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.     

DNA replication-independent silencing in S. cerevisiae

In Saccharomyces cerevisiae, the silent mating loci are repressed by their assembly into heterochromatin. The formation of this heterochromatin requires a cell cycle event that occurs between early S phase and G(2)/M phase, which has been widely assumed to be DNA replication. To determine whether DNA replication through a silent mating-type locus, HMRa, is required for silencing to be established, we monitored heterochromatin formation at HMRa on a chromosome and on a nonreplicating extrachromosomal cassette as cells passed through S phase. Cells that passed through S phase established silencing at both the chromosomal HMRa locus and the extrachromosomal HMRa locus with equal efficiency. Thus, in contrast to the prevailing view, the establishment of silencing occurred in the absence of passage of the DNA replication fork through or near the HMR locus, but retained a cell cycle dependence.     

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.     

Heterochromatin and epigenetic control of gene expression

Eukaryotic DNA is organized into structurally distinct domains that regulate gene expression and chromosome behavior. Epigenetically heritable domains of heterochromatin control the structure and expression of large chromosome domains and are required for proper chromosome segregation. Recent studies have identified many of the enzymes and structural proteins that work together to assemble heterochromatin. The assembly process appears to occur in a stepwise manner involving sequential rounds of histone modification by silencing complexes that spread along the chromatin fiber by self-oligomerization, as well as by association with specifically modified histone amino-terminal tails. Finally, an unexpected role for noncoding RNAs and RNA interference in the formation of epigenetic chromatin domains has been uncovered.     

RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation

Facultative heterochromatin that changes during cellular differentiation coordinates regulated gene expression, but its assembly is poorly understood. Here, we describe facultative heterochromatin islands in fission yeast and show that their formation at meiotic genes requires factors that eliminate meiotic messenger RNAs (mRNAs) during vegetative growth. Blocking production of meiotic mRNA or loss of RNA elimination factors, including Mmi1 and Red1 proteins, abolishes heterochromatin islands. RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. Heterochromatin islands disassemble in response to nutritional signals that induce sexual differentiation. This process involves the antisilencing factor Epe1, the loss of which causes dramatic increase in heterochromatic loci. Our analyses uncover unexpected regulatory roles for mRNA-processing factors that assemble dynamic heterochromatin to modulate gene expression.     

Argonaute2 is the catalytic engine of mammalian RNAi

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.     

Control of genic DNA methylation by a jmjC domain-containing protein in Arabidopsis thaliana

Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.     

Functional genomic analysis of RNA interference in C. elegans

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.     

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.     

Genetic requirements for inheritance of RNAi in C. elegans

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurred through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 were required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent.     

蛋白激酶A催化亚基VdPKAC1对菌丝型大丽轮枝菌V07DF2培养性状及致病力的调控

【目的】通过同源重组敲除技术,解析菌丝型大丽轮枝菌中蛋白激酶A催化亚基基因VdPKAC1在调控微菌核发育、产孢及其致病力方面的作用。【方法】利用Invitrogen公司的Gateway®技术构建VdPKAC1的同源重组敲除质粒。该质粒的潮霉素抗性基因盒两端分别为靶标基因5′和3′端各1 kb的DNA序列。通过农杆菌AGL-1的介导,将该质粒的T-DNA区域整合到来源于棉花的菌丝型大丽轮枝菌V07DF2菌株中。从6个随机选择的含有潮霉素抗性基因片段的转化子中,通过靶标基因的PCR检测,获得了5个VdPKAC1基因敲除突变体。经过Southern杂交检测,选择了3个敲除突变体材料（2B5、C5和HH2）进行突变表型的观察和分析。在液体PDB培养7 d后观察敲除突变体与野生型菌株的黑色素产生情况;在固体PDA培养28 d后,观察敲除突变体与野生菌株的菌丝生长和休眠结构形成情况。此外,利用棉花根提取物对液体Cazpek-Dox培养7 d的各菌株分别进行诱导处理,在处理24、72 h后分别统计分生孢子数量,以此对敲除突变体和野生菌株的分生孢子产生能力进行评估。最后,将孢子浓度为1×107个/mL的突变体和野生菌株的分生孢子液灌根接种2叶期的棉花,测定其致病力。【结果】Southern杂交结果表明,在上述5个敲除突变体中,只有2B5和C5两个菌株为T-DNA单拷贝插入,而其他菌株均存在T-DNA异位整合的情况。在PDA培养条件下,3个敲除突变体（2B5、C5和HH2）与野生型相比具有黑色素合成增加,气生菌丝更加发达的特点。其中,敲除突变体2B5和C5在平板培养条件下还形成了野生型菌株没有的、与典型微菌核形态不同的深褐色“链状休眠菌丝体结构”。在棉花根提取物的诱导处理下,3个敲除突变体产生的分生孢子数量少于野生型菌株。另外,致病力分析结果表明,敲除突变体材料仍然具有一定的致病力,但却显著弱于野生菌株V07DF2。【结论】在大丽轮枝菌中,通过蛋白激酶A介导的环腺苷酸信号途径控制多种重要性状,而VdPKAC1是这一途径中的重要成员。以前的报道证实,VdPKAC1负调控微菌核的数量,而本研究利用菌丝型菌株V07DF2在PDA培养基上不产生微菌核的这一特性,通过同源重组原理敲除该负调控基因,成功获得可以在PDA上产生休眠结构的菌丝型大丽轮枝菌菌株的突变体。这些突变体材料将为大丽轮枝菌微菌核发育信号途径及其调控基因的发掘提供平台和基础。