植物功能着丝粒DNA研究进展

着丝粒（centromere）是真核生物染色体的重要功能结构。在细胞有丝分裂和减数分裂过程中,着丝粒通过招募动粒蛋白行使功能,保障染色体正确分离和传递。真核生物中,含有着丝粒特异组蛋白的CenH3区域被定义为功能着丝粒区,即真正意义上的着丝粒。近年来,借助染色质免疫沉淀技术,人们对功能着丝粒DNA开展了深入研究,揭示其组成、结构及演化特征,并发现功能着丝粒区存在具有转录活性的基因,且部分基因具有重要生物学功能。由于存在大量重复DNA,着丝粒演化之谜一直未能完全揭示。对植物功能着丝粒DNA序列研究进展进行了概述,并重点阐述了着丝粒重复DNA研究的新方法和新进展,以期为深入开展相关研究提供借鉴。     

玉米新着丝粒形成及其表观遗传学

<正>着丝粒是染色体的重要组成部分,功能保守,在细胞分裂过程中确保染色体准确分离。在着丝粒区域的核小体中,有一类组蛋白H3的变异体,在植物中称为CENH3,在动物中称为CENP-A;同时植物中含有在第133位苏氨酸发生磷酸化的组蛋白H2A,这两类组蛋白是活性着丝粒的表观遗传学标记。着丝粒区域的DNA序列经历了高度的进化。除     

植物着丝粒结构及进化的研究进展

植物着丝粒是染色体重要结构域,介导动粒装配。不同物种间着丝粒重复序列快速趋异进化,着丝粒功能保守,确保有丝分裂和减数分裂过程中染色体正确分离和准确传递。伴随染色质免疫共沉淀技术(Chromatin immunoprecipitation, ChIP)、ChIP 与高密度芯片相结合技术(ChIP-chip)、ChIP 与高通量测序相结合技术(ChIP-seq)的应用,植物着丝粒研究获得里程碑式进展:某些模式植物着丝粒DNA 序列、蛋白质结构、功能获得大量新认识;着丝粒基本蛋白质组蛋白H3 被用来界定着丝粒大小和边界;某些非着丝粒区域被激活为新着丝粒,在世代传递中保持稳定性。本文对植物着丝粒结构、功能、进化研究进行了综述,并探讨了植物着丝粒研究存在的问题。     

恶性血液病中着丝粒和着丝粒周染色体重排的研究进展

用染色体特异的卫星DNA探针染色体荧光原位杂交（FISH）分析恶性血液病,发现着丝粒和着丝粒周染色体的重排并非罕见。分子生物学技术和基因组计划的发展,促进了对异染色质分子本质的研究。本就着丝粒和着丝粒周异染色质的分子结构及其重排机制作一综述。     

着丝粒核小体结构研究进展

着丝粒是构成真核生物染色体的必需元件。在细胞有丝分裂或减数分裂时,微管通过动粒与染色体着丝粒连接,参与细胞分裂的染色体分离与分配过程,使染色体平均分配到子细胞中。构成着丝粒的基本单位是着丝粒特异的核小体,与常规核小体不同的是着丝粒核小体中的组蛋白H3被其变种——着丝粒组蛋白H3所替换。最近几年,着丝粒核小体的结构成为细胞生物学研究的热点之一。该文综述了最近在多种真核生物研究中,通过体外和体内实验,提出的着丝粒核小体结构的八聚体、六聚体、同型四聚体以及半八聚体模型,并对着丝粒核小体结构的动态模型与功能的关系进行了探讨。     

小鼠富集着丝粒DNA在小鼠减数分裂粗线期染色体上的原位杂交

本工作用Hoechst 33258及着丝粒特异抗体间接免疫荧光法显示的小鼠粗线期染色体主缢痕区,与以小鼠富集着丝粒(SFA)DNA为探针在粗线期染色体上的原位杂交主缢痕区作了比较。发现SFA DNA探针不仅杂交于全部常染色体联会复合体上的着丝粒区,并且杂交于着丝粒周围的异染色质区;而且,也杂交于X,Y染色体的着丝粒区。由此结论:此富集SFA DNA中含有全套常染色体及X,Y染色体的SFA DNA。     

着丝粒区域DNA的研究进展

着丝粒是染色体的重要结构,在真核生物的细胞分裂中负责染色体的分裂分离。近年来对着丝粒的研究已经成为遗传学的一个热点。本文对着丝粒DNA的重复序列、着丝粒区域的基因及着丝粒的形成机制等作了简要的介绍。     

着丝粒横裂和着丝粒并合及其在高等植物染色体进化中的意义

本文讨论了着丝粒横裂和并合及其在高等植物染色体进化中的意义。着丝粒横裂和着丝粒并合是两个矛盾又辩证统一的过程,是染色体的基本变异形式之一,它们同时影响着植物类群的染色体基数、核型对称性、连锁关系、交叉频率和位点等细胞遗传学的重要方面.从而在高等植物染色体进化中起着重要作用,着丝粒和端粒的复制模型为着丝粒的横裂与并合提供了可能的机理,但尚待直接的生物化学证据的证实,原始基数的确定是判别着丝粒横裂与并合的关键。     

小鼠的克隆着丝粒(SFA)DNA的鉴定

着丝粒是真核染色体上的重要细胞器,是真核染色体作为基因载体行使其遗传功能的关键结构.着丝粒DNA首先是从酵母中分离克隆并被用以构建酵母人工染色体.鉴于真核有丝分裂机制研究和构建高等动物人工染色体研究的需要,从分离和检定过的小鼠着丝粒DNA库中筛选出了6#着丝粒DNA(SFADNA),并用荧光原位杂交法(FISH)对其进行了在染色体上的定位检定.用缺口平移法和PCR法分别标记了SFA DNA和SFA DNA中的小鼠寡份卫星DNA作为探针,分别与小鼠腹水癌细胞和小鼠L929细胞进行原位杂交;并用荧光抗体显示杂交信号的位置.结果,SFA DNA在两种细胞的中期染色体上的杂交信号都位于亚末端的初级缢痕处,表现为单一粗大的斑块.寡份卫星DNA在两种细胞的中期染色体上的杂交信号亦都位于亚末端的初级缢痕处,但极大多数的斑点均表现为成对的细小斑点.初级缢痕正是染色体着丝粒所在的特征性部位.故以上结果说明定位于该部位的克隆的6#SFA DNA,和其中的小鼠寡份卫星DNA都来源于小鼠着丝粒DNA.     

稻属不同染色体组着丝粒BAC克隆的分离和鉴定

着丝粒在真核生物有丝分裂和减数分裂染色体正常的分离和传递中起着重要的作用。通过构建5个稻属二倍体野生种的基因组BAC文库, 采用菌落杂交和FISH技术, 筛选和鉴定了各染色体组着丝粒克隆, 并且分析了这些克隆在不同基因组间的共杂交情况, 结果表明: (1) C染色体组的野生种O. officinalis 和F染色体组的野生种O. brachyantha具有各自着丝粒特异的卫星DNA序列, 并且O. brachyantha着丝粒还具有特异的逆转座子序列; (2) A、B和E染色体组的野生稻O. glaberrima、O. punctata和O. australiensis着丝粒区域都含有与栽培稻着丝粒重复序列CentO和CRR同源的序列; (3) C染色体组野生稻O. officinalis的2条体细胞染色体着丝粒具有CentO的同源序列, 同时也发现其所有着丝粒区域都包含栽培稻CRR的同源序列。这些结果对克隆稻属不同染色体组的着丝粒序列、研究不同染色体组间着丝粒的进化关系和稻属不同着丝粒DNA序列与功能之间的关系均具有重要意义。     

DNA and proteins of plant centromeres

In plants, as in all eukaryotes, centromeres are chromatin domains that govern the transmission of nuclear chromosomes to the next generation of cells/individuals. The DNA composition and sequence organization of centromeres has recently been elucidated for a few plant species. Although there is little sequence conservation among centromeres, they usually contain tandem repeats and retroelements. The occurrence of neocentromeres reinforces the idea that the positions of centromeres are determined epigenetically. In contrast to centromeric DNA, structural and transient kinetochoric proteins are highly conserved among eukaryotes. Candidate sequences have been identified for a dozen putative kinetochore protein homologues, and some have been localized to plant centromeres. The kinetochore protein CENH3, which substitutes histone H3 within centromeric nucleosomes, co-immunoprecipitates preferentially with centromeric sequences. The mechanism(s) of centromere assembly and the functional implication of (peri-)centromeric modifications of chromatin remain to be elucidated.     

An overview of plant centromeres

The centromere is a defining region that mediates chromosome attachment to kinetochore microtubules and proper segregation of the sister chromatids. Intriguingly, satellite DNA and centromeric retrotransposon as major DNA constituents of centromere showed baffling diversification and species-specific. However, the key kinetochore proteins are conserved in both plants and animals, particularly the centromere-specific histone H3-1ike protein （CENH3） in all functional centromeres. Recent studies have highlighted the importance of epigenetic mechanisms in the establishment and maintenance of centromere identity. Here, we review the progress and compendium of research on plant centromere in the light of recent data.     

Maize centromeres: organization and functional adaptation in the genetic background of oat

Centromeric DNA sequences in multicellular eukaryotes are often highly repetitive and are not unique to a specific centromere or to centromeres at all. Thus, it is a major challenge to study the fine structure of individual plant centromeres. We used a DNA fiber-fluorescence in situ hybridization approach to study individual maize (Zea mays) centromeres using oat (Avena sativa)-maize chromosome addition lines. The maize centromere-specific satellite repeat CentC in the addition lines allowed us to delineate the size and organization of centromeric DNA of individual maize chromosomes. We demonstrate that the cores of maize centromeres contain mainly CentC arrays and clusters of a centromere-specific retrotransposon, CRM. CentC and CRM sequences are highly intermingled. The amount of CentC/CRM sequence varies from approximately 300 to >2800 kb among different centromeres. The association of CentC and CRM with centromeric histone H3 (CENH3) was visualized by a sequential detection procedure on stretched centromeres. The analysis revealed that CENH3 is always associated with CentC and CRM but that not all CentC or CRM sequences are associated with CENH3. We further demonstrate that in the chromosomal addition lines in which two CenH3 genes were present, one from oat and one from maize, the oat CENH3 was consistently incorporated by the maize 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.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus

Brachypodium distachyon is a well‐established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere‐specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus.     

Recent advances in plant centromere biology

The centromere, which is one of the essential parts of a chromosome, controls kinetochore formation and chromosome segregation during mitosis and meiosis. While centromere function is conserved in eukaryotes, the centromeric DNA sequences evolve rapidly and have few similarities among species. The histone H3 variant CENH3(CENP-A in human), which mostly exists in centromeric nucleosomes, is a universal active centromere mark in eukaryotes and plays an essential role in centromere identity determination. The relationship between centromeric DNA sequences and centromere identity determination is one of the intriguing questions in studying centromere formation. Due to the discoveries in the past decades, including "neocentromeres" and "centromere inactivation", it is now believed that the centromere identity is determined by epigenetic mechanisms. This review will present recent progress in plant centromere biology.     

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.     

Maize centromeric chromatin scales with changes in genome size

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.