细胞核结构被发现

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) v! B! g5 @5 P8 I1 c& n3 o9 f细胞核结构的调控对基因表达的时间及方式等重要功能有深远的影响。其结构紊乱则导致基因变异的累积，如DNA序列的重复，甚至一条多余的染色体。2006年12月11日美国能源部劳伦斯-伯克利国家实验室消息称：其生命科学部的研究者Gary Karpen 和 Jamy Peng发现了果蝇有两条调控核仁及其他细胞核结构、保持基因稳定性的路径。由于果蝇和人类有很多基因是共同的，因此了解果蝇的细胞核调控有助于认识人类的疾病如先天性疾病，或癌症。这一研究结果发表在最近一期的《自然细胞生物》在线版.$ L# Y0 c2 @1 S, [$ W
    研究人员发现了调控异染色质两个重要功能的分子路径。路径之一在异染色质的内部及外部调控DNA重复序列，另一路径则调节核仁的结构。这是首次报道的对核仁的结构调控的研究。研究者的第一步是确认哪些基因影响了异染色质的组成，然后找出这些基因编码的蛋白，最后探索这些蛋白是怎样影响染色质的。同时，研究人员发现了果蝇的数个基因，如花斑抑制基因（suppressors of variegation，Su(var)）能抑制基因的表达。Su(var)3-9蛋白可以对核小体的某些组蛋白进行化学修饰，如将甲基加在组蛋白H3的第9个氨基酸（赖氨酸）残基上，染色质的甲基化使其盘曲，结构变致密。Su(var)2-5蛋白，使HP1蛋白与甲基化H3K9及Su(var)3-9的复合体结合，使染色质进一步致密化，其中的DNA被关闭，基因不被转录。另一方面，果蝇的E(var)s基因则促进花斑形成。其作用方式之一是使H3组蛋白的第4个氨基酸（也是赖氨酸）残基乙酰化，使染色质在这一部位被打开，而DNA变得易于接近。% i' ]3 e0 O3 n6 R
    Karpen 和 Peng研究了Su(var)3-9基因突变的果蝇，发现Su(var)3-9基因突变引起核糖体DNA基因大量扩增，与其他重复的DNA序列一起无序地满布于细胞核中，细胞中出现了多个核仁，而正常状态是单个形态完好的核仁，其中的核糖体DNA整齐地排列在异染色质上。这与RNAi引起的H3K9甲基化的结果类似。同样，在RNAi路径中起关键作用的编码Dicer-2酶的基因dcr-2的突变也可导致出现多个核仁及异染色质失调的现象。9 Y. }4 i+ S+ w5 K+ `: Q) t
    Su(var)3-9及RNAi突变都使染色体外DNA数量增加，其外观是细胞核中小的重复序列的环。这让Karpen 和 Peng推测H3K9甲基化和RNAi路径通过某种特殊的机制调控细胞核的结构。当H3K9甲基化路径正常时，异染色质处于致密状态，围绕核糖体DNA形成单个核仁。但当Su(var)3-9突变，或编码HP1蛋白的基因突变，或使RNAi功能失活的突变导致H3K9不能被甲基化时，异染色质被打开了，其中的DNA重复序列自由了，DNA重组和修复程序将DNA从染色体上切断，形成染色体外DNA，如果当中有核糖体DNA，它们将聚积成新的核仁。奇怪的是，这种染色体的紊乱并不致死。Karpen说“不过检测到DNA损坏并进行修复，使染色体复制和细胞分裂减慢了。如果果蝇同时有H3K9甲基化及RNAi路径的突变，累积的基因异常将导致死亡。” 这些发现扩展了基因稳定性的含义。研究人员计划调查果蝇中存在的H3K9和RNAi路径是否在人类也起同样的作用，如果是这样，异染色质的失调可能是肿瘤中基因极度不稳定的原因之一。同时还将进一步研究这些路径影响细胞核染色体的机制。 2 z% C% u8 x! g: B! f

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/ l1 x$ t2 E/ i) NFigure 2. Su(var) mutants have dispersed rDNA foci, each of which forms an ectopic nucleolus.
5 y  Y. i+ m* H0 d! S: B2 C9 t! i' @(a) FISH of rDNA (red) and immunfluorescence microscopy of fibrillarin (green) were performed on whole-mount salivary glands from wild-type, Su(var)3-9null, Su(var)3-91699, HP1null and Su(TDA-PEV)1650 homozygous mutants. DAPI, blue. There is a single site of rDNA in >98% of wild-type nuclei, whereas the Su(var) mutant nuclei contain multiple rDNA foci, which are all surrounded by fibrillarin. The scale bars represent 15 m. (b) Combined rDNA FISH (red) and immunofluoresence microscopy of fibrillarin (green) of whole-mount imaginal disc and brain tissues from wild-type and Su(var)3-9null mutant larvae. Wild-type nucleoli contain a single, compact rDNA focus, whereas Su(var)3-9null mutants frequently display multiple rDNA foci. The scale bars represent 3 m. Boxed nuclei are shown at higher magnification to the right of each image. (c) Quantitative analysis of the number of rDNA foci in wild-type and Su(var)3-9null diploid nuclei. 98% of wild-type cells (n = 96) contain one rDNA signal, compared with 67% of Su(var)3-9null nuclei, and the percentages with two, three and four rDNA signals were 24%, 7% and 2%, respectively (average = 1.44 0.73 rDNA foci per mutant nucleus, n = 53, P <0.001).
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原文出处： Nature Cell Biology  December 2006 - Vol 8 No 12 Published online: 10 December 2006; | doi:10.1038/ncb1514 H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability
+ K% d# Y* t; A/ lJamy C. Peng & Gary H. Karpen
* y) Y: M2 \" \  jPublished online: 10 December 2006 | doi:10.1038/ncb1514
& k+ n* F2 S- Z. X  M# y6 eAbstract | Full text | PDF (1,180K)  | Supplementary Information     背景知识： 异染色质的外遗传学特点：
7 ?2 {/ M6 ~3 ?5 i* I2 ]  g# ~    通过细胞核的结构及空间构型的重组来调控细胞及生物的功能，称为外遗传学（epigenetics），其中epi在希腊文中是“上面，外面”的意思，也就是说外遗传学研究的不是对DNA序列的改变，而是对染色质的调整，如基因沉默， 染色体继承等等。 所有细胞核的最显著的特点就是染色体，由DNA和组蛋白所组成的染色质构成，DNA环绕由4个相似的组蛋白分子结合而形成的柱状的芯上，从而形成核小体，核小体之间由连续的DNA链连接，形成串珠样结构，进一步盘曲形成常染色质或异染色质。
7 e% q# Z( D$ f! I- Q* M) C* p    大多数基因位于常染色质，结构相对疏松，DNA易被转录。而异染色质结构致密，所含基因很少，其中的大部份DNA，包括大量的短的重复序列，并不编码蛋白质。
0 I3 A) ?5 M2 m  V4 O1 B    异染色质总是出现在染色体的末端，与端粒相邻。端粒随着细胞的分裂而变短，从而限制细胞分裂的次数。异染色质也出现在着丝点，着丝点位于染色体的中部，在细胞分裂时使染色体的分开。而异染色质其他的功能尚未明了。% v; |2 M1 J0 s8 q( \  |
  相关基因： Su(var)2-HP2 Su(var)2-HP2 [Drosophila melanogaster] Other Aliases: Dmel_CG12864, CG12864, HP2 Other Designations: Su(var)2-HP2 CG12864-PA, isoform A; Su(var)2-HP2 CG12864-PB, isoform B Chromosome: 2R; Location: 51B6-51B6 GeneID: 36621     Dcr-2 Dicer-2 [Drosophila melanogaster] Other Aliases: Dmel_CG6493, CG6493, DCR2, DICER, dcr-2, dicer2 Other Designations: Dicer-2 CG6493-PA Chromosome: 2R; Location: 54C10-54C10 GeneID: 36993     作者简介： Gary Karpen Adjunct Professor of Cell and Developmental Biology University of Californial    Collage of Letters&Sciences Department of Molecular&Cell biology   Research Interests Our studies are focused on understanding inheritance, chromatin structure, gene expression, and the organization of chromosomes in the nucleus. Most of our studies have focused on the fruit fly Drosophila melanogaster as a model for chromosome function in metazoans, which allows us to address mechanisms in animals by synergistically combining molecular, genetic, cell biological and biochemical approaches. However, we have examined the relevance of our findings to human chromosomes, and have demonstrated surprising similarities between these evolutionarily-distant species. Current Projects We are currently pursuing three projects: 1) analysis of the determinants of centromere identity and function, 2) molecular-genetic dissection of proteins that regulate nuclear organization, and 3) sequence analysis of a poorly characterized genomic component, called heterochromatin. Recent results from the centromere project will be described here; access our lab web site for more details about this and other projects. The centromere (CEN) is the chromosomal site associated with kinetochore formation, which is the structure responsible for microtubule attachment to the chromosome, and constitutes an essential component of prometaphase congression, mitotic checkpoint control, anaphase poleward segregation, and cytokinesis. Chromosome gain or loss (aneuploidy) results from having no kinetochore attachments, or multiple attachments. Aneuploidy has catastrophic effects on cells and organisms, and plays a key role in the surprisingly high frequency of human birth defects, and in cancer initiation and progression. How does the cell ensure that one, and only one region of the chromosome attaches to and moves along microtubules?? In other words, how is CEN identity propagated through replication and division? Significant evidence suggests that CEN identity is determined epigenetically, that is by mechanisms that function independent of the primary DNA sequence. Thus, chromatin structure, modification and replication are likely to play a critical role in CEN identity. We have studied a constitutive component of centromeric chromatin (CENP-A), which substitutes for histone H3 in CEN nucleosomes. We have demonstrated that CENP-A is both a structural and a functional foundation for kinetochore formation. Three-dimensional deconvolution microscopy analysis demonstrated that CENP-A chromatin in flies and humans appears as a cylindrical structure, and that kinetochore proteins are wrapped around this structure. As expected from the composition of CEN nucleosomes, H3 appears to be excluded from the CENP-A cylinder. However, surprisingly, in analysis of two-dimensional chromatin preparations we observed that blocks of H3-containing chromatin are interspersed with CENP-A -containing chromatin. How can we reconcile the uniformity of CENP-A and its separation from H3 in the higher order structure with the interspersion of H3 chromatin observed in two dimensions? We propose that the DNA may spiral (Figure 1) or loop through the cylinder, with CENP-A subdomains `stacked' into a cylinder, to the poleward side of a stack of interspersed H3 subdomains. We suggest that the function of this conserved structure may be to `present' centromeric chromatin to the external face of condensed mitotic chromosomes, where it is accessible to recruit kinetochore components and to attach to spindle microtubules. RNA interference in tissue culture cells, live analysis of chromosome segregation after antibody injection into early embryos, and observations of flies with CENP-A null mutations demonstrated that CENP-A is required for all chromosome movements during mitosis and for normal cell cycle progression. Reciprocal epistasis experiments showed that CENP-A is very high in the kinetochore assembly pathway; CENP-A is required to recruit all known kinetochore proteins, but CENP-A localizes to centromeres in the absence of these proteins. We have also demonstrated that failure to form a kinetochore, due to depletion of CENP-A, results in the activation of a cell cycle checkpoint early in mitosis, prior to the time of a previously identified checkpoint known as the Spindle Assembly Checkpoint (SAC). Thus, CENP-A is physically and functionally positioned to be the epigenetic mark for centromere identity, or is closely associated with the mark. If CENP-A is the epigenetic mark for propagation of centromere identity, how is new CENP-A deposited specifically at centromeres in response to replication and division? We hypothesize that `replenishment' in response to replication-generated segregation of CENP-A nucleosomes may be mediated by centromere-specific chromatin assembly complexes (CAFs) or remodeling factors. We are currently focused on in understanding the mechanisms and components responsible for specifically depositing CENP-A into CEN chromatin during or after replication. We have used biochemical and genetic approaches to identify CENP-A interacting proteins, and are determining if these candidates are required for CENP-A deposition. Selected Publications Sequence analysis of a functional Drosophila centromere. [X. Sun, H. Le, J. Wahlstrom, and G.H. Karpen (2003) Genome Research 13, 182-194] Heterochromatic sequences in a Drosophila whole genome shotgun assembly. [R.A. Hoskins, et al. (2002) Genome Biology 3(12), RESEARCH0085] Conserved organization of centromeric chromatin in flies and humans. [M.D. Blower, B.A. Sullivan, and G.H. Karpen (2002) Developmental Cell 2, 319-330] The role of Drosophila CENP-A / CID in kinetochore formation, cell-cycle progression and interactions with heterochromatin. [M.D. Blower and G.H. Karpen (2001) Nature Cell Biology 3, 730-9] The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. [K.L. Hari, K.R. Cook, G.H. Karpen (2001) Genes and Dev. 15, 1334-48