Role of a Putative Histone Demethylase in the Ethylene Signaling Network

描述（申请人提供）：植物激素乙烯调节对植物存活重要的多种形态和生理反应。该提案的目的是定义EIN 6的作用，EIN 6在乙烯信号通路的未解决部分发挥作用。然而，这些实验中提出的工具和方法将对植物生物学的许多领域产生影响。初步序列分析显示，EIN 6含有Jumonji结构域和锌指结构域，表明其作为染色质重塑酶的作用。具体来说，EIN 6包含一个Jumonji结构域，该结构域与哺乳动物组蛋白脱甲基酶的JARID 1家族密切相关，最近研究表明该家族可使组蛋白3赖氨酸4（与主动转录相关的表观遗传标记）脱甲基。组蛋白甲基化是一个特别感兴趣的领域，因为这种表观遗传修饰与癌症相关的细胞畸变有关。我们建议使用染色质免疫沉淀（ChIP）耦合到通过Solexa测序技术的靶DNA鉴定作为一种公正的方法，以确定在基因组水平上的EIN 6靶序列。我们提出的体外试验将确定EIN 6是否具有脱甲基酶活性，这将是植物中组蛋白脱甲基酶的第一个例子，并为乙烯信号转导增加了一个有趣的调节水平。我们努力了解EIN 6影响乙烯反应的机制，包括使用遗传方法来鉴定与EIN 6相互作用的蛋白质，以及在植物中进行遗传筛选来鉴定EIN 6表型的增强子。这些研究将为乙烯信号转导带来新的认识水平，并可能揭示新的信号转导成分。有了更完整的途径，我们可以开始应用我们的知识，农业和经济方面的乙烯信号，包括水果成熟和响应病原体攻击。这里描述的特定目标集中在一个关键的植物突变体，是不敏感的关键生长激素，乙烯。确定这个以前没有特征的基因的作用将提高我们目前对植物乙烯信号的理解。这是我们将我们的知识应用于乙烯信号传导的农业和经济方面的长期目标的重要一步，例如水果成熟和对病原体攻击的反应。这种感兴趣的蛋白质被预测为组蛋白去甲基化酶，这对我们的工作提出了一个有趣的解释，因为这种类型的染色质修饰以前在植物中没有描述过，并且与许多人类疾病包括癌症有关。

DESCRIPTION (provided by applicant): The plant hormone ethylene regulates a variety of morphological and physiological responses important for plant survival. The aims of this proposal serve to define the role of EIN6, which functions in an unresolved section of the ethylene signaling pathway. However, tools and approaches proposed in these experiments will have implications for many areas of plant biology and beyond. Preliminary sequence analysis reveals that EIN6 contains Jumonji domains and zinc-finger domains, suggesting a role as a chromatin remodeling enzyme. Specifically, EIN6 contains a Jumonji domain that is closely related to the JARID1 family of mammalian histone demethylases, which have recently been shown to demethylate Histone 3 Lysine 4 (an epigenetic mark associated with active transcription). Histone methylation is an area of particular interest given that this epigenetic modification is connected to cellular aberrations associated with cancer. We propose the use of chromatin immunoprecipitation (ChIP) coupled to target DNA identification through Solexa sequencing technology as an unbiased approach to identify EIN6 target sequences at the genomic level. Our proposed in vitro assay will determine if EIN6 does possess demethylase activity, which would be the first example of a histone demethylase in plants and add an interesting level of regulation to ethylene signaling. Our efforts to understand the mechanism by which EIN6 influences the ethylene response includes using a genetic approach to identify proteins that interact with EIN6 and a genetic screen in planta to identify enhancers of the ein6 phenotype. These studies will bring a new level of understanding to ethylene signal transduction and may reveal novel signaling components. With a more complete view of the pathway we may begin to apply our knowledge to agricultural and economic aspects of ethylene signaling, including fruit ripening and response to pathogen attack. The Specific Aims described here are centered on a key plant mutant that is insensitive to the critical growth hormone, ethylene. Defining the role of this previously uncharacterized gene will improve our current understanding of ethylene signaling in plants. This is an important step towards our long-term goal of applying our knowledge to agricultural and economic aspects of ethylene signaling, such as fruit ripening and response to pathogen attack. This protein of interest is predicted to function as a histone demethylase, which puts an intriguing spin on our work as this type of chromatin modification has not been previously described in plants and is linked to many human diseases including cancer.