance was also investigated by direct chromosomal and morphological analyses of preimplantation embryos from crosses between karyotypically normal females and male heterozygotes from the 18 stocks with various reciprocal translocations. These analyses revealed that some embryos were arrested in development at the two-cell stage. The karyotype of these two-cell embryos had a common deficiency in a segment of chromosome 1 or chromosome 2. Embryos with partial monosomy including chromosomes 1, 3, 4 and 5 showed arrested development at four- to eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of segments of chromosomes 1p, 1q, 2q, 5q, 7q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between Chinese hamster and mouse chromosomes relating to abnormal embryogenesis at early stages has been partially confirmed from reported maps of chromosomes. The Chinese hamster is useful for further cytogenetic studies during the stages of meiosis and early embryogenesis.     

A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura,Dicroglossidae)

Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.     

Comparative cytogenetic study after treatment of mouse spermatogonia with mitomycin C

Male (101 × C₃H)F₁ hybrid mice, 10–12 weeks old, were injected i.p. with single doses of 2.5, 3.75 or 5.0 mg/kg of mitomycin C (MC). Spermatogonia were sample for mitotic chromosome analyses 6, 18 or 24 h after treatment. Spermatocytes were sample for meiotic chromosome analyses 50 or 60 days after treatment.The maximal yield of chromatid-type aberrations induced in spermatogonia was found 24 h after treatment with 5.0 mg/kg of MC. More than 50% of the cells carried at least one chromatid exchange. The majority (90%) of these were whole-arm exchanges derived from breaks in the centromeric heterochromatin.No translocation multivalents were found in spermatocytes analysed 50 or 60 days after treatment. The discrepancy between the presence of many symmetrical exchanges in spermatogonia and the absence of translocation multivalents in primary spermatocytes may be result of insensitivity of the stem cell spermatogonia against exchange induction by MC or of complete germinal selection against induced translocations before and/or during early meiosis. However, the possibility of missing translocations due to whole-arm exchanges in acrocentric chromosomes during the analysis of diakineses-metaphases I is also discussed.It is emphasized that comparisons of chromatid exchange frequencies in spermatogonia with the yield of translocation multivalents in spermatocytes descended from these spermatogonia as opposed to those from stem cells might provide an estimate of pre-diakinesis germinal selection against chromatid exchanges or the resulting translocations. This estimate is important for the quantitative evaluation of the genetic risk from environmental mutagens.     

Genetic induction of chromosomal rearrangements in barley chromosome 7H added to common wheat

Chromosome 2C of Aegilops cylindrica induces chromosomal rearrangements in alien chromosome addition lines, as well as in euploid lines, of common wheat. To induce chromosomal rearrangements in barley chromosome 7H, reciprocal crosses were made between a mutation-inducing common wheat line that carries a pair of 7H chromosomes and one 2C chromosome and a 7H disomic addition line of common wheat. Many shrivelled seeds were included in the progeny, which was an indication of the occurrence of chromosome mutations. The chromosomal constitution of the viable progeny was examined by FISH (fluorescence in situ hybridization) using the barley subterminal repeat HvT01 as a probe. Structural changes of chromosome 7H were found in about 15% of the progeny of the reciprocal crosses. The aberrant 7H chromosomes were characterized by a combination of N-banding, FISH and genomic in situ hybridization. Mosaicism for aberrant 7H chromosomes was observed in seven plants. In total, 89 aberrant 7H chromosomes were identified in 82 plants, seven of which had double aberrations. More than half of the plants carried a simple deletion: four short-arm telosomes, one long-arm telosome, and 45 terminal deletions (23 in the short arm, 21 in the long arm, and one involving both arms). About 40% of the aberrations represented translocations between 7H and wheat chromosomes. Twenty of the translocations had wheat centromeres, 12 the 7H centromere, with translocation points in the 7HS (five) and in the 7HL (seven), and the remaining four were of Robertsonian type, three involving 7HS and one with 7HL. In addition, one translocation had a barley segment in an intercalary position of a wheat chromosome, and two were dicentric. The breakpoints of these aberrations were distributed along the entire length of chromosome 7H.     

A reciprocal whole-arm translocation, rcp(1;6)(1p6p;1q6q) in a boar, localization of the breakpoints, and reassignment of the genes for glucose phosphate isomerase (GPI) and calcium release channel (CRC).

A reciprocal whole-arm translocation between chromosomes 1 and 6 in a Swiss Large White boar with reduced fertility was identified by the use of different staining techniques in mitotic metaphase cells, synaptonemal complex analyses, and meiotic chromosome preparations. The karyotype of this boar was demonstrated to be 38,XY,rcp(1;6)(1p6p;1q6q). To further localize the breakpoints more precisely and determine the precise gene locations, several in situ hybridization experiments were performed with a chromosome 1 centromere-specific probe and two other gene probes. The breakage and reunion points of both chromosomes were located in the centromeric regions. The genes for glucose phosphate isomerase and calcium release channel were mapped to 6cen----q12.     

Segregation of chromosomes in sperm of reciprocal translocation carriers: a review

Reciprocal translocations, the most frequent structural aberration in humans, are mainly transmitted by one of the parents. In order to analyze the chromosomal content of the spermatozoa from carriers of chromosomal reorganizations, two methods have been used, karyotyping of sperm chromosomes by the human-hamster system and fluorescence in situ hybridization (FISH) in decondensed sperm nuclei. In this work, we review 92 sperm chromosome segregation studies from 85 different reciprocal translocation carriers, including a triple translocation carrier. Using the human-hamster method, a total of 5,818 spermatozoa from 44 reciprocal translocation carriers have been analyzed, 43 of them carrying a single reciprocal translocation and one was a carrier of a double reciprocal translocation. A segregation analysis in a carrier of a t(2;22;11) has been also reported. Carrying out FISH in sperm nuclei, a total of 237,042 spermatozoa from 46 reciprocal translocation carriers have been analyzed. Six of these were also analyzed by the human-hamster system. Taking into account both methods, a total of 76 different reciprocal translocations have been studied. In 74 of these 76 translocations, the reorganization occurs between autosomes, and in the other two, the Y chromosome is involved. Although along general lines, there are similarities between the results obtained by the two methods of analysis, variations are observed when the distribution of the different types of segregations that produce imbalances is compared. As a general rule reciprocal translocation carriers produce more unbalanced sperm than normal or balanced sperm. The results reported also corroborate that the proportion of unbalanced forms depends on the characteristics of the reorganization and that it varies widely. Thus the importance of performing a detailed meiotic behavior analysis for each particular translocation in order to obtain enough information to give adequate genetic counseling is stressed. Aspects as to the possible overestimation of 3:1 segregations or the presence of interchromosomal effects still need to be elucidated.     

Histone Deacetylation is Required for Orderly Meiosis

Histone acetylation is associated with a diversity of chromatin-related processes in mitosis. However, its roles in mammalian oocyte meiosis are largely unknown. In the present study, we first investigated in detail the acetylation changes during porcine oocyte maturation using a panel of antibodies specific for the critical acetylated forms of histone H3 and H4, and showed meiosis stage-dependent and lysine residue-specific patterns of histone acetylation. By using trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), we further determined that selective inhibition of histone deacetylation (thereby maintaining hyperacetylation) delayed the onset of germinal vesicle breakdown and produced a high frequency of lagging chromosomes or chromatin bridges at anaphase and telophase I (AT-I), suggesting that histone deacetylation is required for orderly meiotic resumption and accurate chromosome segregation in porcine oocytes. In addition, we examined the localization and expression of HDAC1 by performing immunofluorescence and immunoblotting analysis. The results showed that subcellular translocation, expression level and phosphorylated modification of HDAC1 were temporally regulated and likely to co-participate in the establishment of histone acetylation profiles in oocyte meiosis.     

A new chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C. Koch) based on FISH analysis of monosomic lines

Fluorescent in situ hybridization (FISH) with multiple probes was used to analyze mitotic and meiotic chromosome spreads of Avena sativa cv ‘Sun II’ monosomic lines, and of A. byzantina cv ‘Kanota’ monosomic lines from spontaneous haploids. The probes used were A. strigosa pAs120a (a repetitive sequence abundant in A-genome chromatin), A. murphyi pAm1 (a repetitive sequence abundant in C-genome chromatin), A. strigosa pITS (internal transcribed spacer of rDNA) and the wheat rDNA probes pTa71 (nucleolus organizer region or NOR) and pTa794 (5S). Simultaneous and sequential FISH employing pairs of these probes allowed the identification and genome assignation of all chromosomes. FISH mapping using mitotic and meiotic metaphases facilitated the genomic and chromosomal identification of the monosome in each line. Of the 17 ‘Sun II’ lines analyzed, 13 distinct monosomic lines were found, corresponding to four monosomes of the A-genome, five of the C-genome and four of the D-genome. In addition, 12 distinct monosomic lines were detected among the 20 ‘Kanota’ lines examined, corresponding to six monosomes of the A-genome, three of the C-genome and three of the D-genome. The results show that 19 chromosomes out of 21 of the complement are represented by monosomes between the two genetic backgrounds. The identity of the remaining chromosomes can be deduced either from one intergenomic translocation detected on both ‘Sun II’ and ‘Kanota’ lines, or from the single reciprocal, intergenomic translocation detected among the ‘Sun II’ lines. These results permit a new system to be proposed for numbering the 21 chromosome pairs of the hexaploid oat complement. Accordingly, the A-genome contains chromosomes 8A, 11A, 13A, 15A, 16A, 17A and 19A; the C-genome contains chromosomes 1C, 2C, 3C, 4C, 5C, 6C and 7C; and the D-genome consists of chromosomes 9D, 10D, 12D, 14D, 18D, 20D and 21D. Moreover, the FISH patterns of 16 chromosomes in ‘Sun II’ and 15 in ‘Kanota’ suggest that these chromosomes could be involved in intergenomic translocations. By comparing the identities of individually translocated chromosomes in the two hexaploid species with those of other hexaploids, we detected different types of intergenomic translocations.