RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins

At the silent mating-type interval of fission yeast, the RNA interference (RNAi) machinery cooperates with cenH, a DNA element homologous to centromeric repeats, to initiate heterochromatin formation. However, in RNAi mutants, heterochromatin assembly can still occur at low efficiency. Here, we report that Atf1 and Pcr1, two ATF/CREB family proteins, act in a parallel mechanism to the RNAi pathway for heterochromatin nucleation. Deletion of atf1 or pcr1 alone has little effect on silencing at the mating-type region, but when combined with RNAi mutants, double mutants fail to nucleate heterochromatin assembly. Moreover, deletion of atf1 or pcr1 in combination with cenH deletion causes loss of silencing and heterochromatin formation. Furthermore, Atf1 and Pcr1 bind to the mating-type region and target histone H3 lysine-9 methylation and the Swi6 protein essential for heterochromatin assembly. These analyses link ATF/CREB family proteins, involved in cellular response to environmental stresses, to nucleation of constitutive heterochromatin.     

Requirement of heterochromatin for cohesion at centromeres

Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin. Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.     

植物着丝粒区串联重复序列的研究进展

着丝粒是细胞染色体的重要结构组成,控制姊妹染色单体的结合、动粒的组装和纺锤丝的附着,确保真核生物细胞在有丝分裂和减数分裂过程中染色体的正常分离及遗传信息的稳定传递。植物着丝粒DNA序列主要由反转录转座子和串联重复序列构成。串联重复序列在着丝粒功能实现和基因组进化过程中起重要作用。随着测序技术的成熟,近年来对串联重复序列的研究取得了很大的进展。综述了植物串联重复序列结构、分析方法及在进化中的作用,以期为相关研究提供参考。     

高等植物着丝粒序列的结构与特征研究

着丝粒是染色体的重要组成成分之一,在细胞有丝分裂和减数分裂中行使重要的生物学功能,着丝粒在不同的物1种中保持着一致的功能,即在细胞分裂中期,着丝粒在纺锤丝的牵引下使染色体向两级运动,从而完成细胞的分裂。如此保守的功能,是乎暗示着丝粒序列不同物种间具有一定的保守性,然而随着测序技术的发展,越来越多的物种的基因组序列被释放,分析发现着丝粒序列主要由重复序列和反转录转座子组成,然而不同物种着丝粒序列比对发现,它们之间的同源性极低,此外染色体的着丝粒的序列的组成和大小都相差甚远。文中回顾了近些年在着丝粒研究方面所取得的进展,探讨维持着丝粒功能稳定性的序列结构特征。     

Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing

The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.     

甘蓝型油菜丙酮酸激酶基因RNA干扰载体的构建

采用基因芯片技术,从油菜中鉴定了一个油分积累优势表达基因即丙酮酸激酶（Pyruvate kinase, PK）基因。为构建甘蓝型油菜PK基因的RNA干扰（RNAi）载体,通过设计引物克隆了 PK基因长498 bp的 siRNA靶序列,连接到 pEASY-T1载体上,采用 NotI和XhoI酶切,酶切产物连接到本课题组新近改造的 RNAi 平台载体 pHurricane 的 NotI 和XhoI位点,通过多重 PCR 鉴定证实载体构建成功,然后将上述 PK基因反向重复框克隆到加入 napin启动子的 pCAMBIA1390表达载体相应的酶切位点上,构建具有种子特异性表达的PK基因的RNAi载体。限制性内切酶酶切证实载体构建成功,为进一步研究 PK基因参与决定油菜油分积累的分子机理和代谢调控奠定了基础。     

Heterochromatin integrity affects chromosome reorganization after centromere dysfunction

The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference-dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.     

Establishment and maintenance of a heterochromatin domain

The higher-order assembly of chromatin imposes structural organization on the genetic information of eukaryotes and is thought to be largely determined by posttranslational modification of histone tails. Here, we study a 20-kilobase silent domain at the mating-type region of fission yeast as a model for heterochromatin formation. We find that, although histone H3 methylated at lysine 9 (H3 Lys9) directly recruits heterochromatin protein Swi6/HP1, the critical determinant for H3 Lys9 methylation to spread in cis and to be inherited through mitosis and meiosis is Swi6 itself. We demonstrate that a centromere-homologous repeat (cenH) present at the silent mating-type region is sufficient for heterochromatin formation at an ectopic site, and that its repressive capacity is mediated by components of the RNA interference (RNAi) machinery. Moreover, cenH and the RNAi machinery cooperate to nucleate heterochromatin assembly at the endogenous mat locus but are dispensable for its subsequent inheritance. This work defines sequential requirements for the initiation and propagation of regional heterochromatic domains.     

RNAi技术及其应用

RNAi主要通过双链RNA(dsRNA)被核酸酶切割成21~23 nt的干涉性小的RNA,即siRNA,由siRNA介导识别并靶向切割同源性靶mRNA分子而实现。目前RNAi技术在基因功能和基因治疗等方面的研究有了广泛的应用,RNAi技术有望成为后基因组时代基因功能分析的有力工具。     

RNA干涉原理及其应用

RNA干涉是指外源dsRNA引发生物体内的基因的同源序列降解，从而表现出的基因转录后的沉默现象，它与植物中的共抑制和真菌中的基因压制可能具有相同的作用机制。在这一过程中，需要eIF2c类似蛋白因子、RNA螺旋酶、RNA依赖性RNA多聚酶、核糖核酸酶、ATP和转膜蛋白参与。RNA干涉可以用于功能基因组学研究，也可用于克服转基因生物的基因沉默现象，使外源基因在遗传改良生物中能更好地表达，还用于基因治疗，抑制有害基因的表达等。     

Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants.     

RNA干涉技术研究进展

RNA干涉是由双链RNA引起的转录后基因沉默机制.RNAi作为一门新的基因沉默技术,具有序列特异性和高效性等许多优点,在基因功能研究、疾病治疗、药物开发等方面具有广泛的应用.综述RNAi现象的发现、发生机制及其应用,并展望未来的研究.     

RNAi及其在哺乳动物中的应用*

 RNA干扰(RNAi)是由特定双链RNA(dsRNA)引发的转录后基因沉默,广泛存在于真菌、植物和动物中,它是近年来迅速发展起来的高效、特异、易操作的基因阻断技术,在功能基因组的研究中有着广阔的应用前景。研究表明,断裂dsRNA产生的小干扰RNA(siRNA)可抑制哺乳动物基因表达。从哺乳动物整体水平应用研究的角度,对RNAi的分子机制、生物学功能和特点,siRNA导入体内的方法以及应用等方面的研究作一综述。