Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment.

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.

Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T.

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

Higher-order unfolding of satellite heterochromatin is a consistent and early event in cell senescence.

Epigenetic changes to chromatin are thought to be essential to cell senescence, which is key to tumorigenesis and aging. Although many studies focus on heterochromatin gain, this work demonstrates large-scale unraveling of peri/centromeric satellites, which occurs in all models of human and mouse senescence examined. This was not seen in cancer cells, except in a benign senescent tumor in vivo. Senescence-associated distension of satellites (SADS) occurs earlier and more consistently than heterochromatin foci formation, and SADS is not exclusive to either the p16 or p21 pathways. Because Hutchinson Guilford progeria syndrome patient cells do not form excess heterochromatin, the question remained whether or not proliferative arrest in this aging syndrome involved distinct epigenetic mechanisms. Here, we show that SADS provides a unifying event in both progeria and normal senescence. Additionally, SADS represents a novel, cytological-scale unfolding of chromatin, which is not concomitant with change to several canonical histone marks nor a result of DNA hypomethylation. Rather, SADS is likely mediated by changes to higher-order nuclear structural proteins, such as LaminB1.

[Gytogenetics of Chironomus riparius L. from the fishpond in Borok village (Diptera, Chironomidae)].

We have found three inherited inversions in Chironomus riparius populations from the Borok fishpond, namely: (A3d-B1a) in the arm A (C5a-C6a) in the arm D and (B3b-4d/e) in the arm F. Increase of heterochromatin in some bands of chromosome F (B3h, B3h + B3c--C1a) and puffs appearance in the arms C, D and E have been observed. We saw also changes in functional activity of nucleolar organizer (N) and Balbiani rings (BRe/BRb). It has been found that some of inversion breakpoints coincide with the Alu and Hinf satellite DNA localization sites.

Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells.

A human diploid lung fibroblast cell strain, TIG-7, has a heteromorphic chromosome 15 with an extra short arm carrying a homogeneously staining region (15p+hsr). We demonstrated previously that the 15p+hsr consists of an inactive and G+C-rich rDNA cluster characterized by fluorescence in situ hybridization (FISH) and various chromosome banding techniques. Thus, it was suggested that the region could contain highly methylated DNA. To observe methylation status on the target region directly under the microscope, we used a demethylating agent, 5-azacytidine (5-azaC), to induce decondensation of the chromatin containing methylated DNA. At 24 h after treatment with 0.5 microM 5-azaC, marked decondensation of the 15p+hsr was observed in almost all of the metaphases. Furthermore, we observed micronuclei, which were equivalent to the rDNA of the 15p+hsr demonstrated by FISH in the same preparation. In contrast, the DNA cross-linking agent mitomycin C (MMC) preferentially induced 15p+hsr-negative micronuclei. These findings indicated that chromatin decondensation and subsequent DNA strand breakage induced by the demethylating effect of 5-azaC led specifically to 15p+hsr-positive micronuclei.

High-resolution organization of mouse telomeric and pericentromeric DNA.

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.

[Position of the satellite DNA relative to heterochromatin in the interphase nuclei in cultured cells]. / Polozhenie satellitnykh DNK po otnosheniiu k geterokhromatinu v interfaznykh iadrakh kletok v kul'ture.

It is considered that centromeric (CEN) regions play the leading role in the formation of chromocentres predominantly consisting of satellite DNA. Cloned mouse and human satellite DNA sequences from CEN and periGEN regions were used in order to trace their positions relative to chromocentres. Methods of fluorescent in situ hybridization (FISH) and immunoFISH with antibodies against CENP-B, known is a marker of prekinetochore, were employed. Peripheral position of the signals was observed at the chromocentres, but a combined signal of GEN and periGEN satellite DNAs never covered the whole brightly DAPI stained regions. A reasonable amount of the chromocentre body is not a satellite DNA. We suppose that the matrix-associated regions (MAR) of structural genes and, probably, the heavy methylated coding sequences of the genes, which are not expressed in the given cell type, are included in the chromocentres in addition to satellite DNAs.

A comparative study of chromosome morphology among the nine annual species of Cicer L.

Thirty-six accessions, representing the full complement of all the nine annual Cicer L. species, viz C. arietinum, C. reticulatum, C. echinospermum, C. pinnatifidum, C. judaicum, C. bijugum, C. chorassanicum, C. yamashitae and C. cuneatum, were subjected to karyotype analysis for the first time in a single comprehensive study. The detailed karyotype of C. chorassanicum was also investigated for the first time. A 12 h cold water pretreatment and 13 min 60 degrees C 1 N HCl hydrolysis confirmed a somatic chromosome number of 2n = 16 in all the species. Within species interchromosomal size variation was observed to be quite large in C. arietinum, C. reticulatum and C. echinospermum, but not in the remaining six species. Individual chromosome size ranged from 3.77 microns in C. echinospermum to 1.32 microns in C. arietinum while the haploid genome length ranged from 20.65 microns in C. echinospermum to 14.92 microns in C. cuneatum. Ample rearrangement of chromatin among chromosomes within a species was implied to have played a role in Cicer genome evolution. The nine species were classified in two groups based on karyotypic similarity, with the first group comprising the inter-crossable species C. arietinum, C. reticulatum and C. echinospermum, while the remaining species forming the second group. The first group species are also genetically close to each other as deduced by other morphological, biochemical and DNA based studies. Circumstantial evidence has lead to the speculation that perhaps karyotypic similarity and interspecific crossability are positively related to each other.

Nucleoli in a pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes.

OBJECTIVE: To investigate the arrangement of chromosomes within pronuclei-stage mouse zygotes. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Location of major alpha-satellite DNA, centromeres, and telomeres, and relative location of chromosomes. RESULT(S): Chromosomes appeared to be oriented inward by centromeres and to be interconnected by major alpha-satellite DNA, which appeared to be the sole DNA component of the nucleoli. This chromosomal arrangement persisted throughout interphase. Chromosomal painting failed to identify chromosomal ordering within pronuclei. CONCLUSION(S): Pronuclear nucleoli are represented by alpha-satellite sequences of interconnecting chromosomes that hold all chromosomes together during interphase. Chromosomes within the pronucleus are randomly positioned relative to each other.

Searching for a common centromeric structural motif: Drosophila centromeric satellite DNAs show propensity to form telomeric-like unusual DNA structures.

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophila centromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation.

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.

Polymorphic curvature of satellite DNA in three subspecies of the beetle Pimelia sparsa.

The curvature of the monomeric repeats of satellite DNAs from three subspecies of the beetle Pimelia sparsa (Coleoptera, Tenebrionidae) has been analysed. Evidence of curvature was inferred from their retarded migration in native polyacrylamide gels, which was confirmed by direct electron microscopy visualisation. Sequence-comparison analysis, which included sequence alignments and modelling studies, was used to reveal the patterns of local bending and curvature. The effects of the minor-groove-binding drugs distamycin and berenil on the curvature were assayed, and analysed with respect to their (A+T)-rich sequence preferences. Since our study deals with satellite DNAs from closely related organisms (three subspecies), we correlated the differences in sequence, and also the high similarity conserved in the (A+T)-rich regions, with the changes in the patterns of curvature.

Scaffold attachment regions in centromere-associated DNA.

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with 32P-labeled fragments from the human 1.9 kb centromeric alpha-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric alpha-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the alpha-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 alpha-satellite region using computer and base ratio analysis, to address the question as to why some alpha-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region.

Centromeric dodeca-satellite DNA sequences form fold-back structures.

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G.A pairs as well as regular Watson-Crick G.C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75 degrees C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

Human alpha-satellite DNAs are not susceptible to undercondensation after 5-azacytidine treatment.

Localization of alphoid human satellite DNAs using fluorescence in situ hybridization (FISH) of metaphase chromosomes following treatment with 5-azacytidine to produce undercondensation showed that human alpha-satellite DNA is not sensitive to the condensation-inhibition effect of 5-azacytidine. The difference in heterochromatic DNA subsets was particularly evident in the constitutive heterochromatin of chromosomes 1 and 9. Comparison of the results obtained after FISH with those obtained from electron microscopy and G-banding enabled accurate localization of this DNA domain.

5-azacytidine produces differential undercondensation of alpha, beta and classical human satellite DNAs.

Fluorescence in situ hybridization employing human alphoid, beta and classical satellite DNA probes was performed on 5-azacytidine treated and untreated chromosomes obtained from human lymphocytes. The individual used in this study presented a polymorphism of constitutive heterochromatin of chromosomes 1 and 9 as revealed by in situ digestion with the restriction endonuclease Alul. Neither the alphoid nor the beta satellite DNA domains were susceptible to condensation-inhibition by 5-azacytidine. Only the classical satellite localized on chromosome 9 was affected. The constitutive heterochromatin size polymorphism was shown to depend mainly on variations of the classical satellite DNA domain. Therefore, condensation-inhibition, as a phenomenon which may modify the natural folding of the chromatin fibre, regionally affects human constitutive heterochromatin and seems to be dependent on the heterochromatic family.

On the tertiary structure of the Citrus ichangensis satellite DNA.

A hexamer of the repeating unit of Citrus ichangensis satellite DNA was cloned. Polyacrylamide gel electrophoresis demonstrated that the shape of cloned hexamer is other than linear. As the computing of tertiary coordinates made by Eckdahl and Anderson's BEN program proved, the hexamer is a solenoid consisting of two turns termed coiled double helix (CDH)-form. An electron microscopic analysis revealed small diameter circles in the hexamer under investigation. Unlike the hexamer control molecules are s-shaped. It is concluded that the CDH-form is a characteristic of the satellite DNA of Citrus ichangensis.

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin.

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.

Unusual distribution of chromosome 12 in a testicular germ-cell tumor cell line (833K) and its cisplatin-resistant derivative (64CP9).

The distribution of chromosome 12 in a cisplatin-sensitive testicular germ-cell tumor (TGCT) cell line (833K), and its cisplatin-resistant derivative (64CP9), was studied by fluorescent in situ hybridization (FISH) using DNA alpha satellite and whole chromosome painting probes for chromosome 12. Chromosomes 12 and i(12p) in these cell lines were readily identified. However, chromosome 12-derived chromatin was also observed in acrocentric- and nonacrocentric-derived chromosomes. Several of the chromosome 12 painted regions resembled satellites and were separated from the chromosome by nonstaining stalk-like regions. In each cell line, different chromosomes were involved. In the 833K TGCT cell line these were a der(8), a der(14) with a 12-labeled "satellite" on the q arm and a der(14) with a 12-labeled "satellite" on the p arm. In the 64CP9 TGCT cell line, these were a der(5), a der(17), and a der(8). The der(8) observed in 64CP9 was different from the der(8) seen in 833K. These derived chromosomes were characterized using sequential FISH, Wright staining, and silver staining for nucleolar organizing regions (AgNORs). The non-staining stalk-like regions were AgNOR positive, indicating "ectopic" NORs. Chromosome 22 distribution in these cell lines was also studied. FISH, using a chromosome 22-specific painting probe, identified a small metacentric marker and a der(12)t(12;22) in each cell line. It is not known whether the differences in distribution of chromosome 12-derived chromatin between the two cell lines are related to cisplatin resistance. Our study shows that the distribution of chromosome 12 in the two TGCT cell lines is much more extensive than could be identified in GTG-banded karyotypes. FISH allows characterization of unidentified chromatin and thus is a valuable adjunct to traditional cytogenetic techniques.

Heteromorphism of human chromosome 18 detected by fluorescent in situ hybridization.

The individual variations of pericentromeric heterochromatin in human chromosome 18 were analysed using the C-banding techniques and nonradioactive fluorescence in situ hybridization (FISH) with chromosome 18 alpha-satellite DNA probe. FISH analysis shows heteromorphisms almost undetectable by C-banding and which regularly segregate in a family.