Transcriptional Gene Silencing in Chlamydomonas and Arabidopsis

本项目研究植物表观遗传转录沉默的分子机制。在单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）中，单拷贝转基因的沉默发生在引入DNA没有检测到甲基化的情况下。这一过程需要含有WD40-repeat的蛋白（Mut11p），该蛋白与真菌转录共抑制因子Tup1p的c端结构域同源。类似的蛋白广泛分布于真核生物中，但其功能尚不清楚。衣藻中不依赖甲基化的转基因沉默也需要丝氨酸/苏氨酸蛋白激酶（Mut9p），这似乎是植物界特有的。这两个基因也是抑制转座因子、正常细胞生长和耐受诱导DNA双链断裂的模拟辐射剂所必需的。Mut9p和Mut11p的同源基因在拟南芥基因组中编码，尽管它们是小的多基因家族的成员。有趣的是，对拟南芥EST数据库的分析表明，在一个给定的家族中，一些基因在生殖组织中优先表达。我们很容易推测，在多细胞植物中，一些家族成员在每个细胞中都表达，并在基础细胞功能中发挥作用，如转座子抑制。相比之下，其他成员可能在特定的发育阶段专门调节特定的基因组。本研究的近期目标将包括：(1)确定Mut9p和Mut11p在莱茵衣藻中的分子作用。这一目标将通过各种蛋白质组学方法分离与克隆基因产物相互作用的蛋白质来实现。该研究还将通过分析突变型和野生型菌株的差异基因表达，确定由沉默机制调节的潜在目标位点。(2) Mut9p或Mut11p同源基因在拟南芥发育中的作用。在一个给定的多基因家族中，所有同源物的表达模式将在不同的发育阶段进行检查。主要在生殖组织中表达的基因突变将通过筛选T-DNA标记系来鉴定。这些突变体的表型有望为不同家族成员可能的功能差异提供信息。(3)一个衣藻突变体（mutt -39）的鉴定，并克隆了该突变基因，该基因在多拷贝、甲基化转基因的转录沉默中存在缺陷。由于这些转基因基因的结构类似于异染色质旋钮，这一发现可能有助于我们对植物中异染色质成分的理解。在真核生物中，表观遗传过程在不改变DNA序列的情况下导致基因表达的可遗传变化，在控制发育以及细胞对病毒、类病毒、转座因子和转基因的反应中发挥重要作用。因此，理解表观遗传基因沉默的机制可能会为基因调控和防御“基因组寄生虫”的基本特征提供相对未被探索的见解。这些发现也可能在农业和医学上有实际应用，例如改进转基因技术或增强我们对表观遗传现象在恶性疾病（如癌症）中所起作用的理解。

This project addresses the molecular mechanisms responsible for epigenetic transcriptional silencing in plants. In the unicellular green alga Chlamydomonas reinhardtii, silencing of single-copy transgenes occurs without detectable methylation of the introduced DNA. A WD40-repeat containing protein (Mut11p), with homology to the C-terminal domain of the fungal transcriptional co-repressor Tup1p, is required for this process. Similar proteins are widely distributed among eukaryotes but their function is unknown. Methylation-independent transgene silencing in Chlamydomonas also requires a serine/threonine protein kinase (Mut9p) that appears to be specific to the plant kingdom. Both genes are also necessary for the repression of transposable elements, normal cellular growth, and tolerance to radiomimetic agents inducing DNA double strand breaks. Close homologs of Mut9p and Mut11p are encoded in the Arabidopsis thaliana genome, albeit as members of small multigene families. Intriguingly, an analysis of the Arabidopsis EST databases suggests that some genes, in a given family, are expressed preferentially in reproductive tissues. It is tempting to speculate that in multicellular plants some family members are expressed in every cell and play a role in basal cellular functions, such as transposon repression. In contrast, other members may have specialized to regulate specific sets of genes at a certain developmental stage. The immediate goals of the study will include: (1) Characterization of the molecular role(s) of Mut9p and Mut11p in Chlamydomonas reinhardtii. This objective will be achieved through the isolation of proteins interacting with the cloned gene products, by a variety of proteomic approaches. The research will also identify potential target loci regulated by the silencing machinery, by an analysis of differential gene expression in mutant and wild-type strains. (2) Characterization of the role(s) of homologs of Mut9p or Mut11p in Arabidopsis development. The expression pattern of all homologs in a given multigene family will be examined at different stages of development. Mutants in genes predominantly expressed in reproductive tissues will be identified by screening T-DNA tagged lines. The phenotype(s) of these mutants is expected to provide information on the possible functional divergence of the various family members. (3) Characterization of one Chlamydomonas mutant (Mut-39), and cloning of the disrupted gene, defective in the transcriptional silencing of multiple copy, methylated transgenes. Since the structure of these transgenes resembles a heterochromatic knob, the findings may contribute to our understanding of heterochromatin components in plants.In eukaryotes, epigenetic processes, which result in heritable changes in gene expression without modifications in DNA sequence, play important roles in the control of development as well as in the cellular responses to viruses, viroids, transposable elements, and transgenes. Thus, understanding the mechanisms of epigenetic gene silencing may provide insights into relatively unexplored but essential features of gene regulation and of defense responses against "genomic parasites." These findings may also have practical applications in agriculture and medicine, such as improving transgenic technology or enhancing our understanding of the role that epigenetic phenomena play in malignant diseases such as cancer.