Transcriptional Gene Silencing in Chlamydomonas and Arabidopsis

衣藻和拟南芥中的转录基因沉默

• 批准号: 0131357
• 负责人: Heriberto Cerutti
• 金额: $ 39万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0131357&HistoricalAwards=false
• 关键词: Transcriptional; Gene; Silencing; Chlamydomonas; Arabidopsis

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.

该项目研究了植物中表观遗传转录沉默的分子机制。 在单细胞绿藻莱茵衣藻中，单拷贝转基因发生沉默，而引入的 DNA 没有可检测到的甲基化。 此过程需要包含 WD40 重复序列的蛋白 (Mut11p)，该蛋白与真菌转录共阻遏物 Tup1p 的 C 端结构域具有同源性。 类似的蛋白质广泛分布于真核生物中，但其功能尚不清楚。 衣藻中不依赖于甲基化的转基因沉默还需要丝氨酸/苏氨酸蛋白激酶 (Mut9p)，该激酶似乎是植物界特有的。 这两个基因对于转座元件的抑制、正常细胞生长以及对诱导 DNA 双链断裂的拟放射剂的耐受性也是必需的。 Mut9p 和 Mut11p 的密切同源物在拟南芥基因组中编码，尽管它们是小型多基因家族的成员。 有趣的是，对拟南芥 EST 数据库的分析表明，特定家族中的某些基因优先在生殖组织中表达。 人们很容易推测，在多细胞植物中，一些家族成员在每个细胞中表达，并在基础细胞功能中发挥作用，例如转座子抑制。 相比之下，其他成员可能在某个发育阶段专门调节特定的基因组。 该研究的近期目标包括： (1) 表征莱茵衣藻中 Mut9p 和 Mut11p 的分子作用。 这一目标将通过多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株和野生型菌株中的差异基因表达来确定受沉默机制调节的潜在目标位点。 (2) Mut9p 或 Mut11p 同源物在拟南芥发育中的作用的表征。 将在不同的发育阶段检查给定多基因家族中所有同源物的表达模式。 主要在生殖组织中表达的基因突变体将通过筛选 T-DNA 标记系来鉴定。 这些突变体的表型有望提供有关各个家族成员可能的功能差异的信息。 (3) 一种衣藻突变体 (Mut-39) 的表征，以及被破坏基因的克隆，该基因在多拷贝、甲基化转基因的转录沉默中存在缺陷。 由于这些转基因的结构类似于异染色质旋钮，这些发现可能有助于我们了解植物中的异染色质成分。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，在控制中发挥着重要作用。发育以及细胞对病毒、类病毒、转座元件和转基因的反应。 因此，了解表观遗传基因沉默的机制可能有助于深入了解基因调控和针对“基因组寄生虫”的防御反应的相对未探索但基本的特征。 这些发现也可能在农业和医学方面有实际应用，例如改进转基因技术或增强我们对表观遗传现象在癌症等恶性疾病中所起作用的理解。