生物谷報道：細(xì)胞核結(jié)構(gòu)的調(diào)控對基因表達(dá)的時間及方式等重要功能有深遠(yuǎn)的影響,。其結(jié)構(gòu)紊亂則導(dǎo)致基因變異的累積,，如DNA序列的重復(fù),，甚至一條多余的染色體,。2006年12月11日美國能源部勞倫斯-伯克利國家實(shí)驗(yàn)室消息稱：其生命科學(xué)部的研究者Gary Karpen 和 Jamy Peng發(fā)現(xiàn)了果蠅有兩條調(diào)控核仁及其他細(xì)胞核結(jié)構(gòu),、保持基因穩(wěn)定性的路徑,。由于果蠅和人類有很多基因是共同的,，因此了解果蠅的細(xì)胞核調(diào)控有助于認(rèn)識人類的疾病如先天性疾病,，或癌癥。這一研究結(jié)果發(fā)表在最近一期的《自然細(xì)胞生物》在線版.
研究人員發(fā)現(xiàn)了調(diào)控異染色質(zhì)兩個重要功能的分子路徑,。路徑之一在異染色質(zhì)的內(nèi)部及外部調(diào)控DNA重復(fù)序列,，另一路徑則調(diào)節(jié)核仁的結(jié)構(gòu)。這是首次報道的對核仁的結(jié)構(gòu)調(diào)控的研究,。研究者的第一步是確認(rèn)哪些基因影響了異染色質(zhì)的組成,，然后找出這些基因編碼的蛋白，最后探索這些蛋白是怎樣影響染色質(zhì)的,。同時,，研究人員發(fā)現(xiàn)了果蠅的數(shù)個基因，如花斑抑制基因（suppressors of variegation,，Su(var)）能抑制基因的表達(dá),。Su(var)3-9蛋白可以對核小體的某些組蛋白進(jìn)行化學(xué)修飾，如將甲基加在組蛋白H3的第9個氨基酸（賴氨酸）殘基上,，染色質(zhì)的甲基化使其盤曲,，結(jié)構(gòu)變致密。Su(var)2-5蛋白,，使HP1蛋白與甲基化H3K9及Su(var)3-9的復(fù)合體結(jié)合,，使染色質(zhì)進(jìn)一步致密化，其中的DNA被關(guān)閉,，基因不被轉(zhuǎn)錄,。另一方面，果蠅的E(var)s基因則促進(jìn)花斑形成,。其作用方式之一是使H3組蛋白的第4個氨基酸（也是賴氨酸）殘基乙?；谷旧|(zhì)在這一部位被打開,，而DNA變得易于接近,。
Karpen 和 Peng研究了Su(var)3-9基因突變的果蠅,，發(fā)現(xiàn)Su(var)3-9基因突變引起核糖體DNA基因大量擴(kuò)增，與其他重復(fù)的DNA序列一起無序地滿布于細(xì)胞核中,，細(xì)胞中出現(xiàn)了多個核仁，而正常狀態(tài)是單個形態(tài)完好的核仁,，其中的核糖體DNA整齊地排列在異染色質(zhì)上,。這與RNAi引起的H3K9甲基化的結(jié)果類似。同樣,，在RNAi路徑中起關(guān)鍵作用的編碼Dicer-2酶的基因dcr-2的突變也可導(dǎo)致出現(xiàn)多個核仁及異染色質(zhì)失調(diào)的現(xiàn)象,。
Su(var)3-9及RNAi突變都使染色體外DNA數(shù)量增加，其外觀是細(xì)胞核中小的重復(fù)序列的環(huán),。這讓Karpen 和 Peng推測H3K9甲基化和RNAi路徑通過某種特殊的機(jī)制調(diào)控細(xì)胞核的結(jié)構(gòu),。當(dāng)H3K9甲基化路徑正常時，異染色質(zhì)處于致密狀態(tài),，圍繞核糖體DNA形成單個核仁,。但當(dāng)Su(var)3-9突變，或編碼HP1蛋白的基因突變,，或使RNAi功能失活的突變導(dǎo)致H3K9不能被甲基化時,，異染色質(zhì)被打開了，其中的DNA重復(fù)序列自由了,，DNA重組和修復(fù)程序?qū)NA從染色體上切斷,，形成染色體外DNA，如果當(dāng)中有核糖體DNA,，它們將聚積成新的核仁,。奇怪的是，這種染色體的紊亂并不致死,。Karpen說“不過檢測到DNA損壞并進(jìn)行修復(fù),，使染色體復(fù)制和細(xì)胞分裂減慢了。如果果蠅同時有H3K9甲基化及RNAi路徑的突變,，累積的基因異常將導(dǎo)致死亡,。”
這些發(fā)現(xiàn)擴(kuò)展了基因穩(wěn)定性的含義。研究人員計(jì)劃調(diào)查果蠅中存在的H3K9和RNAi路徑是否在人類也起同樣的作用,，如果是這樣,，異染色質(zhì)的失調(diào)可能是腫瘤中基因極度不穩(wěn)定的原因之一。同時還將進(jìn)一步研究這些路徑影響細(xì)胞核染色體的機(jī)制,。
Figure 2. Su(var) mutants have dispersed rDNA foci, each of which forms an ectopic nucleolus.
(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).
原文出處：
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
Jamy C. Peng & Gary H. Karpen
Published online: 10 December 2006 | doi:10.1038/ncb1514
Abstract | Full text | PDF (1,180K) | Supplementary Information
背景知識：
異染色質(zhì)的外遺傳學(xué)特點(diǎn)：
通過細(xì)胞核的結(jié)構(gòu)及空間構(gòu)型的重組來調(diào)控細(xì)胞及生物的功能,，稱為外遺傳學(xué)（epigenetics），其中epi在希臘文中是“上面,，外面”的意思,，也就是說外遺傳學(xué)研究的不是對DNA序列的改變,，而是對染色質(zhì)的調(diào)整，如基因沉默,， 染色體繼承等等,。
所有細(xì)胞核的最顯著的特點(diǎn)就是染色體，由DNA和組蛋白所組成的染色質(zhì)構(gòu)成,，DNA環(huán)繞由4個相似的組蛋白分子結(jié)合而形成的柱狀的芯上,，從而形成核小體，核小體之間由連續(xù)的DNA鏈連接,，形成串珠樣結(jié)構(gòu),，進(jìn)一步盤曲形成常染色質(zhì)或異染色質(zhì)。
大多數(shù)基因位于常染色質(zhì),，結(jié)構(gòu)相對疏松,，DNA易被轉(zhuǎn)錄。而異染色質(zhì)結(jié)構(gòu)致密,，所含基因很少,，其中的大部份DNA，包括大量的短的重復(fù)序列,，并不編碼蛋白質(zhì),。
異染色質(zhì)總是出現(xiàn)在染色體的末端，與端粒相鄰,。端粒隨著細(xì)胞的分裂而變短,，從而限制細(xì)胞分裂的次數(shù)。異染色質(zhì)也出現(xiàn)在著絲點(diǎn),，著絲點(diǎn)位于染色體的中部,，在細(xì)胞分裂時使染色體的分開。而異染色質(zhì)其他的功能尚未明了,。
相關(guān)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
作者簡介：
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]
相關(guān)報道：
著絲粒的異染色質(zhì)區(qū)對黏附的作用
