应用HeLa细胞染色体(簇)和蛙卵提取物进行细胞核体外重建试验

将HeLa细胞中期染色体(簇)、非洲爪蟾卵提取物和ATP再生体系混合温育,能够促使细胞核自发重建。在此非细胞体系中重建的细胞核处于一般细胞核大小范围,具有典型的双层核膜,核孔复合体、染色质、核纤层、核骨架等结构,核重建具有一个明显的过程;发现环形片层通过与核膜融合方式参与核膜和核孔复合体组装。     

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.     

Structure and Stability of Telocentric Chromosomes in Wheat

In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring telocentric chromosomes (telosomes) with a terminal centromere are rare but do exist. Telosomes arise through misdivision of centromeres in normal chromosomes, and their cytological stability depends on the structure of their kinetochores. The instability of telosomes may be attributed to the relative centromere size and the degree of completeness of their kinetochore. Here we test this hypothesis by analyzing the cytogenetic structure of wheat telosomes. We used a population of 80 telosomes arising from the misdivision of the 21 chromosomes of wheat that have shown stable inheritance over many generations. We analyzed centromere size by probing with the centromere-specific histone H3 variant, CENH3. Comparing the signal intensity for CENH3 between the intact chromosome and derived telosomes showed that the telosomes had approximately half the signal intensity compared to that of normal chromosomes. Immunofluorescence of CENH3 in a wheat stock with 28 telosomes revealed that none of the telosomes received a complete CENH3 domain. Some of the telosomes lacked centromere specific retrotransposons of wheat in the CENH3 domain, indicating that the stability of telosomes depends on the presence of CENH3 chromatin and not on the presence of CRW repeats. In addition to providing evidence for centromere shift, we also observed chromosomal aberrations including inversions and deletions in the short arm telosomes of double ditelosomic 1D and 6D stocks. The role of centromere-flanking, pericentromeric heterochromatin in mitosis is discussed with respect to genome/chromosome integrity.     

Epigenetics regulate centromere formation and kinetochore function

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function.     

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.     

Electron microscopical observations on nuclear division inMicrasterias americana

Each stage of nuclear division inMicrasterias americana was investigated by electron microscopy. Some chromosomes in metaphase had two or more centromeres on them, that is, they were polycentric. The centromere was roundish, moderately dense, and partially embedded in the chromosomes. Many microtubules of the spindle fibers were attached to the centromere. Abundant granules of high electron density, derived from dictyosomes in the cytoplasm, were seen in the metaphase spindle. Only the chromosomes moved towards the poles in anaphase, while these granules remained at the equatorial plate. Many nucleoli appeared in early telophase in one or more regions in almost all chromosomes. These nucleoli fused and enlarged during telophase.     

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three- dimensional optical sectioning microscopy. Time-lapse, three- dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high- resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.     

CENH3 interacts with the centromeric retrotransposon <Emphasis Type="Italic">cereba</Emphasis> and GC-rich satellites and locates to centromeric substructures in barley

The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.     

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.     

Demonstration of membranous patches on isolated chromosomes

High resolution scanning electron microscopy of isolated Chinese hamster ovary metaphase chromosomes revealed “membranous patches” at telomeric and juxtatelomeric regions of the chromosomes. The “membranous patches” remained bound to the chromosomes during centrifugation through dense sucrose, but not after treatment with detergents. These membrane fragments on isolated purified chromosomes may represent a component that binds the chromosome to the inner portion of the nuclear envelope up to late stages of prophase. These chromosome associated membranous patches may represent sites of reformation of the nuclear envelope at telophase.     

Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Evidence for centromere drive in the holocentric chromosomes of Caenorhabditis

In monocentric organisms with asymmetric meiosis, the kinetochore proteins, such as CENH3 and CENP-C, evolve adaptively to counterbalance the deleterious effects of centromere drive, which is caused by the expansion of centromeric satellite repeats. The selection regimes that act on CENH3 and CENP-C genes have not been analyzed in organisms with holocentric chromosomes, although holocentrism is speculated to have evolved to suppress centromere drive. We tested both CENH3 and CENP-C for positive selection in several species of the holocentric genus Caenorhabditis using the maximum likelihood approach and sliding-window analysis. Although CENP-C did not show any signs of positive selection, positive selection has been detected in the case of CENH3. These results support the hypothesis that centromere drive occurs in Nematoda, at least in the telokinetic meiosis of Caenorhabditis.     

Recognition of <Emphasis Type="Italic">A. thaliana</Emphasis> centromeres by heterologous CENH3 requires high similarity to the endogenous protein

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.     

Loading of the centromeric histone H3 variant during meiosis–how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.     

Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability.     

The cotton centromere contains a Ty3-gypsy-like LTR retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.     

Centromeric histone H3 protein: from basic study to plant breeding applications

Centromere is the defining unit of a chromosome where kinetochore complex assembles and facilitates chromosome segregation. Centromeres contain unique repetitive sequences and are enriched with transposons and retrotransposons. Although how centromere is determined is still not clearly understood, binding of a key protein, namely, the Centromeric Histone H3 (CENH3) to centromeric repetitive DNA sequences has been found to be critical for the specification of centromere. Hence, centromeres are said to be epigenetically specified by CENH3. Despite considerable variation in size and sequence, CENH3 protein shows significant conservation of structure and function. CENH3 disruption or overexpression shows severe defects in spindle fiber attachment and ultimately leads to embryo lethality. Basic studies on complementation of CENH3 in Arabidopsis thaliana have led to the development of a novel method of haploid production through selective elimination of one set of parental chromosomes in the zygote. These findings have also shed new light on selective loss of chromosomes in interspecific crosses of Hordeum vulgare × H. bulbosum. Here, we briefly review unique features of CENH3 and discuss the new plant breeding opportunities that have emerged from the study of CENH3.     

Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres

While the approximate chromosomal position of centromeres has been identified in many species, little is known about the dynamics and diversity of centromere positions within species. Multiple lines of evidence indicate that DNA sequence has little or no impact in specifying centromeres in maize and in most multicellular organisms. Given that epigenetically defined boundaries are expected to be dynamic, we hypothesized that centromere positions would change rapidly over time, which would result in a diversity of centromere positions in isolated populations. To test this hypothesis, we used CENP-A/cenH3 (CENH3 in maize) chromatin immunoprecipitation to define centromeres in breeding pedigrees that included the B73 inbred as a common parent. While we found a diversity of CENH3 profiles for centromeres with divergent sequences that were not inherited from B73, the CENH3 profiles from centromeres that were inherited from B73 were indistinguishable from each other. We propose that specific genetic elements in centromeric regions favor or inhibit CENH3 accumulation, leading to reproducible patterns of CENH3 occupancy. These data also indicate that dramatic shifts in centromere position normally originate from accumulated or large-scale genetic changes rather than from epigenetic positional drift.